Expression profiling and Ingenuity biological function analyses of interleukin-6- versus nerve growth factor-stimulated PC12 cells

  • Dieter Kunz1Email author,

    Affiliated with

    • Gaby Walker2Email author,

      Affiliated with

      • Marc Bedoucha3,

        Affiliated with

        • Ulrich Certa4,

          Affiliated with

          • Pia März-Weiss2,

            Affiliated with

            • Beatrice Dimitriades-Schmutz1 and

              Affiliated with

              • Uwe Otten1

                Affiliated with

                BMC Genomics200910:90

                DOI: 10.1186/1471-2164-10-90

                Received: 04 December 2008

                Accepted: 24 February 2009

                Published: 24 February 2009

                Abstract

                Background

                The major goal of the study was to compare the genetic programs utilized by the neuropoietic cytokine Interleukin-6 (IL-6) and the neurotrophin (NT) Nerve Growth Factor (NGF) for neuronal differentiation.

                Results

                The designer cytokine Hyper-IL-6 in which IL-6 is covalently linked to its soluble receptor s-IL-6R as well as NGF were used to stimulate PC12 cells for 24 hours. Changes in gene expression levels were monitored using Affymetrix GeneChip technology. We found different expression for 130 genes in IL-6- and 102 genes in NGF-treated PC12 cells as compared to unstimulated controls. The gene set shared by both stimuli comprises only 16 genes.

                A key step is upregulation of growth factors and functionally related external molecules known to play important roles in neuronal differentiation. In particular, IL-6 enhances gene expression of regenerating islet-derived 3 alpha (REG3A; 1084-fold), regenerating islet-derived 3 beta (REG3B/PAPI; 672-fold), growth differentiation factor 15 (GDF15; 80-fold), platelet-derived growth factor alpha (PDGFA; 69-fold), growth hormone releasing hormone (GHRH; 30-fold), adenylate cyclase activating polypeptide (PACAP; 20-fold) and hepatocyte growth factor (HGF; 5-fold). NGF recruits GDF15 (131-fold), transforming growth factor beta 1 (TGFB1; 101-fold) and brain-derived neurotrophic factor (BDNF; 89-fold). Both stimuli activate growth-associated protein 43 (GAP-43) indicating that PC12 cells undergo substantial neuronal differentiation.

                Moreover, IL-6 activates the transcription factors retinoic acid receptor alpha (RARA; 20-fold) and early growth response 1 (Egr1/Zif268; 3-fold) known to play key roles in neuronal differentiation.

                Ingenuity biological function analysis revealed that completely different repertoires of molecules are recruited to exert the same biological functions in neuronal differentiation. Major sub-categories include cellular growth and differentiation, cell migration, chemotaxis, cell adhesion, small molecule biochemistry aiming at changing intracellular concentrations of second messengers such as Ca2+ and cAMP as well as expression of enzymes involved in posttranslational modification of proteins.

                Conclusion

                The current data provide novel candidate genes involved in neuronal differentiation, notably for the neuropoietic cytokine IL-6. Our findings may also have impact on the clinical treatment of peripheral nerve injury. Local application of a designer cytokine such as H-IL-6 with drastically enhanced bioactivity in combination with NTs may generate a potent reparative microenvironment.

                Background

                Interleukin-6 (IL-6) is the prototype member of the IL-6 cytokine family, also termed neuropoietic cytokines, including IL-6, IL-11, IL-27, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), oncostatin M, cardiotrophin-1 (CT-1), cardiotrophin-like cytokine (CLC; also known as novel neurotrophin 1, NNT1), neuropoietin and B cell stimulatory factor 3 (BSF3) [1, 2]. A common feature of all family members is the signaling through a specific receptor that is associated to the intracellularly located transduction component gp130. Subsequently, the Janus-activated kinase-signal transducer, activator of transcription (JAK-STAT) and mitogen-activated protein kinase (MAPK) signal transduction pathways are activated. Neuropoietic cytokines display multiple functions in the peripheral (PNS) and central nervous systems (CNS), including the developing and adult brain, synaptic plasticity as well as the brain's response to injury and disease. In particular these molecules control cell fate and differentiation of neural stem and progenitor cells during development; due to their neurotrophic and regenerative actions they crucially affect injury-induced neurogenesis, neuronal survival and regeneration; moreover, these molecules can also influence neuronal activity and are implicated in long-term potentiation (LTP; reviewed in [2]).

                Cellular functions of IL-6 are mediated by two specific receptors, the membrane-bound 80 KDa IL-6 receptor (IL-6R) or the soluble form of IL-6R (s-IL-6R) which can be generated either by shedding of IL-6R or by alternative splicing of the IL-6R mRNA [3, 4]. Using s-IL-6R, IL-6 responsiveness may be conferred to cells expressing the transduction component gp130, but are devoid of membrane-bound IL-6R in the process of transsignaling [57]. The transsignaling mechanism led to the development of a fusion protein in which IL-6 is covalently linked to s-IL-6R thereby creating a unimolecular protein with enhanced biological activities. The fusion protein, termed Hyper-IL-6 (H-IL-6), turned out to be fully active at 100–1000-fold lower concentrations as compared to the combination of the two separate molecules [8, 9].

                The neurotrophin (NT) family of growth factors including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and NT-4/5 is important for development, maintenance and survival of many different cell types in the PNS and the CNS [10]. NTs are also involved in regulating adult neurogenesis [11, 12], learning and memory [13, 14]. NTs are synthesized as proNT precursors that may be processed to mature NTs intra- and extracellulary by specific proteases [15]. NTs exert their effects via two different types of cellular receptors: pan-neurotrophin receptor p75 (p75NTR) which binds all NTs with a similar affinity, and the family of high affinity tyrosine kinase receptors (Trk). The interactions of proNTs and NTs with the NT-receptors comprise a complex signaling system thus generating a broad variety of biological effects [16, 17].

                In the first report of IL-6 actions on neural cells rat pheochromocytoma cells (PC12), a well characterised cellular model for neuronal differentiation, were incubated for up to 6 days with B-cell stimulatory factor BSF-2/IL-6 thereby inducing significant neurite outgrowth [18]. PC12 cells that were differentiated either using irradiation [19] or the well-known hypoxia mimetic agent CoCl2 [20] require IL-6 expression. We have demonstrated that primary sympathetic neurons [21] and PC12 cells [22] can strongly respond to IL-6 by transsignaling, and that the potential of IL-6 to induce neuronal differentiation in PC12 cells is in close correlation to the availability of s-IL-6R [22, 23]. PC12 cell differentiation is accompanied by enhanced expression of GAP-43 mRNA at 24 hours after stimulation with IL-6/s-IL-6R [22]. Moreover, we found that the fusion protein H-IL-6 is a highly active molecule in inducing survival of cultured sympathetic neurons, comparable to the effects of NGF [21, 22]. Recently, IL6RIL6, a fusion protein in which IL-6 is directly linked to the extracellular domain of the IL-6 specific receptor, has been used for expression profiling studies in primary cultures of dorsal root ganglia. In these cells, IL6RIL6 strongly increases axonal network and expression of neural genes [24].

                A significant problem in the clinical treatment of peripheral nerve injury is that the currently used therapeutic approaches do not allow complete neuronal recovery [25]. Mixtures comprising neuropoietic cytokines, glial cell-line derived neurotrophic factor ligands (GFLs) and NTs are being tested for the suitability to generate a microenvironment with a high reparative potential upon local administration at the site of the lesion [26].

                In the present study we monitored changes in neuronal gene expression induced by incubation of PC12 cells for 24 hours with H-IL-6 as well as NGF, and compared the genetic programs utilized by these stimuli for neuronal differentiation.

                Results

                Overall changes in gene expression patterns in IL-6- and NGF-stimulated PC12 cells

                Affymetrix Gene Chip U34A arrays were used to analyse global changes in gene transcripts using a cutoff in the change of gene expression of > 2-fold. In PC12 cells stimulated for 24 h with 10 ng/ml H-IL-6, we found 130 differently expressed genes as compared to unstimulated controls. Of them, 94 genes were upregulated with gene expression values from 2-fold to 1085-fold, whereas 36 genes were found to be downregulated in the range from -2-fold to -61-fold. The genes are further classified into major functional categories including cytokines (2 up-regulated/0 down-regulated), enzymes (20/8), G-protein coupled receptors (2/3), growth factors (7/1), ion channels (2/0), kinases (4/4), nuclear receptors (2/1), peptidases (3/1), phosphatases (0/2), transcription regulators (8/4), transmembrane receptors (5/0), transporters (8/3) and molecules with other functions (31/9; Table 1).
                Table 1

                List of gene set regulated by IL-6 in PC12 cells

                Gene

                 

                Accession no.

                Fold change

                Subcellular location

                Cytokines

                    

                   chemokine ligand 13

                CXCL13

                AF044196

                43

                Extracellular Space

                   chemokine ligand 10

                CXCL10

                U17035

                7

                Extracellular Space

                Enzymes

                    

                   cytochrome P450, 4f16

                CYP4F16

                U39207

                424

                Cytoplasm

                   ceruloplasmin

                CP

                AF202115

                191

                Extracellular Space

                   peptidyl arginine deiminase, type III

                PADI3

                D88034

                142

                Cytoplasm

                   acyl-CoA synthetase, member 1

                ACSL1

                D90109

                102

                Cytoplasm

                   transglutaminase 1

                TGM1

                M57263

                93

                Plasma Membrane

                   nitric oxide synthase 2A

                NOS2A

                U03699

                58

                Cytoplasm

                   ornithine carbamoyltransferase

                OTC

                M11266

                43

                Cytoplasm

                   Similar to Lysophospholipase

                LOC374569

                AB009372

                37

                Unknown

                   trehalase

                TREH

                AF038043

                35

                Plasma Membrane

                   kynureninase

                KYNU

                U68168

                25

                Cytoplasm

                   nitric oxide synthase 3

                NOS3

                AJ011115

                21

                Cytoplasm

                   glycine amidinotransferase

                GATM

                U07971

                14

                Cytoplasm

                   guanine nucleotide binding protein, alpha z

                GNAZ

                U77485

                14

                Plasma Membrane

                   ST6 galactosamide alpha-2,6-sialyltranferase 1

                ST6GAL1

                M83143

                14

                Cytoplasm

                   aldo-keto reductase, 1C1

                AKR1C1

                BAA92883

                12

                Cytoplasm

                   myxovirus resistance 1

                MX1

                P20591

                9

                Nucleus

                   aldolase C

                ALDOC

                X06984

                3

                Cytoplasm

                   2',5'-oligoadenylate synthetase 1

                OAS1

                Z18877

                3

                Cytoplasm

                   protein disulfide isomerise, A2

                PDIA2

                AAC50401

                3

                Cytoplasm

                   RNA (guanine-7-) methyltransferase

                RNMT

                BAA82447

                3

                Nucleus

                   polymerase, alpha 2

                POLA2

                AJ245648

                -2

                Nucleus

                   steroid-5-alpha-reductase, alpha 1

                SRD5A1

                J05035

                -2

                Cytoplasm

                   aminolevulinate, delta-, synthase 2

                ALAS2

                D86297

                -3

                Cytoplasm

                   glutathione S-transferase A3

                GSTA3

                X78847

                -3

                Cytoplasm

                   UDP glycosyltransferase 8

                UGT8

                BC075069

                -3

                Cytoplasm

                   cell division cycle 42

                CDC42

                U37720

                -4

                Cytoplasm

                   cysteine dioxygenase, type I

                CDO1

                M35266

                -4

                Cytoplasm

                   ST8 alpha-2,8-sialyltransferase 3

                ST8SIA3

                X80502

                -5

                Cytoplasm

                G-protein coupled receptors

                    

                   adrenergic receptor, alpha-2B

                ADRA2B

                M32061

                26

                Plasma Membrane

                   arginine vasopressin receptor 2

                AVPR2

                AAB87678

                5

                Plasma Membrane

                   vasoactive intestinal peptide receptor 1

                VIPR1

                M86835

                -2

                Plasma Membrane

                   cholinergic receptor, muscarinic 3

                CHRM3

                AB017656

                -3

                Plasma Membrane

                   cholinergic receptor, muscarinic 4

                CHRM4

                M16409

                -10

                Plasma Membrane

                Growth factors

                    

                   regenerating islet-derived 3 alpha

                REG3A

                L10229

                1084

                Extracellular Space

                   regenerating islet-derived 3 beta

                REG3B

                S43715

                672

                Extracellular Space

                   growth differentiation factor 15

                GDF15

                AJ011970

                80

                Extracellular Space

                   platelet-derived growth factor alpha

                PDGFA

                M29464

                69

                Extracellular Space

                   nudix-type motif 6

                NUDT6

                AF188995

                22

                Extracellular Space

                   jagged 2

                JAG2

                U70050

                5

                Extracellular Space

                   hepatocyte growth factor

                HGF

                X84046

                4

                Extracellular Space

                   macrophage stimulating 1

                MST1

                X95096

                -4

                Extracellular Space

                Ion channels

                    

                   glutamate receptor, ionotropic, delta 2

                GRID2

                U08256

                91

                Plasma Membrane

                   purinergic receptor P2X

                P2RX2

                Y10475

                11

                Plasma Membrane

                Kinases

                    

                   fyn-related kinase

                FRK

                U02888

                122

                Nucleus

                   Janus kinase 2

                JAK2

                U13396

                120

                Cytoplasm

                   phosphatidylinositol 4-kinase beta

                PI4KB

                D84667

                2

                Cytoplasm

                   pim-3 oncogene

                PIM3

                AF086624

                2

                Unknown

                   fer tyrosine kinase

                FER

                X13412

                -2

                Cytoplasm

                   mitogen-activated protein kinase kinase 5

                MAP2K5

                U37462

                -2

                Cytoplasm

                   fibroblast growth factor receptor 1

                FGFR1

                S54008

                -3

                Plasma Membrane

                   activin receptor, type IIA

                ACVR2A

                S48190

                -4

                Plasma Membrane

                Nuclear receptors

                    

                   retinoic acid receptor alpha

                RARA

                U15211

                20

                Nucleus

                   nuclear receptor, *C2

                NR3C2

                M36074

                8

                Nucleus

                   vitamin D receptor

                VDR

                J03630

                -4

                Nucleus

                Peptidases

                    

                   complement component 1s

                C1S

                D88250

                230

                Extracellular Space

                   caspase 1

                CASP1

                U14647

                40

                Cytoplasm

                   proteasome subunit, alpha 1

                PSMA1

                M29859

                5

                Cytoplasm

                   kallikrein-related peptidase 8

                KLK8

                AJ005641

                -5

                Extracellular Space

                Phosphatases

                    

                   pyruvate dehydrogenase phosphatase 2

                PDP2

                AF062741

                -4

                Cytoplasm

                   protein tyrosine phosphatase receptor D

                PTPRD

                U57502

                -9

                Plasma Membrane

                Transcription regulators

                    

                   signal transducer and activator of transcription 1

                STAT1

                AF205604

                579

                Nucleus

                   Kruppel-like factor 6

                KLF6

                AF072403

                249

                Nucleus

                   HIV-1 Tat interacting protein

                HTATIP

                AAB18236

                159

                Nucleus

                   HIV enhancer binding protein 2

                HIVEP2

                D37951

                65

                Nucleus

                   upstream transcription factor 1

                USF1

                U41741

                22

                Nucleus

                   early growth response 1

                EGR1

                M18416

                3

                Nucleus

                   interferon regulatory factor 1

                IRF1

                M34253

                3

                Nucleus

                   signal transducer and activator of transcription 2

                STAT2

                AF206162

                3

                Nucleus

                   breast cancer 1

                BRCA1

                U36475

                -2

                Nucleus

                   D site of albumin promoter binding protein

                DBP

                J03179

                -2

                Nucleus

                   nuclear factor I/B

                NFIB

                Y07685

                -2

                Nucleus

                   transcription elongation factor A 2

                TCEA2

                D12927

                -5

                Nucleus

                Transmembrane receptors

                    

                   oxidized low density lipoprotein receptor 1

                OLR1

                AB018097

                587

                Plasma Membrane

                   histocompatibility 2, Q region locus 10

                H2-Q10

                M31018

                160

                Plasma Membrane

                   insulin-like growth factor 2 receptor

                IGF2R

                NM_000876

                39

                Plasma Membrane

                   Fc fragment of IgG receptor IIa (CD32)

                FCGR2A

                M64368

                16

                Plasma Membrane

                   growth hormone receptor

                GHR

                Z83757

                12

                Plasma Membrane

                Transporters

                    

                   cadherin 17

                CDH17

                X78997

                273

                Plasma Membrane

                   solute carrier family 6, member 3

                SLC6A3

                M80570

                90

                Plasma Membrane

                   nucleoporin 153kDa

                NUP153

                L06821

                83

                Nucleus

                   solute carrier family 9, member 2

                SLC9A2

                L11004

                32

                Plasma Membrane

                   cadherin 17

                CDH17

                L46874

                13

                Plasma Membrane

                   lipocalin 2

                LCN2

                X13295

                9

                Extracellular Space

                   syntaxin 4

                STX4

                L20821

                3

                Plasma Membrane

                   secretory carrier membrane protein 2

                SCAMP2

                AF295405

                2

                Cytoplasm

                   solute carrier family 12, member 5

                SLC12A5

                U55816

                -3

                Plasma Membrane

                   solute carrier family 30, member 2

                SLC30A2

                U50927

                -5

                Plasma Membrane

                   syntaxin 5

                STX5

                U87971

                -8

                Cytoplasm

                Others

                    

                   regenerating islet-derived 1 alpha

                REG1A

                J05722

                796

                Extracellular Space

                   TIMP metallopeptidase inhibitor 1

                TIMP1

                L31883

                210

                Extracellular Space

                   calcitonin-related polypeptide beta

                CALCB

                M11596

                195

                Extracellular Space

                   fibrinogen gamma chain

                FGG

                J00734

                164

                Extracellular Space

                   trans-golgi network protein 2

                TGOLN2

                X53565

                113

                Cytoplasm

                   LIM and senescent cell antigen-like domains 1

                LIMS1

                AAA20086

                94

                Plasma Membrane

                   alpha-2-HS-glycoprotein

                AHSG

                M29758

                80

                Extracellular Space

                   ribosomal protein L3-like

                RPL3L

                AAC50777

                60

                Unknown

                   collagen, type IV, alpha 5

                COL4A5

                AB041350

                59

                Extracellular Space

                   parvalbumin

                LOC4951

                J02705

                58

                Unknown

                   YTH domain containing 1

                YTHDC1

                AF144731

                39

                Cytoplasm

                   growth hormone releasing hormone

                GHRH

                Z34092

                31

                Extracellular Space

                   annexin A1

                ANXA1

                M19967

                29

                Plasma Membrane

                   collagen, type XII, alpha 1

                COL12A1

                U57362

                26

                Extracellular Space

                   regenerating islet-derived 3 gamma

                REG3G

                L20869

                24

                Extracellular Space

                   adenylate cyclase activating polypeptide 1

                ADCYAP1

                S83513

                20

                Extracellular Space

                   heat shock protein 90 kDa, alpha B 1

                HSP90AB1

                S45392

                20

                Cytoplasm

                   luteinizing hormone beta

                LHB

                U25653

                17

                Extracellular Space

                   galectin 5

                LGALS5

                L36862

                8

                Extracellular Space

                   myocilin

                MYOC

                AF093567

                8

                Cytoplasm

                   prolactin family 8a81

                PRL8A8

                AB000107

                8

                Extracellular Space

                   troponin C type 2

                TNNC2

                J05598

                8

                Unknown

                   ribosomal protein L18a

                RPL18A

                X14181

                7

                Cytoplasm

                   fibrinogen beta chain

                FGB

                U05675

                6

                Extracellular Space

                   tropomyosin 3

                TPM3

                X72859

                4

                Cytoplasm

                   tubulin, beta

                TUBB

                AB011679

                4

                Cytoplasm

                   extracellular proteinase inhibitor

                EXPI

                X13309

                3

                Extracellular Space

                   growth associated protein 43

                GAP43

                M16736

                3

                Plasma Membrane

                   galectin 9

                LGALS9

                U72741

                3

                Extracellular Space

                   tubulin, alpha 4a

                TUBA4A

                M13444

                3

                Cytoplasm

                   BCL2-like 11

                BCL2L11

                AF136927

                2

                Cytoplasm

                   integrin alpha 7

                ITGA7

                X65036

                -2

                Plasma Membrane

                   syndecan 2

                SDC2

                M81687

                -2

                Plasma Membrane

                   zinc finger protein 260

                ZNF260

                U56862

                -2

                Nucleus

                   filamin C

                FLNC

                AF119148

                -3

                Cytoplasm

                   metallothionein 3

                MT3

                S65838

                -3

                Cytoplasm

                   arginine vasopressin

                AVP

                M25646

                -4

                Extracellular Space

                   fasciculation and elongation protein zeta 1

                FEZ1

                U63740

                -4

                Cytoplasm

                   crystallin, alpha B

                CRYAB

                U04320

                -6

                Nucleus

                   neurofascin

                NFASC

                U81036

                -7

                Plasma Membrane

                Gene description names, gene symbols are from IPA Tool; accession numbers are from GenBank

                In PC12 cells stimulated for 24 hours with 50 ng/ml NGF, we identified 102 differently expressed genes as compared to unstimulated controls. Of them, 71 genes were upregulated with gene expression values from 2-fold to 303-fold, whereas 31 genes were found to be downregulated by -2-fold to -20-fold. Major functional categories include enzymes (18 up-regulated/9 down-regulated), G-Protein coupled receptors (2/2), growth factors (3/1), ion channels (7/2), kinases (6/2), peptidases (4/1), phosphatases (2/1), transcription regulators (0/2), transmembrane receptors (1/0), transporters (9/2) and molecules with other functions (21/9; Table 2).
                Table 2

                List of gene set regulated by NGF in PC12 cells

                Gene

                 

                Accession no.

                Fold change

                Subcellular location

                Enzymes

                    

                   rat senescence marker protein 2A gene

                SMP2A

                X63410

                303

                Cytoplasm

                   myosin, heavy chain 3

                MYH3

                K03468

                133

                Cytoplasm

                   lecithin-cholesterol acyltransferase

                LCAT

                X54096

                101

                Extracellular Space

                   UDP glucuronosyltransferase 2, polypeptide A1

                UGT2A1

                X57565

                63

                Cytoplasm

                   contactin 4

                CNTN4

                U35371

                44

                Plasma Membrane

                   phosphodiesterase 4B,

                PDE4B

                J04563

                37

                Cytoplasm

                   gulonolactone (L-) oxidase

                GULO

                J03536

                34

                Cytoplasm

                   superoxide dismutase 3

                SOD3

                Z24721

                28

                Extracellular Space

                   fibronectin 1

                FN1

                X15906

                28

                Plasma Membrane

                   acetylcholinesterase

                ACHE

                S50879

                28

                Plasma Membrane

                   tryptophan hydroxylase 1

                TPH1

                X53501

                24

                Cytoplasm

                   aldo-keto reductase family 1, member C1

                AKR1C1

                BAA92883

                10

                Cytoplasm

                   guanine nucleotide binding protein, alpha z

                GNAZ

                U77485

                9

                Plasma Membrane

                   aminoadipate aminotransferase

                AADAT

                Z50144

                5

                Cytoplasm

                   phospholipase D2

                PLD2

                D88672

                4

                Cytoplasm

                   N-deacetylase/N-sulfotransferase 1

                NDST1

                M92042

                3

                Cytoplasm

                   phosphate cytidylyltransferase 2

                PCYT2

                AF080568

                2

                Cytoplasm

                   peptidylprolyl isomerase A

                PPIA

                M19533

                -2

                Cytoplasm

                   Rab geranylgeranyltransferase alpha

                RABGGTA

                L10415

                -2

                Unknown

                   glutathione S-transferase A3

                GSTA3

                X78847

                -3

                Cytoplasm

                   cytochrome P450, 4F4

                CYP4F4

                U39206

                -3

                Cytoplasm

                   3-hydroxyanthranilate 3,4-dioxygenase

                HAAO

                D28339

                -3

                Cytoplasm

                   stearoyl-Coenzyme A desaturase 2

                SCD2

                AB032243

                -4

                Cytoplasm

                   aldo-keto reductase family 1, member C3

                AKR1C3

                L32601

                -6

                Cytoplasm

                   myxovirus resistance 2

                MX2

                X52711

                -10

                Nucleus

                   serine dehydratase

                SDS

                M38617

                -11

                Cytoplasm

                G-protein coupled receptors

                    

                   calcitonin/calcitonin-related polypeptide alpha

                CALCA

                V01228

                136

                Plasma Membrane

                   angiotensin II receptor 1

                AGTR1

                NM_009585

                50

                Plasma Membrane

                   cholinergic receptor, muscarinic 3

                CHRM3

                AB017656

                -2

                Plasma Membrane

                   parathyroid hormone receptor 1

                PTHR1

                M77184

                -3

                Plasma Membrane

                Growth factors

                    

                   growth differentiation factor 15

                GDF15

                AJ011970

                131

                Extracellular Space

                   transforming growth factor beta 1

                TGFB1

                X52498

                101

                Extracellular Space

                   brain-derived neurotrophic factor

                BDNF

                X67108

                89

                Extracellular Space

                   neuregulin 1

                NRG1

                U02324

                -3

                Extracellular Space

                Ion channels

                    

                   calcium channel, voltage-dependent, beta 2

                CACNB2

                M80545

                90

                Plasma Membrane

                   glutamate receptor, ionotropic, delta 2

                GRID2

                U08256

                78

                Plasma Membrane

                   sodium channel, voltage-gated, type II, beta

                SCN2B

                U37147

                73

                Plasma Membrane

                   potassium inwardly-rectifying channel J4

                KCNJ4

                X87635

                51

                Plasma Membrane

                   solute carrier family 9 member 3

                SLC9A3

                M85300

                40

                Plasma Membrane

                   purinergic receptor P2X, ligand-gated ion channel 2

                P2RX2

                Y10475

                13

                Plasma Membrane

                   sodium channel, voltage-gated, type I, alpha

                SCN1A

                M22253

                12

                Plasma Membrane

                   purinergic receptor P2X-like 1

                P2RXL1

                X92070

                -2

                Plasma Membrane

                   gamma-aminobutyric acid A receptor gamma 2

                GABRG2

                X56313

                -19

                Plasma Membrane

                Kinases

                    

                   G protein-coupled receptor kinase 5

                GRK5

                NM_005308

                131

                Plasma Membrane

                   protein kinase, cGMP-dependent, type II

                PRKG2

                Z36276

                68

                Cytoplasm

                   mitogen-activated protein kinase kinase kinase kinase 1

                MAP4K1

                Y09010

                25

                Cytoplasm

                   calcium/calmodulin-dependent serine protein kinase

                CASK

                U47110

                3

                Plasma Membrane

                   discs, large homolog 1

                DLG1

                U14950

                3

                Plasma Membrane

                   phosphatidylinositol 4-kinase beta

                PI4KB

                D84667

                3

                Cytoplasm

                   discoidin domain receptor family member 1

                DDR1

                L26525

                -8

                Plasma Membrane

                   non-metastatic cells 6

                NME6

                AF051943

                -14

                Extracellular Space

                Peptidases

                    

                   carboxypeptidase A3

                CPA3

                U67914

                5

                Extracellular Space

                   ADAM metallopeptidase domain 17

                ADAM17

                AJ012603

                4

                Plasma Membrane

                   Proteasome subunit alpha 1

                PSMA1

                M29859

                3

                Cytoplasm

                   protein disulfide isomerase family A member 3

                PDIA3

                D63378

                2

                Cytoplasm

                   caspase 1

                CASP1

                U14647

                -5

                Cytoplasm

                Phosphatases

                    

                   dual specificity phosphatase 6

                DUSP6

                U42627

                53

                Cytoplasm

                   protein phosphatase 1 subunit 1A

                PPP1R1A

                AJ276593

                18

                Cytoplasm

                   protein tyrosine phosphataser type 11

                PTPN11

                U09307

                -2

                Cytoplasm

                Transcription regulators

                    

                   jun dimerization protein 2

                JDP2

                U53449

                -2

                Nucleus

                   cAMP responsive element modulator

                CREM

                Z15158

                -4

                Nucleus

                Transmembrane receptors

                    

                   cholinergic receptor, nicotinic, beta 1

                CHRNB1

                X74833

                39

                Plasma Membrane

                Transporters

                    

                   solute carrier family 1 member 1

                SLC1A1

                U21104

                238

                Plasma Membrane

                   solute carrier family 22, member 3

                SLC22A3

                AF055286

                95

                Plasma Membrane

                   gap junction protein, beta 2

                GJB2

                X51615

                55

                Plasma Membrane

                   solute carrier family 1, member 3

                SLC1A3

                S59158

                6

                Plasma Membrane

                   solute carrier family 22, member 6

                SLC22A6

                AF008221

                6

                Plasma Membrane

                   vacuolar protein sorting 33 homolog B

                VPS33B

                U35245

                4

                Cytoplasm

                   solute carrier family 30, member 1

                SLC30A1

                U17133

                3

                Plasma Membrane

                   syntaxin 4

                STX4

                L20821

                2

                Plasma Membrane

                   murinoglobulin 1

                MUG1

                J03552

                -2

                Extracellular Space

                   ATPase, Cu++ transporting, beta polypeptide

                ATP7B

                AF120492

                -6

                Cytoplasm

                Others

                    

                   BCL2/adenovirus E1B interacting protein 3

                BNIP3

                AF243515

                216

                Cytoplasm

                   natriuretic peptide precursor C

                NPPC

                D90219

                109

                Extracellular Space

                   trans-golgi network protein 2

                TGOLN2

                X53565

                106

                Cytoplasm

                   fibrillin 2

                FBN2

                L39790

                105

                Extracellular Space

                   amyloid P component, serum

                APCS

                M83177

                85

                Extracellular Space

                   zinc finger, matrin type 3

                ZMAT3

                Y13148

                84

                Nucleus

                   LIM and senescent cell antigen-like domains 1

                LIMS1

                AAA20086

                75

                Plasma Membrane

                   CD44 molecule

                CD44

                U96138

                61

                Plasma Membrane

                   common salivary protein 1

                LOC171161

                U00964

                54

                Extracellular Space

                   selectin P

                SELP

                L23088

                44

                Plasma Membrane

                   collagen, type XI, alpha 1

                COL11A1

                AJ005396

                39

                Extracellular Space

                   collagen, type XII, alpha 1

                COL12A1

                U57362

                28

                Extracellular Space

                   nucleosome assembly protein 1-like 4

                NAP1L4

                AJ002198

                22

                Nucleus

                   spermine binding protein

                SBP

                J02675

                20

                Unknown

                   ribosomal protein L35

                RPL35

                M34331

                6

                Cytoplasm

                   connector enhancer of kinase suppressor of Ras 2

                CNKSR2

                AF102854

                5

                Plasma Membrane

                   prolactin family 8, subfamily a, member 81

                PRL8A8

                AB000107

                4

                Extracellular Space

                   extracellular proteinase inhibitor

                EXPI

                X13309

                3

                Extracellular Space

                   fibrinogen gamma chain

                FGG

                J00735

                3

                Extracellular Space

                   smooth muscle alpha-actin

                ACTA2

                X06801

                2

                Unknown

                   tropomyosin 1 alpha

                TPM1

                M34134

                2

                Cytoplasm

                   calcineurin binding protein 1

                CABIN1

                AF061947

                -2

                Nucleus

                   crystallin, gamma E

                CRYGE

                J00716

                -2

                Unknown

                   follistatin-like 1

                FSTL1

                M91380

                -2

                Extracellular Space

                   secreted phosphoprotein 2

                SPP2

                U19485

                -2

                Extracellular Space

                   tachykinin, precursor 1

                TAC1

                M15191

                -2

                Extracellular Space

                   myosin light chain 9

                MYL9

                S77900

                -3

                Cytoplasm

                   ubiquitin B

                UBB

                X51703

                -3

                Cytoplasm

                   golgin B1 protein

                GOLGB1

                D25543

                -6

                Cytoplasm

                   lysosomal-associated membrane protein 1

                LAMP1

                X14765

                -11

                Plasma Membrane

                Gene description names, gene symbols are from IPA Tool; accession numbers are from GenBank

                Only a small overlapping gene subset is shared by IL-6 and NGF comprising a total of 16 genes and including the major functional categories enzymes (3 genes), G-Protein coupled receptors (1), growth factors (1), ion channels (2), kinases (1), peptidases (2), transporters (1) and molecules with other functions (5; Table 3). All genes are regulated in a parallel fashion except for caspase 1 with an opposite expression pattern of IL-6 (40-fold) as compared to NGF (-5-fold; Table 3). Tables 1, 2, 3 summarize gene description names, Genbank accession numbers and changes in expression levels derived from the Chip analyses, gene symbols and abbreviations derived from the IPA Tool.
                Table 3

                Set of genes commonly regulated by IL-6 and NGF in PC12 cells

                Gene

                 

                Fold change

                  

                IL-6

                NGF

                Enzymes

                   

                   guanine nucleotide binding protein, alpha z

                GNAZ

                14

                9

                   glutathione S-transferase A3

                GSTA3

                - 3

                - 3

                   aldo-keto reductase family 1, member C1

                AKR1C1

                12

                10

                G-protein coupled receptors

                   

                   cholinergic receptor, muscarinic 3

                CHRM3

                - 3

                - 2

                Growth factors

                   

                   growth differentiation factor 15

                GDF15

                80

                131

                Ion channels

                   

                   glutamate receptor, ionotropic, delta 2

                GRID2

                91

                78

                   purinergic receptor P2X, ligand-gated ion channel

                P2RX2

                11

                13

                Kinases

                   

                   phosphatidylinositol 4-kinase beta

                PI4KB

                2

                3

                Peptidases

                   

                   caspase 1

                CASP1

                40

                - 5

                   proteasome subunit alpha 1

                PSMA1

                5

                3

                Transporters

                   

                   syntaxin 4

                STX4

                3

                2

                Others

                   

                   trans-golgi network protein 2

                TGOLN2

                113

                106

                   LIM and senescent cell antigen-like domains 1

                LIMS1

                94

                75

                   fibrinogen gamma chain

                FGG

                94

                3

                   collagen, type XII, alpha 1

                COL12A1

                26

                28

                   extracellular proteinase inhibitor

                EXPI

                3

                3

                Gene description names, gene symbols are from IPA Tool

                Exemplary validation of microarray data using LightCycler quantitative RT-PCR analyses (qRT-PCR) on GAP-43 and REG3B mRNA expression

                For an exemplary validation of the microarray data, qRT-PCR using LightCycler was performed on GAP-43 and REG3B mRNA expression. In the microarray analyses, GAP-43 mRNA was found to be upregulated 3-fold by IL-6 (Table 1), whereas qRT-PCR revealed an induction of about 20-fold (Figure 1, left). In NGF-treated PC12 cells, GAP-43 mRNA was found to be upregulated by < 2-fold and therefore did not meet the exclusion criteria applied in the current work. However, qRT-PCR analyses revealed a 10-fold induction of GAP-43 mRNA levels induced by NGF in PC12 cells (Figure 2). Thus, PC12 cells treated with IL-6 or NGF undergo substantial neuronal differentiation. REG3B mRNA expression in the microarray analysis was found to be induced to 672-fold by IL-6 (Table 1), whereas qRT-PCR revealed an induction of REG3B mRNA by about 955-fold (Figure 1, right). In NGF-treated PC12 cells, neither microarray nor qRT-PCR analyses revealed changes in RGE3B expression.
                http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-10-90/MediaObjects/12864_2008_Article_1974_Fig1_HTML.jpg
                Figure 1

                Changes in expression of GAP-43- and REG3B mRNA levels in IL-6-stimulated PC12 cells determined by qRT-PCR versus GeneChip. Affymetrix Genechip- and qRT-PCR analyses were performed as described in the Methods section.

                http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-10-90/MediaObjects/12864_2008_Article_1974_Fig2_HTML.jpg
                Figure 2

                Changes in expression of GAP-43- mRNA levels in IL-6- versus NGF-stimulated PC12 cells. qRT-PCR analyses were performed as described in the Methods section.

                Ingenuity biological functional analyses of the gene sets regulated by IL-6 and NGF in PC12 cells

                The criteria applied for the search of major biological function categories were maximum number of genes and the p-value of significance. As shown in Table 4, top biological functions found to be regulated by IL-6 include cancer (61 genes), cellular growth and proliferation (54 genes), cell death (47 genes), cell-to-cell signalling and interaction (46 genes), tissue development (45 genes) and others. A further gene set is involved in nervous system development and function (24 genes). The p-values in the range of 2.26 × 10-7 to 3.77 × 10-3 indicate statistical significance.
                Table 4

                Top high-level functions identified by Ingenuity global function analysis of regulated genes in IL-6-versus NGF- stimulated PC 12 cells

                Biological function classification

                Number of genes

                Significance (p-value)

                IL-6-regulated genes

                  

                Cancer

                61

                2.98 × 10-6 to 5.16 × 10-3

                Cellular Growth and Proliferation

                54

                1.14 × 10-6 to 5.16 × 10-3

                Cell Death

                47

                4.54 × 10-6 to 5.16 × 10-3

                Cell-to-Cell Signalling and Interaction

                46

                2.26 × 10-7 to 5.16 × 10-3

                Tissue Development

                45

                2.26 × 10-7 to 5.15 × 10-3

                Cellular Movement

                39

                9.19 × 10-6 to 5.16 × 10-3

                Cellular Development

                38

                8.56 × 10-6 to 4.85 × 10-3

                Small Molecule Biochemistry

                37

                1.32 × 10-5 to 4.47 × 10-3

                ...

                  

                Nervous system development and function

                24

                2.83 × 10-5 to 3.77 × 10-3

                NGF-regulated genes

                  

                Cellular growth and proliferation

                37

                7.86 × 10-5 to 8.88 × 10-3

                Cell-to-cell signalling and interaction

                31

                1.03 × 10-4 to 7.43 × 10-3

                Molecular transport

                30

                8.89 × 10-6 to 8.70 × 10-3

                Cancer

                30

                1.03 × 10-4 to 7.43 × 10-3

                Cellular movement

                29

                2.41 × 10-5 to 8.70 × 10-3

                Cell death

                29

                2.73 × 10-5 to 8.77 × 10-3

                Neurological diseases

                29

                1.07 × 10-4 to 8.70 × 10-3

                Nervous system development and function

                29

                1.60 × 10-4 to 8.70 × 10-3

                p-values are from IPA Tool

                Similarly, in NGF-treated PC12 cells top biological functions deal with the overall topics on cellular growth and proliferation (37 genes), cell-to-cell signalling and interaction (31 genes), molecular transport (30 genes), cancer (30 genes), cellular movement (29 genes) and others. One gene set is involved in nervous system development and function (29 genes). The p-values in the range from 8.89 × 10-6 to 7.43 × 10-3 indicate statistical significance (Table 4).

                More detailed analyses for functional sub-categories are summarized in Table 5. Both stimuli utilize different repertoires of genes to exert the same biological functions that are all crucial for neuronal differentiation and nervous system development. Among others, important functional sub-categories include cellular growth (IL-6, 33 genes; NGF, 24 genes), differentiation (IL-6, 45 genes; NGF, 16 genes), cell movement (IL-6, 39 genes; NGF, 27 genes), chemotaxis (IL-6, 13 genes; NGF, 13 genes), adhesion of cells (IL-6, 26 genes; NGF, 18 genes), cellular signalling and small molecule biochemistry aiming at changing intracellular concentrations of second messengers such as Ca2+ (IL-6, 16 genes; NGF, 16 genes) as well as cAMP (IL-6, 12 genes; NGF, 9 genes) as well as expression of posttranslational processing enzymes (IL-6, 23 genes; NGF, 15 genes). Table 5 (bottom) summarizes genes involved in specialized sub-categories of nervous system and development as far as they are represented in the IPKB.
                Table 5

                Ingenuity biological function analyses of IL-6-versus NGF-regulated genes in PC12 cells (selected)

                 

                IL-6 regulated genes in PC12 cells

                NGF-regulated genes in PC12 cells

                Category

                p-value

                Molecules

                p-value

                Molecules

                Sub-Category or Function annotation

                    

                Cellular Growth and Proliferation

                    

                Growth of cells

                2.27 × 10-4

                ACVR2A, AHSG, ANXA1, BCL2L11, BRCA1, CASP1, CDC42, CHRM3, CXCL10, EGR1, FGFR1, GAP43, GDF15, GHR, GRID2, HGF, IGF2R, IRF1, ITGA7, JAK2, MAP2K5, MST1, MT3, MX1, NOS3, NOS2A, PIM3, RARA, SCAMP2, SDC2, STAT1, TIMP1, VDR

                8.82 × 10-3

                ACHE, AGTR1, BDNF, BNIP3, CASP1, CD44, CHRM3, CREM, DDR1, DUSP6, FBN2, FN1, GDF15, GJB2, GRID2, MYL9, NRG1, PDIA3, PTPN11, SLC30A1, TGFB1, TPM1, VPS33B, ZMAT3

                Proliferation of cells

                9.06 × 10-7

                ACVR2A, ADCYAP1, ANXA1, AVP, BCL2L11, BRCA1, CALCB, CDC42, CHRM3, CHRM4, CRYAB, CXCL10, EGR1, FGFR1, FRK, GDF15, GHR, GHRH, HGF, IGF2R, IRF1, JAG2, JAK2, KLF6, KLK8, LCN2, MAP2K5, MT3, NFIB, NOS3, NOS2A, NR3C2, PDGFA, RARA, REG1A, REG3A, RNMT, SDC2, ST6GAL1, STAT1, TIMP1, TPM3, USF1, VDR, VIPR1

                3.82 × 10-3

                AGTR1, AKR1C3, BDNF, CALCA, CD44, CHRM3, DDR1, FN1, GDF15, GRK5, NPPC, NRG1, PPIA, PTPN11, TAC1, TGFB1

                Cellular Movement

                    

                Cell movement

                2.18 × 10-8

                ADCYAP1, ANXA1, CASP1, CDC42, CHRM3, CHRM4, CXCL10, CXCL13, EGR1, FCGR2A, FER, FGB, FGFR1, GNAZ, GRID2, HGF, HLA-G, HSP90AB1, IGF2R, JAK2, LCN2, LGALS9, LIMS1, MAP2K5, MST1, NOS3, NOS2A, OLR1, PDGFA, RARA, REG3A, SDC2, ST6GAL1, STAT1, TIMP1, TPM3, TUBB, VDR, VIPR1

                7.96x10-5

                ADAM17, AGTR1, APCS, BDNF, CALCA, CASP1, CD44, CHRM3, DDR1, FN1, GJB2, GNAZ, GRID2, LCAT, LIMS1, NAP1L4, NPPC, NRG1, PDE4B, PPIA, PTPN11, SCN2B, SELP, SLC1A3, TAC1, TGFB1, TPM1

                Chemotaxis

                4.05 × 10-4

                ANXA1, CDC42, CXCL10, CXCL13, FCGR2A, FER, FGFR1, GNAZ, HGF, IGF2R, LGALS9, NOS3, VIPR1

                6.29x10-5

                AGTR1, BDNF, CALCA, CD44, FN1, GNAZ, NAP1L4, PDE4B, PPIA, PTPN11, SCN2B, TAC1, TGFB1

                Cell-To-Cell Signaling and Interaction

                    

                Adhesion of cells

                1.47 × 10-7

                ANXA1, CDC42, CDH17, CXCL10, EGR1, FCGR2A, FER, FEZ1, FGB, FGFR1, FGG, GRID2, HGF, IGF2R, ITGA7, JAG2, LGALS9, LIMS1, NOS3, OLR1, REG3A, SDC2, ST6GAL1, STAT1, STX4, TIMP1

                1.34x10-4

                ACHE, ADAM17, CASK, CD44, CNTN4, DDR1, DLG1, FGG, FN1, GRID2, LIMS1, NRG1, PTPN11, SELP, STX4, TAC1, TGFB1, TPH1

                Cell Signaling

                    

                Quantity of calcium

                3.25 × 10-3

                ADCYAP1, AVP, CHRM3, CXCL10, CXCL13, FCGR2A, GHRH, HGF, NOS3, NOS2A, VDR

                8.89x10-6

                AGTR1, BDNF, CALCA, CHRM3, FN1, GRK5, NPPC, PLD2, PPIA, PTHR1, PTPN11, SELP, TAC1, TGFB1

                Production of nitric oxide

                1.33 × 10-3

                IRF1, JAK2, MST1, NOS3, NOS2A, STAT1

                -

                -

                Flux of calcium

                1.67 × 10-3

                ADCYAP1, ANXA1, AVP, CHRM3, CXCL10, CXCL13, FCGR2A, P2RX2

                2.20x10-3

                CALCA, CHRM3, FN1, NPPC, P2RX2, PPIA, TGFB1

                Cell surface receptor linked signal transduction

                1.45 × 10-3

                ACVR2A, ANXA1, CDC42, CXCL10, FCGR2A, FGFR1, ITGA7, JAK2, KLF6, LIMS1, PDGFA, PTPRD, STAT1

                -

                -

                Small Molecule Biochemistry

                    

                Quantity of cyclic AMP

                1.00 × 10-5

                ADCYAP1, AVP, CHRM4, CXCL10, GAP43, GHRH, GNAZ, NOS3, VIPR1

                6.03x10-3

                BDNF, CALCA, GNAZ, NPPC, PTHR1

                Production of cyclic AMP

                2.17 × 10-4

                ADCYAP1, AVP, GHRH, GNAZ, NOS3, NOS2A, VIPR1

                  

                Accumulation of cyclic AMP

                1.21 × 10-3

                ADCYAP1, AVP, AVPR2, CHRM3, GHRH, VIPR1

                4.35 × 10-4

                CALCA, CHRM3, GRK5, PTHR1, TAC1, TGFB1

                Formation of cyclic AMP

                1.28 × 10-4

                ADCYAP1, AVP, AVPR2, GHRH, GANZ

                7.26 × 10-4

                CALCA, GNAZ, PTHR1, TAC1

                Release of Ca2+

                9.82 × 10-5

                ANXA1, AVP, CHRM3, FCGR2A, FGB, FGG

                -

                -

                Quantity of cholesterol

                -

                -

                2.85 × 10-3

                ATP7B, BDNF, CALCA, GULO, LCAT

                Post-Translational Modification

                    

                Modification of protein

                1.57 × 10-5

                AVP, BRCA1, CASP1, CHRM3, FCGR2A, FER, FGFR1, GRID2, HSP90AB1, HTATIP, JAK2, LHB, MST1, NOS3, NOS2A, PDGFA, PDIA2, PDP2, PIM3, PTPRD, ST6GAL1, STAT1, TGM1

                4.47 × 10-3

                APCS, CASP1, CD44, CHRM3, DUSP6, FN1, GRID2, NDST1, NRG1, PDIA3, PPIA, PTPN11, RABGGTA, TAC1, UBB

                Nervous system development and function

                    

                growth of neurites

                8.02 × 10-3

                ADCYAP1, CDC42, GAP43, HGF, TPM3

                -

                -

                survival of neurons

                3.60 × 10-3

                ADCYAP1, BCL2L11, GDF15, HGF, RARA, REG3A

                -

                -

                development of synapse

                6.57 × 10-3

                GRID2, NFASC

                -

                -

                fasciculation of axons

                3.14 × 10-2

                GAP43

                -

                -

                complexity of dendritic trees

                1.25 × 10-2

                HGF

                -

                -

                long-term potentiation of dentate gyrus

                1.25 × 10-2

                EGR1

                -

                -

                neurological process of synapse

                -

                -

                1.60 × 10-4

                BDNF, CHRM3, CHRNB1, NRG1, PPP1R1A

                synaptic transmission

                -

                -

                2.88 × 10-4

                BDNF, CACNB2, CHRM3, CHRNB1, GABRG2, P2RX2, SCN2B, SLC1A1, SLC1A3

                neurological process of axons, neurites

                -

                -

                4.79 × 10-4

                BDNF, CNTN4, GRID2, NRG1, PDIA3, UBB

                activation of nerves

                -

                -

                7.73 × 10-4

                CALCA, TAC1

                binding of neurites

                -

                -

                7.73 × 10-4

                BDNF, CD44

                size of cell body

                -

                -

                7.73 × 10-4

                ACHE, BDNF

                survival of neurons

                -

                -

                8.92 × 10-4

                BDNF, GDF15, NRG1, PDIA3, SLC1A3, TGFB1

                development of neurites

                -

                -

                2.83 × 10-3

                ACHE, BDNF, GRID2, NRG1, PDIA3, PTPN11

                migration of nervous tissue cell lines

                -

                -

                3.38 × 10-3

                NRG1, TGFB1

                proliferation of nervous tissue cell lines

                -

                -

                6.67 × 10-3

                NPPC, TGFB1

                -, no subcategories found in IPA Tool; p-values and gene symbols are from IPA Tool

                Discussion

                In a previous study, we have used PC12 cells to examine the effects of IL-6/s-IL6R on neuronal differentiation in comparison to NGF [22]. Already after 24 hours of exposure to IL-6/s-IL-6R or NGF PC12 cells are highly active in cellular growth and proliferation displaying pronounced formation of extending neurites. Combined incubation with IL-6/s-IL-6 plus NGF drastically enhanced cell number and neurite outgrowth arguing for an additive effect of both stimuli on neuronal differentiation. In the current study we have chosen this time point to perform microarray analyses in order to monitor changes in gene expression and to compare the genetic programs utilized for neuronal differentiation by IL-6 versus NGF.

                An important aspect in gene expression profiling using microarrays is the accuracy of the measurements in the relative changes in mRNA expression. Thus, alternative technologies such as qRT-PCR are used for the validation of microarray data [27]. Several systematic studies comparing the changes in gene expression obtained from oligonucleotide- or cDNA arrays to data from qRT-PCR revealed that a good correlation exists for genes exhibiting fold-change differences in expression of > 2-fold [28, 29]. Therefore, in our datasets all genes displaying changes in expression levels of < 2-fold were excluded. Moreover, our exemplary validation data on GAP-43- and REG3B-expression are in line with other previous reports confirming that it is rather the magnitude of fold change varying between qRT-PCR and Affymetrix-analysis, but not the direction.

                Detailed Ingenuity biological function analyses reveal that IL-6 and NGF activate gene sets that regulate the same process in neuronal differentiation and nervous system development, however, utilizing completely distinguished sets of individual molecules. This may explain our previous observation that combined application of IL-6/s-IL-6R plus NGF generates an additive effect on PC12 cell differentiation. Important processes in neuronal differentiation and nervous tissue development include cellular growth and proliferation in order to enhance cell number. Neurite outgrowth and network generation requires migration of neurons or nerve growth cones. Neuronal navigation is guided by the interaction of the neuron with its local environment, in particular by chemotaxis as the key mechanism. This process involves three major steps including directional sensing along a gradient of chemotactic factors, cellular motility i.e. the cell's movement by changes in cytoskleletal organisation and cellular adhesion and cellular polarisation [3032]. Certainly, a key step in the regulation of these processes is the increased gene expression of growth factors and functionally related external molecules, indicating convergence of several different signaling pathways (Table 5). In IL-6 stimulated PC12 cells these tasks may be taken by growth differentiation factor 15 (GDF15), platelet-derived growth factor alpha (PDGFA), hepatocyte growth factor (HGF), regenerating islet-derived 3 alpha (REG3A), regenerating islet-derived 3 beta/pancreatitis-associated protein I (REG3B/PAPI), growth hormone releasing hormone (GHRH) and adenylate cyclase activating polypeptide (PACAP). NGF recruits GDF15 (131-fold), transforming growth factor beta 1 (TGFB1; 101-fold) and brain-derived neurotrophic factor (BDNF; 89-fold). TGFB1 is the prototype member of the TGFB-superfamily comprising multifunctional growth factors with numerous cell and tissue functions such as cell cycle control, regulation of early development, differentiation, extracellular matrix (ECM) formation and chemotaxis. In the nervous system, TGFB1 has been shown to regulate neuroprotection against glutamate cytotoxicity, ECM production, and cell migration in the cerebral cortex, control of neuronal death as well as survival of neurons (reviewed in [33]). GDF15 is a member of the TGFB- superfamily and has been shown to be a potent trophic factor in the brain (reviewed in [34]). Hepatocyte growth factor (HGF) is a chemoattractant and a survival factor for embryonic motor neurons. In addition, sensory and sympathetic neurons and their precursors respond to HGF with increased differentiation, survival and axonal outgrowth [35]. Moreover, HGF may synergize with other neurotrophic factors to potentiate the response of developing neurons to specific signals [36]. Platelet derived growth factor (PDGF) has been suggested to support neuronal differentiation [37], and has previously been reported to act as a mitogen for immature neurons [38] and neural progenitor cells [39]. REG3A and REG3B/PAPI are members of the regenerating protein (REG)/pancreatitis-associated protein (PAP) family representing a complex group of small secretory proteins which display many different functions, among them growth factor activity for neural cells [40]. So far, only limited knowledge is available about the role and function of PAP/REG-proteins in the nervous system. REG3B/PAPI expression is induced in spinal motor neurons as well as subsets of the dorsal root ganglion neurons [41]. Moreover, in vitro REG3B/PAPI has a mitogenic effect on Schwann cells [42]. In a hypoglossal nerve injury model in rats, expression of REG3B/PAPI mRNA was found to be enhanced in injured motor neurons after axotomy and a marked induction of REG3G/PAPIII mRNA was observed in the distal part of the injured nerve [43]. More recently, REG3G/PAPIII has been identified as a macrophage chemoattractant that is induced in and released from injured nerves [44]. With REG1A/PSP and REG3G/PAPIII, two further members of the REG/PAP family are induced by IL-6 in PC12 cells. It is noteworthy that these genes are up-regulated at the highest levels obtained in the entire dataset for IL-6. In NGF-treated PC12 cells, no up-regulation of the PAP/REG protein genes was observed. The results in our study are in line with an earlier report demonstrating up-regulation of PAP/REG gene family members in PC12 cells upon stimulation with IL-6/s-IL-6R [45].

                So far various studies have investigated gene expression profiles in NGF-treated PC12 cells applying different experimental protocols in respect to time points and periods of NGF administration [4651]. From most studies, it is obvious that PC12 cells require at least 3 to 5 days of NGF-treatment to obtain the fully differentiated neuronal phenotype. The most significant morphological changes occur within the first 2 days, reaching a plateau phase at day 3 [51]. Redundant data sets as well as unique genes have been identified and followed. Our study provides novel candidate genes activated in the early phase of the differentiation process and thus may enlarge the repertoire of known NGF-regulated genes.

                The current study reveals novel aspects of IL-6 action, notably that it applies several major routes to direct PC12 cell differentiation. Besides up-regulation of growth factors known to act in autocrine and paracrine fashion to take over further tasks in the differentiation process, these include induction of PACAP, a pleiotropic molecule with a broad spectrum of biological functions. Among them are actions as a neurotrophic factor similar to NGF as well as induction of transcription factors known to be of key importance in neuronal differentiation [52].

                Upregulation of PACAP could have an important impact on IL-6-induced PC12 cell differentiation. A recent report provided data from microarray analyses of PACAP-regulated gene transcripts in primary cultures of sympathetic neurons at 6 hours and 92 hours of stimulation [53]. A comparison with our data reveals that many gene families that are activated by PACAP in primary sympathetic neurons are also induced by IL-6 in PC12 cells (Table 6). Thus, many of the effects of IL-6 on PC12 cells are likely to be mediated by the intermediate autocrine and/or paracrine action of PACAP. PACAP is a member of a family of neuropetides known to activate class II G-protein coupled receptors (GPCRs; reviewed in [54]). Other family members include growth hormone releasing hormone (GHRH) and calcitonin-related peptide beta (CALCB) which are activated by IL-6 in PC12 cells by 31-and 195-fold, respectively. All members of the class II GPCR superfamily regulate intracellular cAMP-levels by receptor coupling to the Gs-adenylate cyclase-cAMP signaling pathway [54]. A further mechanism of PACAP action in PC12 cells could be a transactivation of TrkA receptors [55]. However, in light that the overlap in the datasets of IL-6 versus NGF is rather small, TrkA activation may not be a primary event at all or at the time point of our study.
                Table 6

                Comparison of commonly regulated gene families in PACAP-stimulated sympathetic primary neurons versus IL-induced PC12 cells (data derived from [53])

                PACAP-stimulated sympathetic neurons (data are from [53])

                  

                IL-6-stimulated PC12 cells

                Gene family

                    

                Gene abbreviation

                9 hours

                96 hours

                Gene abbreviation

                24 hours

                Pituitary adenylate cyclase activating polypeptide

                    

                ADCYAP1

                +

                +

                ADCYAP1

                +

                BCL2-like protein

                    

                BCL2L11

                +

                n.c.

                BCL2L11

                +

                Chemokine Ligands

                    

                CXCL1

                +

                +

                  
                   

                CXCL10

                +

                   

                CXCL13

                +

                Cytochrome P450 proteins

                    

                CYP1B1

                +

                +

                  
                   

                CYP4F16

                +

                Early growth response

                    

                EGR1

                +

                n.c.

                EGR1

                +

                Glutathione S-transferase

                    

                GSTA3

                +

                n.c.

                GSTA3

                -

                Heat shock proteins

                    

                HSP27B1

                +

                n.c.

                HSP90B1

                +

                Janus kinase

                    

                JAK2

                 

                +

                JAK2

                +

                Kruppel-like factors

                    

                KLF4

                +

                n.c.

                KLF6

                +

                KLF9

                +

                n.c.

                  

                Nuclear factors

                    

                NFIA

                +

                n.c.

                NFIB

                +

                Nuclear receptors

                    

                NR4A3

                +

                n.c.

                  

                NR4A2

                +

                n.c.

                  

                NR4A1

                +

                n.c.

                  
                   

                NR3C2

                +

                Sialytransferases

                    

                ST8SIA1

                +

                +

                ST8SIA3

                -

                ST6GAL1

                +

                +

                ST6GAL1

                +

                Solute carrier proteins

                    

                SLC1A3

                +

                n.c.

                  

                SLC2A1

                 

                +

                  

                SLC2A3

                +

                +

                  
                   

                SLC6A3

                +

                SLC7A1

                +

                +

                  

                SLC7A3

                 

                +

                  
                   

                SLC12A5

                -

                SLC18A2

                +

                +

                  
                   

                SLC30A2

                -

                SLC24A2

                 

                +

                  

                Tubulins

                    

                TUBA1

                -

                n.c.

                  
                   

                TUBB

                +

                Tissue Inhibitor of metalloproteinase

                    

                TIMP1

                +

                +

                TIMP1

                +

                +, upregulated -, downregulated; n.c., not changed from control cultures; gene symbols are from IPA Tool

                A further key step in IL-6 actions on PC12 cell differentiation is the induction of RARA and EGR-1/Zif268, two transcription factors known to be of crucial importance in neuronal differentiation. Among the genes regulated by retinoic acid is GAP-43, a neuron specific protein frequently used as a marker of neuronal differentiation as it is expressed in most neurons during neuronal development, nerve regeneration and LTP [5660]. The data herein are confirmative to our previous study in which we have found induction of GAP-43 mRNA upon stimulation of PC12 cells with IL-6/s-IL-6R [22]. EGR-1/Zif268 is induced in nearly every model of long-lasting synaptic plasticity in the CNS [6164] and suppression of Zif268 prevents neurite outgrowth in PC12 cells [65]. Recently candidate target genes of Zif268 in PC12 cells were identified suggesting that a key component of the long-lasting effects of Zif268 on CNS plasticity is the regulation of proteasome activity [66, 67].

                Signal transducer and activator of transcription 1/2 (STAT1/2), two members of the STAT family of transcriptions factors involved in signaling by Interferons (IFN) [68] are activated by stimulation of the PC12 cells with IL-6. As we could not detect changes in IFN gene expression, an autocrine action of PDGF is the most likely candidate for upregulation of STAT1/2 as described for neural progenitor cells [39]. STAT1/2 may upregulate interferon regulatory factor 1(IRF1)-expression, a further transcription factor of IFN-signaling. Breast cancer 1 (BRCA1) encodes a tumour suppressor gene whose germ line mutations in women are associated with a genetic predisposition to breast and ovarian cancer. STAT1 transcriptional activity is decreased by a physical interaction with BRCA1 as a key step in the regulation of IFN-induced cellular growth arrest [69]. By the action of IL-6, BRCA1 gene expression is down-regulated thus supporting STAT1 mediated PC12 cell growth. We failed to detect STAT3 expression, the key transcription factor of IL-6 signaling. This is most likely due to the fact that STAT3 gene transcription occurs very early in IL-6-stimulation and is already terminated at the time point of the analysis, or the expression levels are below 2-fold and thus did not meet the exclusion criteria.

                The morphological changes during nervous system development are controlled by interactions of individual neurons with the ECM. Signals from the ECM into a particular neuron are mediated by integrins via associated adapter molecules. In this way growth factor induced receptor tyrosine kinase (RTK)- and integrin-mediated signalling determine the fate of a particular cell, notably differentiation, cell shape, adhesion, polarity, migration, as well as proliferation versus apoptotic cell death (reviewed in [70]). LIM and senescent cell antigen-like domains1/PINCH (LIMS1/PINCH) is an intracellular adaptor molecule providing the molecular link of an integrin-RTK network. LIMS1 physically connects integrin-linked kinase (ILK) to non-catalytic (region of) tyrosine kinase adaptor protein 2 (Nck2), an adapter molecule of the growth factor receptor (RTK) [70]. LIMS1 is activated by IL-6 as well as NGF and thus is one of few genes regulated in the common subset. In contrast to IL-6, NGF simultaneously up-regulates major components of the ECM including collagen, type XI, alpha1 (COL11A1), COL12A1, fibronectin1 (FN1) as well as fibrillin2 (FN2) (Table 2).

                In contrast to NGF, only one publication provided expression profiling data analysing gene sets regulated by IL-6 upon neuronal differentiation. Primary cultures of rat dorsal root ganglia (DRG) were treated with IL6RIL6 for 2 and 4 days, respectively. A detailed comparison reveals that only a small number of commonly regulated genes may be identified in the datasets that are regulated in parallel or opposite direction. These include Egr-1 (upregulated in PC12 cells; downregulated in DRG cells), TGFA (upregulated in PC12 cells and DRG cells), TGFB (upregulated in PC12 cells; downregulated in DRG cells), PDGFA (upregulated in PC12 cells; downregulated in DRG cells) and IRF-1 (upregulated in PC12 cells and in DRG cells) [24].

                The results obtained from our study may also have impact into clinical treatments of injured peripheral nerves which, in contrast to central nerves, have the ability to recover from damage. Currently the therapy of choice is the use of autologous grafts where the defect is bridged with a section of autologous nerve tissue, mostly a sensory nerve [71]. Alternatively, nerve conduits or decellularized nerve grafts can be used; however, no therapy could yield a satisfactory functional recovery [72]. Various combinations of NTs, neuropoietic cytokines and GFLs have been shown to generate a microenvironment suitable to improve nerve repair [26]. The results of our study may provide novel aspects for the treatment of peripheral nerve injury as the local application of a designer cytokine such as H-IL-6 with a strongly enhanced bioactivity on neuronal development and neurite outgrowth in combination with NTs and/or GFLs may create a microenvironment with a strong reparative potency.

                Conclusion

                IL-6 and NGF utilize different genetic programs to exert the same biological functions in neuronal differentiation. An important step is the recruitment of many growth factors that may act in autocrine and/or paracrine fashion and may control the long-term effects on growth, neuronal differentiation or survival.

                Methods

                Reagents, buffers and cells

                DMEM medium, horse serum, fetal bovine serum and other cell culture supplements were obtained from GibcoBRL. TRIZOL reagent and Superscript reverse transcriptase were purchased Life Technologies. PC12 cells were obtained from ATCC, Manassas (VA), USA. Hyper-IL-6 was generated as described [8]. The LightCycler PCR kit was from Roche Diagnostics, Mannheim, Germany.

                Cell culture

                PC12 cells were cultured in DMEM medium containing 10% fetal bovine serum and 100 U/ml penicillin and streptomycin at 37°C in humidified 5% CO2/95% air. For stimulation confluent cells were washed once with PBS and cultured in cell culture medium containing 10 ng/ml H-IL-6 or 50 ng/ml recombinant human NGF for 24 hours. Control cells were incubated in cell culture medium alone for 24 hours.

                RNA Preparation

                Total RNA from unstimulated (control), H-IL-6- and NGF- stimulated PC12 cells was isolated using TRIZOL reagent according to the manufacturer's instructions. RNA was quantified spectrophotometrically by measuring the absorbance at 260 nm and the integrity was checked by formaldehyde agarose gel electrophoresis. The extracted RNA was stored at -80°C.

                GeneChip analysis

                20 μg of total RNA was used for each experiment and the target cRNA for Affymetrix Gene Chip analysis was prepared according to the manufacturer's instructions. Affymetrix GeneChip Rat Genome U34A arrays containing each 8'799 probes including full-length or annotated rat genes and several thousands of rat EST clusters consisting of redundant probes spanning an identical transcript were hybridized with the target cRNAs at 45°C for 16 h, washed and stained by using the Gene Chip Fluidics Station. The arrays were scanned with the Gene Array scanner (Affymetrix), and the fluorescence images obtained were processed by the Expression Analysis algorithm in Affymetrix Microarray Suite (ver. 4.0) and Microsoft Excel. Data were imported into GeneSpring® analysis software (ver. 4.1.3, Silicon Genetics, Redwood City, CA) for further analysis. Genes that showed substantial up- or down-regulation after stimulation by fold changes > 2 were selected from three independent experiments. Genes whose fold change was < 2 and expressed sequence tags (ESTs) that were not fully identified were excluded from the gene list. Thus, only genes with a change fold cutoff > 2 were considered to be significantly differentially regulated. Values are given as round off numbers. For each condition (unstimulated control- and H-IL-6-simulated PC12 cells or unstimulated control and NGF-stimulated PC12 cells) 3 independent microarray analyses (n = 3) were performed using RNA samples derived from independently prepared cell culture batches.

                Quantitative Real Time PCR (qRT-PCR)

                Total RNA (10 μg) from individual samples cultured separately from those used for microarray analyses was reverse-transcribed using Superscript II Reverse Transcriptase (GibcoBRL) according to the manufacturer's instructions.

                PCR reactions were performed in glass capillaries with the LightCycler thermal cycler system (Software version 3.5; Roche Diagnostics, Mannheim, Germany) using the LightCycler DNA Master SYBR Green I kit (Roche Diagnostics, Mannheim) according to the manufacturer's instructions. The primers used for RT-PCR analyses were rat S12 forward: 5'-GGC ATA GCT GCT GGA GGT GTA A-3'; rat S12 reverse: 5'-CCT TGG CCT GAG ATT CTT TGC-3'; rat REG3B forward: 5'-GGT TTG ATG CAG AAC TGG CCT-3'; rat REG3B reverse: 5'-TGA CAA GCT GCC ACA GAA TCC-3'; rat GAP-43 forward: 5'-CGT TGC TGA TGG TGT GGA GAA-3'; rat GAP-43 reverse: 5'-GCA GGC ACA TCG GCT TGT TTA-3'. PCR conditions were: 50 cycles with denaturation at 95°C for 8 seconds, annealing at 57°C for 8 seconds, and extension at 72°C for 14 seconds. Negative controls without cDNA (non-template controls; ntc) were run concomitantly. Specificity of amplified PCR products was confirmed by melting curve analysis after completion of the PCR run. Each PCR was performed in 3 independent experiments (n = 3) using different cell-culture batches.

                Quantification of LightCycler qRT-PCR data

                Quantification of data was performed with the LightCycler software 3.3 (Roche Diagnostics) using the ΔΔCp method. The difference between the crossing points (CPs; ΔCp values) for the target mRNA samples and reference S12 RNA samples (ΔΔCp) was used to calculate the expression values of the target mRNAs (2-Δ(ΔCp)).

                Ingenuity global functional analyses

                To investigate possible biological interactions of differently regulated genes, datasets representing genes with altered expression profile derived from microarray analyses were imported into the Ingenuity Pathway Analysis Tool (IPA Tool; Ingenuity®Systems, Redwood City, CA, USA; http://​www.​ingenuity.​com). The basis of the IPA-program consists of the Ingenuity Pathway Knowledge Base (IPKB) which is derived from known functions and interactions of genes published in the literature. Thus, the IPA Tool allows the identification of biological networks, global functions and functional pathways of a particular dataset. The complete dataset containing gene identifiers (Genbank accession numbers) and corresponding expression values was uploaded into the application. Each gene identifier is mapped to its corresponding gene object in the IPKB. Each gene product is assigned to functional (e.g. "cellular growth and proliferation") and sub-functional (e.g. "colony formation") categories. The biological functions that are most significant to the dataset are identified by the use of Fischer's exact test to calculate a p-value that determines the probability that each biological function assigned to that data set is due to chance alone.

                Statistical analysis

                Differences were tested by Welch's t-test based on three independent experiments, and p-values less than 0.05 were considered statistically significant. Values are expressed as means ± SEM.

                Declarations

                Acknowledgements

                The authors would like to thank Prof. Dr. Stefan Rose-John, University of Kiel, Germany, for kindly providing recombinant H-IL-6. This work was supported by a grant of the Swiss National Science Foundation (SNF; grant nr.3200BO-100730).

                Authors’ Affiliations

                (1)
                Department of Biomedicine, Institute of Physiology, University of Basel
                (2)
                Molecular Medicine Laboratories (MML), Hoffmann-La Roche Ltd.
                (3)
                Discovery Research (PRBD), Hoffmann-La Roche Ltd.
                (4)
                Non-Clinical Drug Safety (NCS), Hoffmann-La Roche Ltd.

                References

                1. Taga T, Kishimoto T: gp 130 and the interleukin-6 family of cytokines. Annu Rev Immunol 1997, 15:797–819.View ArticlePubMed
                2. Bauer S, Kerr BJ, Patterson PH: The neuropoietic cytokine family in development, plasticity, disease and injury. Nat Rev Neurosci 2007, 8:221–232.View ArticlePubMed
                3. Rose-John S, Heinrich P: Soluble receptors for cytokines and growth factors: generation and biological function. Biochem J 1994, 300:281–290.PubMed
                4. Müllberg J, Althoff K, Jostock T, Rose-John S: The importance of shedding of membrane proteins for cytokine biology. Eur Cytokine Netw 2000,11(1):27–38.PubMed
                5. McLoughlin RM, Jenkins BJ, Grail D, Williams AS, Fielding CA, Parker CR, Ernst M, Topley N, Jones SA: IL-6 trans-signaling via STAT3 directs T cell infiltration in acute inflammation. Proc Natl Acad Sci USA 2005, 102:9589–9594.View ArticlePubMed
                6. Rose-John S: Coordination of interleukin-6 biology by membran-bound and soluble receptors. Adv Exp Med Biol 2001, 495:145–151.PubMed
                7. Jones SA, Richards PJ, Scheller J, Rose-John S: IL-6 Transsignaling: The In Vivo Consequences. J Interferon Cytokine Res 2005,25(5):241–253.View ArticlePubMed
                8. Fischer M, Goldschmitt J, Peschel C, Brakenhoff JP, Kallen KJ, Wollmer A, Grotzinger J, Rose-John S: A bioactive designer cytokine for human hematopoietic progenitor cell expansion. Nat Biotechnol 1997, 15:142–145.View ArticlePubMed
                9. Peters M, Blinn G, Solem F, Fischer M, Meyer zum Buschenfelde KH, Rose-John S: In vivo and in vitro activities of the gp130-stimulating designer cytokine Hyper-IL-6. J Immunol 1998, 161:3575–3581.PubMed
                10. Skaper SD: The biology of neurotrophins, signalling pathways, and functional peptide mimetics of neurotrophins and their receptors. CNS Neurol Disord Drug Targets 2008,7(1):46–62.View ArticlePubMed
                11. Lee FS, Rajagopal R, Chao MV: Distinctive features of Trk neurotrophin receptor transactivation by G protein-coupled receptors. Cytokine Growth Factor Rev 2002, 13:11–17.View ArticlePubMed
                12. Rossi C, Angelucci A, Costantin L, Braschi C, Mazzantini M, Babbini F, Fabbri ME, Tessarollo L, Maffei L, Berardi N, Caleo M: Brain-derived neurotrophic factor (BDNF) is required for the enhancement of hippocampal neurogenesis following environmental enrichment. Eur J Neurosci 2006,24(7):1850–1856.View ArticlePubMed
                13. Gorski JA, Balogh SA, Wehner JM, Jones KR: Learning deficits in forebrain-restricted brain-derived neurotrophic factor mutant mice. Neuroscience 2003, 121:341–354.View ArticlePubMed
                14. Pang PT, Teng HK, Zaitsev E, Woo NT, Sakata K, Zhen S, Teng KK, Yung WH, Hempstead BL, Lu B: Cleavage of proBDNF by tPA/plasmin is essential for long-term hippocampal plasticity. Science 2004, 306:487–491.View ArticlePubMed
                15. Chao MV, Bothwell M: Neurotrophins: to cleave or not to cleave. Neuron 2002, 33:9–12.View ArticlePubMed
                16. Huang EJ, Reichardt LF: Trk receptors: roles in neuronal signal transduction. Annu Rev Biochem 2003, 72:609–642.View ArticlePubMed
                17. Lu B, Pang PT, Woo NH: The yin and yang of neurotrophin action. Nat Rev Neurosci 2005, 6:603–614.View ArticlePubMed
                18. Satoh T, Nakamura S, Taga T, Matsuda T, Hirano T, Kishimoto T, Kaziro Y: Induction of neuronal differentiation in PC12 cells by B-cell stimulatory factor 2/interleukin 6. Mol Cell Biol 1988, 8:3546–3549.PubMed
                19. Abeyama K, Kawano K, Nakajima T, Takasaki I, Kitajima I, Maruyama I: Interleukin 6 mediated differentiation and rescue of cell redox in PC12 cellsexposed to ionizing radiation. FEBS Lett 1995, 364:298–300.View ArticlePubMed
                20. Kotake-Nara E, Takizawa S, Quan J, Wang H, Saida K: Cobalt chloride induces neurite outgrowth in rat pheochromocytoma PC12 cells through regulation of endothelin-2/vasoactive intestinal contractor. J Neurosci Res 2005, 81:563–571.View ArticlePubMed
                21. März P, Cheng JC, Gadient RA, Patterson P, Stoyan T, Otten U, Rose-John S: Sympathetic neurons can produce and respond to interleukin-6. Proc Natl Acad Sci USA 1998, 95:3251–3256.View ArticlePubMed
                22. März P, Herget Th, Lang E, Otten U, Rose-John S: Activation of gp130 by IL-6/soluble IL-6 receptor induces neuronal differentiation. Eur J Neurosci 1998,10(5):2765–2773.
                23. März P, Otten U, Rose-John S: Neuronal activities of IL-6 type cytokines often depend on soluble cytokine receptors. Eur J Neurosci 1999, 11:2995–3004.View ArticlePubMed
                24. Zhang PL, Levy AM, Ben-Simchon L, Haggiag S, Chebath J, Revel M: Induction of neuronal and myelin-mediated gene expression by IL6receptor/IL-6: A study on embryonic dorsal root ganglia cells and isolated Schwann cells. Exp Neurol 2007, 208:285–296.PubMed
                25. Gordon Boyd GJ, Gordon T: Neurotrophic Factors and Their Receptors in Axonal Regeneration and Functional Recovery After Peripheral Nerve Injury. Mol Neurobiol 2003, 27:277–324.View ArticlePubMed
                26. Deister C, Schmidt CE: Optimizing neurotrophic factor combinations for neurite outgrowth. J Neural Eng 2006, 3:172–179.View ArticlePubMed
                27. Mimmack ML, Brooking J, Bahn S: Quantitative polymerase chain reaction: validation of microarray results from postmortem brain studies. Biol Psychiatry 2004, 55:337–345.View ArticlePubMed
                28. Yuen T, Wurmbach E, Pfeffer RL, Ebersole BJ, Sealfon SC: Accuracy and calibration of commercial oligonucleotide and custom cDNA microarrays. Nucleic Acids Res 2002, 30:e48.View ArticlePubMed
                29. Dallas PB, Gottardo NG, Firth MJ, Beesley AH, Hoffmann K, Terry PA, Freitas JR, Boag JM, Cummings AJ, Kees UR: Gene expression levels assessed by oligonucleotide microarray analysis and quantitative real-time RT-PCR- how well do they correlate? BMC Genomics 2002,6(1):59.View Article
                30. Song HJ, Poo Mm: The cell biology of neuronal navigation. Nat Cell Biol 2001, 3:E81-E88.View ArticlePubMed
                31. Van Haastert PJM, Devreotes PN: Chemotaxis: Siganlling the way forward. Nat Rev Mol Cell Bio 2004,5(8):626–634.View Article
                32. Mortimer D, Fothergill T, Pujic Z, Richards LJ, Goodhill GJ: Growth cone chemotaxis. Trends Neurosci 2008, 31:90–98.View ArticlePubMed
                33. Gomes FC, Sousa Vde O, Romão L: Emerging roles for TGF-beta1 in nervous system development. Int J Dev Neurosci 2005, 23:413–424.View ArticlePubMed
                34. Kriegelstein K, Strelau J, Schober A, Sullivan A, Unsicker K: TGF-beta and the regulation of neuron survival and death. J Physiol Paris 2002, 96:25–30.View Article
                35. Kato M, Yoshimura S, Kokuzawa J, Kitajima H, Kaku Y, Iwama T, Shinoda J, Kunisada T, Sakai N: Hepatocyte growth factor promotes neuronal differentiation of neural stem cells derived from embryonic stem cells. javascript:AL_get(this, 'jour', 'Neuroreport.'). Neuroreport 2004, 15:5–8.View ArticlePubMed
                36. Thompson J, Dolcet X, Hilton M, Tolcos M, Davies AM: HGF promotes survival and growth of maturing sympathetic neurons by PI-3 kinase- and MAP kinase-dependent mechanisms. Mol Cell Neurosci 2004, 27:441–452.View ArticlePubMed
                37. Williams B, Park J, Alberta J, Muhlebach SG, Hwang GY, Roberts TM, Stiles CD: A PDGF-regulated immediate early gene response initiates neuronal differentiation in ventricular zone progenitor cells. Neuron 1997, 18:553–562.View ArticlePubMed
                38. Erlandsson A, Enarsson M, Forsberg-Nilsson K: Immature neurons from CNS stem cells proliferate in response to PDGF. J Neurosci 2001, 21:3483–3491.PubMed
                39. Erlandsson A, Braennvall M, Gustafsdottir S, Westermark B, Forsberg-Nilsson K: Autocrine/Paracrine Platelet-Derived Growth Factor Regulates Proliferation of Neural Progenitor Cells. Cancer Res 2006, 66:8042–8048.View ArticlePubMed
                40. Zhang YW, Ding LS, Lai MD: Reg gene family and human diseases. World J Gastroenterol 2003, 9:2635–2641.PubMed
                41. Livesey FJ, O-Brien JA, Li M, Smith AG, Murphy LJ, Hunt SP: A Schwann cell mitogen accompanying regeneration of motor neurons. Nature 1997, 390:614–618.View ArticlePubMed
                42. Averill S, Davis DR, Shortland PJ, Priestley JV, Hunt SP: Dynamic pattern of reg-2 expression in rat sensory neurons after peripheral nerve injury. J Neurosci 2002, 22:7493–7501.PubMed
                43. Namikawa K, Fukushima M, Murakami K, Suzuki A, Takasawa S, Okamoto H, Kiyama H: Expression of Reg/PAP family members during motor nerve regeneration in rat. Biochem Biophys Res Commun 2005, 332:126–134.View ArticlePubMed
                44. Namikawa K, Okamoto T, Suzuki A, Konishi H, Kiyama H: Pancreatitis-associated protein-III is a novel macrophage chemoattractant implicated in nerve regeneration. J Neurosci 2006, 26:7460–7467.View ArticlePubMed
                45. Broekaert D, Eyckerman S, Lavens D, Verhee A, Waelput W, Vandekerckhove J, Tavernier J: Comparison of leptin- and interleukin-6-regulated expression of the rPAP gene family: evidence for differential co-regulatory signals. Eur Cytokine Netw 2002, 13:78–85.PubMed
                46. Lee NH, Weinstock KG, Kirkness EF, Earle-Hughes JA, Fuldner RA, Marmaros S, Glodek A, Gocayne JD, Adams MD, Kerlavage AR, Fraser CM, Venter JC: Comparative expressed-sequence-tag analysis of differential gene expression profiles in PC-12 cells before and after nerve growth factor treatment. Proc Natl Acad Sci USA 1995, 92:8303–8307.View ArticlePubMed
                47. Mayumi K, Yaoi T, Kawai J, Kojima S, Watanabe S, Suzuki H: Improved restriction landmark cDNA scanning and its application to global analysis of genes regulated by nerve growth factor in PC12 cells. Biochim Biophys Acta 1998, 1399:10–18.PubMed
                48. Brown AJH, Hutchings C, Burke JF, Mayne LV: Application of a rapid method (targeted display) for the identification of differentially expressed mRNAs following NGF-induced neuronal differentiation in PC12 cells. Mol Cell Neurosci 1999, 13:119–130.View ArticlePubMed
                49. Angelastro JM, Klimaschewski L, Tang S, Vitolo OV, Weissman TA, Donlin LT, Shelanski ML, Greene LA: Identification of diverse nerve growth factor-regulated genes by serial analysis of gene expression (SAGE) profiling. Proc Natl Acad Sci USA 2000, 97:10424–10429.View ArticlePubMed
                50. Lee KH, Ryu CJ, Hong HJ, Kim J, Lee EH: CDNA microarray analysis of nerve growth factor-regulated gene expression profile in rat PC12 cells. Neurochem Res 2005, 30:533–540.View ArticlePubMed
                51. Dijkmans TF, van Hooijdonk LW, Schouten TG, Kamphorst JT, Vellinga AC, Meerman JH, Fitzsimons CP, de Kloet ER, Vreugdenhil E: Temporal and functional dynamics of the transcriptome during nerve growth factor-induced differentiation. J Neurochem 2008, in press.
                52. Ravni A, Bourgault S, Lebon A, Chan P, Galas L, Fournier A, Vaudry H, Gonzalez B, Eiden LE, Vaudry D: The neurotrophic effects of PACAP in PC12 cells: control by multiple transduction pathways. J Neurochem 2006, 98:321–329.View ArticlePubMed
                53. Braas KM, Schutz KC, Bond JP, Vizzard MA, Girard BM, May V: Microarray analyses of pituitary adenylate cyclase activating polypeptide (PACAP)-regulated gene targets in sympathetic neurons. Peptides 2007, 28:1856–1870.View ArticlePubMed
                54. Martin B, de Maturana RL, Brenneman R, Walent T, Mattson MP, Maudsley S: Class II G Protein-coupled receptors and their ligands in neuronal function and protection. Neuromolecular Med 2005,7(1–2):3–36.View ArticlePubMed
                55. Shah BH, Catt KJ: GPCR-mediated transactivation of RTKs in the CNS: mechanisms and consequences. Trends Neurosci 2004, 27:48–53.View ArticlePubMed
                56. Saunders DE, Hannigan JH, Zajac CS, Wappler NL: Reversal of alcohol's effects on neurite extension and on neuronal GAP-43/B50, N-myc, and c-myc protein levels by retinoic acid. Brain Res Dev Brain Res 1995, 86:16–23.View ArticlePubMed
                57. Routtenberg A, Cantallops I, Zaffuto S, Serrano P, Namgung U: Enhanced learning after genetic overexpression of a brain growth protein. Proc Natl Acad Sci USA 2000, 97:7657–7662.View ArticlePubMed
                58. Benowitz LI, Routtenberg A: GAP-43: an intrinsic determinant of neuronal development and plasticity. Trends Neurosci 1997, 20:84–91.View ArticlePubMed
                59. Oestreicher AB, De Graan PN, Gispen WH, Verhaagen J, Schrama LH: B-50, the growth associated protein-43: modulation of cell morphology and communication in the nervous system. Prog Neurobiol 1997, 53:627–686.View ArticlePubMed
                60. Rekart J, Meiri K, Routtenberg A: Hippocampal-Dependent Memory Is Impaired in Heterozygous GAP-43 Knockout Mice. Hippocampus 2005, 15:1–7.View ArticlePubMed
                61. Bozon B, Davis S, Laroche S: Regulated transcription of the immediate-early gene Zif268: mechanisms and gene dosage-dependent function in synaptic plasticity and memory formation. Hippocampus 2002, 12:570–577.View ArticlePubMed
                62. Bozon B, Kelly A, Josselyn SA, Silva AJ, Davis S, Laroche S: MAPK, CREB and Zif268 are all required for the consolidation of recognition memory. Philos Trans R Soc Lond B Biol Sci 2003, 358:805–814.View ArticlePubMed
                63. Davis S, Bozon B, Laroche S: How necessary is the activation of the immediate early gene Zif268 in synaptic plasticity and learning? Behav Brain Res 2003, 142:17–30.View ArticlePubMed
                64. Jones MW, Errington ML, French PJ, Fine A, Bliss TV, Garel S, Charnay P, Bozon B, Laroche S, Davis S: A requirement for the immediate early gene Zif268 in the expression of late LTP and long-term memories. Nat Neurosci 2001, 4:289–296.View ArticlePubMed
                65. Levkovitz Y, Baraban JM: A dominant negative Egr inhibitor blocks nerve growth factor-induced neurite outgrowth by suppressing c-Jun activation: role of an Egr/c-Jun complex. J Neurosci 2002, 22:3845–3854.PubMed
                66. James AB, Conway AM, Morris BJ: Genomic profiling of the neuronal target genes of the plasticity-related transcription factor - Zif268. J Neurochem 2005, 95:796–810.View ArticlePubMed
                67. James AB, Conway AM, Morris BJ: Regulation of the neuronal proteasome by Zif268 (Egr1). J Neurosci 2006, 26:1624–1634.View ArticlePubMed
                68. van Boxel-Dezaire AH, Stark GR: Cell type-specific signaling in response to interferon-gamma. Curr Top Microbiol Immunol 2007, 316:119–154.View ArticlePubMed
                69. Mullan PB, Quinn JE, Harkin DP: The role of BRCA1 in transcriptional regulation and cell cycle control. Oncogene 2006, 25:5854–5863.View ArticlePubMed
                70. Hehlgans S, Haase M, Cordes N: Signalling via integrins: Implications for cell survival and anticancer stratgies. Biochim Biophys Acta 2007, 1775:163–180.PubMed
                71. Lee SK, Wolfe SW: Peripheral nerve injury and repair. J Am Acad Orthop Surg 2000, 8:243–252.PubMed
                72. Lundborg G: A 25-year perspective of peripheral nerve surgery: evolving neuroscientific concepts and clinical significance. J Hand Surg [Am] 2000,25(3):391–414.View Article

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                © Kunz et al. 2009

                This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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