Open Access

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

  • Dieter Kunz1Email author,
  • Gaby Walker2,
  • Marc Bedoucha3,
  • Ulrich Certa4,
  • Pia März-Weiss2,
  • Beatrice Dimitriades-Schmutz1 and
  • Uwe Otten1
Contributed equally
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.
https://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.

https://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.

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© Kunz et al; licensee BioMed Central Ltd. 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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