Identification of novel target genes of nerve growth factor (NGF) in human mastocytoma cell line (HMC-1 (V560G c-Kit)) by transcriptome analysis
- Priyanka Dutta†1,
- Alexandra Koch†1,
- Bjoern Breyer1,
- Heike Schneider1,
- Oliver Dittrich-Breiholz1,
- Michael Kracht2 and
- Teruko Tamura1Email author
© Dutta et al; licensee BioMed Central Ltd. 2011
Received: 7 December 2010
Accepted: 18 April 2011
Published: 18 April 2011
Nerve growth factor (NGF) is a potent growth factor that plays a key role in neuronal cell differentiation and may also play a role in hematopoietic differentiation. It has been shown that NGF induced synergistic action for the colony formation of CD34 positive hematopoietic progenitor cells treated with m acrophage-c olony s timulating f actor (M-CSF or CSF-1), or s tem c ell f actor (SCF). However, the exact role of NGF in hematopoietic system is unclear. It is also not clear whether NGF mediated signals in hematopoietic cells are identical to those in neuronal cells.
To study the signal transduction pathways induced by NGF treatment in hematopoietic cells, we utilized the mastocytoma cell line HMC-1(V560G c-Kit) which expresses the NGF receptor, t ropomyosin-r eceptor-k inase (Trk)A, as well as the constitutively activated SCF receptor, V560G c-Kit, which can be inhibited completely by treatment with the potent tyrosine kinase inhibitor imatinib mesylate (imatinib). NGF rescues HMC-1(V560G c-Kit) cells from imatinib mediated cell death and promotes proliferation. To examine the NGF mediated proliferation and survival in these cells, we compared the NGF mediated upregulated genes (30 and 120 min after stimulation) to the downregulated genes by imatinib treatment (downregulation of c-Kit activity for 4 h) by transcriptome analysis. The following conclusions can be drawn from the microarray data: Firstly, gene expression profiling reveals 50% overlap of genes induced by NGF-TrkA with genes expressed downstream of V560G c-Kit. Secondly, NGF treatment does not enhance expression of genes involved in immune related functions that were down regulated by imatinib treatment. Thirdly, more than 55% of common upregulated genes are involved in cell proliferation and survival. Fourthly, we found Kruppel-like factor (KLF) 2 and Smad family member 7 (SMAD7) as the NGF mediated novel downstream genes in hematopoietic cells. Finally, the downregulation of KLF2 gene enhanced imatinib induced apoptosis.
NGF does not induce genes which are involved in immune related functions, but induces proliferation and survival signals in HMC-1(V560G c-Kit) cells. Furthermore, the current data provide novel candidate genes, KLF2 and SMAD7 which are induced by NGF/TrkA activation in hematopoietic cells. Since the depletion of KLF2 causes enhanced apoptosis of HMC-1(V560G c-Kit), KLF2 may play a role in the NGF mediated survival signal.
Nerve growth factor (NGF) is a member of the family of neurotrophins and is essential for the survival and differentiation of neurons in central and peripheral nerve systems . The binding of NGF to its high affinity receptor, t ropomyosin-r eceptor-k inase(Trk)A, causes activation of the receptor associated tyrosine kinase and participates in the control of mitogenic, survival or differentiation pathways. It has been suggested that NGF and its receptor may also be involved in hematopoietic cell development [2, 3]. In those studies NGF induced synergistic action for the colony formation of CD34 positive hematopoietic progenitor cells treated with the macrophage colony stimulating factor (M-CSF, or CSF-1) , or stem cell factor (SCF) . However, the exact role of TrkA in hematopoietic cell differentiation remains unclear.
The receptor for SCF, c-Kit tyrosine kinase plays a key role in hematopoietic stem cell and mast cell survival, mitogenesis, proliferation, differentiation, adhesion, homing, migration, and functional activation. Despite diversity in the mechanisms of their activation by growth factor ligands , most receptor tyrosine kinases induce signals through the same pathways to typically enhance proliferation and prolong viability. These pathways include activation of the Ras/Raf/Erk, activation of signal transducers and activators of transcription (STATs), and phosphatidylinositol 3 kinase (PI3K). Indeed, c-Kit activation induces all of these pathways, while activated TrkA induces Ras/Raf/Erk, and PI3K pathways but does not cause tyrosine phosphorylation of endogenous STATs [5, 6], suggesting that SCF and NGF not only induce common signal pathways, but also induce unique signal pathways. However, the differences between a set of genes which are upregulated by NGF and those upregulated by SCF in hematopoietic cells has not yet been studied.
The rat pheochromocytoma cell line, PC12, is one of the most thoroughly established systems to study the NGF mediated signal transduction pathway followed by neuronal differentiation. Various studies have investigated gene expression profiles in NGF-treated PC12 cells [7–14], however whether these upregulated genes are similar to genes in the hematopoietic system is not clear. Interestingly, leukemogenic mutant TrkA  does not induce tumor formation, but induces the differentiation of PC12 cells (Koch and Breyer, unpublished data), suggesting that NGF/TrkA signaling is different in neuronal and hematopoietic cells. We have previously shown that NGF-TrkA signaling partially rescues TrkA expressing Bcr-Abl transformed chronic myelogenous leukemia (CML) cells, such as K562, and Meg-01, from cell death induced by a potent inhibitor of Bcr-Abl tyrosine kinase, imatinib mesylate (imatinib). However, the effects of NGF on imatinib treated CML cells are modest. In the presence of NGF, the number of living K562 cells treated with imatinib increased by only 1.5-fold within 4 days and Meg-01 cells did not grow, but just survived for a longer period . A dramatic effect of NGF treatment was observed in oncogenic c-Kit (V560G c-Kit) transformed human mastocytoma cells (HMC-1 (V560G c-Kit)) which are also induced to undergo apoptosis by treatment with imatinib. HMC-1 (V560G c-Kit) cells continue to grow nearly normally in the presence of both imatinib and NGF .
In this paper, using HMC-1 (V560G c-Kit) cells we compared NGF and SCF signaling in the same cell system. HMC-1  expresses the activated SCF receptor, V560G and/or D816V c-Kit [17, 18] and TrkA [19, 20]. The kinase activity of V560G c-Kit can be inhibited completely by treatment with imatinib [21, 22] and cells died within 3 days. NGF rescues HMC-1 (V560G c-Kit) cells from imatinib mediated cell death and promotes proliferation , indicating that NGF can take over mitogenic signaling in these cells. Therefore, we compared the NGF mediated upregulated genes (30 and 120 min after stimulation) to the downregulated genes by imatinib treatment (downregulation of c-Kit for 4 h) by transcriptome analysis. We found Kruppel-like factor (KLF) 2 and Smad family member 7 (SMAD7) as the NGF mediated novel down stream genes in hematopoietic cells and KLF2 may be involved in NGF mediated survival of imatinib treated cells.
NGF rescues HMC-1 (V560G c-Kit) cells from imatinib mediated cell death and promotes proliferation
NGF induced immediate and delayed early genes in imatinib treated HMC-1(V560G c-Kit) cells including several known NGF responsive immediate early genes such as the early growth response (EGR) family EGR1, 2 and 4, c-FOS, and JUNB being upregulated after 30 min of NGF treatment, followed by induction of delayed early genes such as NGFI-A binding protein 2 (NAB2), hairy and enhancer of split, (Drosophila) (HES1), Kruppel-like factor (KLF)10, and activating transcription factors 3 (ATF3) after 2 h.
List of gene set regulated by NGF in HMC-1 (V560G c-Kit) cells.
Gene accession ID
cardiotrophin-like cytokine factor 1
Fas ligand (TNF superfamily, member 6)
interleukin 1, beta
leukemia inhibitory factor (cholinergic differentiation factor)
lymphotoxin beta (TNF superfamily, member 3)
growth differentiation factor 15
ADP-ribosylation factor-like 5B
cytochrome c, somatic
UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 4 (GalNAc-T4)
GTP binding protein overexpressed in skeletal muscle
LFNG O-fucosylpeptide 3-beta-N-acetylglucosaminyltransferase
phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase
Ras-related associated with diabetes
telomerase-associated protein 1
ubiquitin-conjugating enzyme E2E 1 (UBC4/5 homolog, yeast)
cDNA FLJ16758 fis, clone BRACE3038687, moderately similar to Homo sapiens apoptosis-associated tyrosine kinase (AATK)
Homo sapiens mRNA; cDNA DKFZp547J0513 (from clone DKFZp547J0513).
polo-like kinase 3 (Drosophila)
protein kinase, cAMP-dependent, regulatory, type II, alpha
serum/glucocorticoid regulated kinase
tribbles homolog 1 (Drosophila)
LIGAND DEPENDENT NUCLEAR RECEPTOR
nuclear receptor subfamily 4, group A, member 1
nuclear receptor subfamily 4, group A, member 2
nuclear receptor subfamily 4, group A, member 3
G-PROTEIN COUPLED RECEPTORS
chemokine (C-X-C motif) receptor 4 (CXCR4), transcript variant 1
histamine receptor H1
actinin, alpha 2
aryl hydrocarbon receptor nuclear translocator-like 2
Homo sapiens activating transcription factor 3 (ATF3), transcript variant 2
AXIN1 up-regulated 1 (AXUD1)
early growth response 1
early growth response 2
early growth response 3
early growth response 4
ets variant 5
FBJ murine osteosarcoma viral oncogene homolog
Homo sapiens FBJ murine osteosarcoma viral oncogene homolog B (FOSB)
FOS-like antigen 1
FOS-like antigen 2
hairy and enhancer of split 1, (Drosophila)
jun B proto-oncogene
Kruppel-like factor 10 (KLF10), transcript variant 1
Kruppel-like factor 2 (lung)
v-maf musculoaponeurotic fibrosarcoma oncogene homolog F (avian) (MAFF), transcript variant 1
v-myc myelocytomatosis viral oncogene homolog (avian)
NGFI-A binding protein 2 (EGR1 binding protein 2)
nucleolar complex associated 2 homolog (S. cerevisiae)
pre-B-cell leukemia homeobox 2
mRNA for KIAA1452 protein, partial cds.
RING1 and YY1 binding protein
splicing factor 1
SMAD family member 7
suppression of tumorigenicity 18 (breast carcinoma) (zinc finger protein)
zinc finger protein 36, C3H type, homolog (mouse)
zinc finger protein 36, C3H type-like 1
adaptor-related protein complex 1, sigma 1 subunit (AP1S1), transcript variant 2
apolipoprotein L, 6
golgi-associated, gamma adaptin ear containing, ARF binding protein 1
myeloid cell leukemia sequence 1 (BCL2-related)
solute carrier family 2 (facilitated glucose transporter), member 14, mRNA (cDNA clone MGC:71510 IMAGE:5297510), complete cds. [BC060766]
solute carrier family 2 (facilitated glucose transporter), member 3
syntaxin 1A (brain) (STX1A)
transmembrane and coiled-coil domains 5 (TMCO5)
dual specificity phosphatase 4
dual specificity phosphatase 5
dual specificity phosphatase 6
eyes absent homolog 4 (Drosophila)
cancer susceptibility candidate 5 (CASC5), transcript variant 1
coiled-coil domain containing 71
cyclin-dependent kinase inhibitor 1A (p21, Cip1)
ceroid-lipofuscinosis, neuronal 8 (epilepsy, progressive with mental retardation)
pleckstrin homology, Sec7 and coiled-coil domains 4 (PSCD4),
Homo sapiens DNA-damage-inducible transcript 4 (DDIT4),
family with sequence similarity 40, member B (FAM40B)
F-box and leucine-rich repeat protein 17, mRNA (cDNA clone IMAGE:4215262), partial cds. [BC018548]
guanine nucleotide binding protein-like 3 (nucleolar)-like
heterogeneous nuclear ribonucleoprotein A0 (HNRPA0)
immediate early response 2
immediate early response 3
leucine rich repeat containing 8 family, member B (LRRC8B)
leucine zipper protein 1 (LUZP1)
metastasis associated lung adenocarcinoma transcript 1 (non-protein coding)
mediator of RNA polymerase II transcription, subunit 18 homolog (S. cerevisiae) (MED18)
methyltransferase like 7A
myeloma overexpressed (in a subset of t(11;14) positive multiple myelomas)
nucleoporin 188kDa, mRNA (cDNA clone IMAGE:3461492), partial cds. [BC005407]
period homolog 2 (Drosophila)
pleckstrin homology-like domain, family A, member 1
pleckstrin homology-like domain, family A, member 2
phorbol-12-myristate-13-acetate-induced protein 1
mRNA for KIAA0277 gene, partial cds. [D87467]
ring finger protein 125
ribosomal protein L23, mRNA (cDNA clone MGC:34067 IMAGE:5186030), complete cds. [BC034378]
serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1
SH2B adaptor protein 3
SH2 domain protein 2A (SH2D2A)
sprouty-related, EVH1 domain containing 1
sprouty-related, EVH1 domain containing 2
sprouty homolog 2 (Drosophila)
transmembrane protein 49
transmembrane, prostate androgen induced RNA (TMEPAI), transcript variant 1
tumor necrosis factor receptor superfamily, member 12A
zinc finger CCCH-type containing 7B
zinc finger, CCHC domain containing 2
zinc finger protein 295 (ZNF295)
villin 2 (ezrin) (VIL2)
NGF-TrkA activation does not enhance expression of genes involved in immune related functions that were downregulated by imatinib treatment
PANTHER analyses of c-Kit (downregulated genes by imatinib treatment) versus NGF-regulated genes which are involved in immune related function in HMC-1 (V560G c-Kit) cells (selected).
PANTHER Protein Class
V560G c-KIT (P-value)
Cytokine/ interleukin superfamily
Receptor/ cytokine receptor
receptor /type I cytokine receptor
More than 67% of NGF-TrkA upregulated genes are involved in cell survival and proliferation
Ingenuity biological function analyses of NGF regulated genes in HMC-1 (V560G c-Kit) cells (selected).
sub category or function annotation
NGF induced molecules
1. CELL DEATH
AATK, ADM, ATF3, CCND1, CDKN1A, CLCF1, CLN8, CXCR4, CYCS, DDIT4, DUSP4, DUSP6, EGR1, EGR2, EGR3, EGR4, EZR, FASLG, FOS, FOSB, FOSL1, FST, GDF15, HES1, IER3, IL1B, KLF2, KLF10, LIF, LMNB1, LTB, MCL1, MYC, NOC2L, NR4A1, NR4A2, NR4A3, PHLDA1, PHLDA2, PLK3, PMAIP1, PMEPA1, PRNP, RYBP, SERPINE1, SGK1, SKIL, SLC2A3, SMAD7, SPRY2, STX1A, TNFRSF12A, TRIB1, ZFP36
CCND1, CDKN1A, CLCF1, DUSP5, DUSP6, EGR3, FASLG, GDF15, HES1, IL1B, LIF, MCL1, MYC, NR4A1, PRNP, SERPINE1, SGK1, ZFP36
2. CELL GROWTH AND PROLIFERATION
ADM, ARNTL2, ATF3, CCND1, CDKN1A, CXCR4, DUSP5, DUSP6, EGR1, EGR2, EZR, FASLG, FOS, FOSL1, FST, GDF15, GEM, GGA1, HNRNPA0, IER3, IL1B, JUNB, KLF2, KLF10, LIF, MAFF, MCL1, MYC, NR4A2, NR4A3, PHLDA1, PHLDA2, PMEPA1, PRNP, RRAD, SERPINE1, SF1, SGK1, SH2B3, SKIL, SMAD7, SPRY2, TMEM49, TNFRSF12A
ADM, CCND1, CDKN1A, CLCF1, CTSZ, CXCR4, EGR1, EGR2, EGR3, FASLG, FOS, FOSB, FOSL1, FOSL2, FST, GDF15, HES1, HRH1, IER3, IL1B, JUNB, KLF2, KLF10, LIF, MYC, NAB2, NR4A1, NR4A3, PMAIP1, PRNP, SERPINE1, SF1, SH2B3, SH2D2A, SKIL, SMAD7, SPRED1, SPRY2, TNFRSF12A, TRIB1, ZFP36, ZFP36L1
3. CELL DEVELOPMENT
Development of blood cells
ADM, CCND1, CDKN1A, CLCF1, CXCR4, DUSP5, EGR1, EGR2, EGR3, EZR, FASLG, FOS, FOSL1, HRH1, IER3, IL1B, JUNB, KLF2, KLF10, LFNG, LIF, LTB, MYC, NR4A1, PRNP, SH2B3, SH2D2A, SMAD7, SPRED2, TNFRSF12A, ZFP36
Development of tumor cell lines
ADM, ATF3, CCND1, CDKN1A, CXCR4, DUSP5, EGR1, EGR2, FOS, FOSL1, FST, GDF15, GEM, HES1, IER3, IL1B, KLF2, LIF, MCL1, MYC, NAB2, NR4A2, PMEPA1, PRNP, RRAD, SERPINE1, SMAD7, SPRY2, TMEM49
Differentiation of cells
ATF3, CCND1, CDKN1A, CLCF1, CXCR4, DUSP5, EGR1, EGR2, EGR3, FASLG, FOS, FOSL1, FOSL2, FST, HES1, HRH1, IL1B, JUNB, KLF10, LIF, MAFF, MCL1, MYC, NAB2, NR4A1, NR4A2, NR4A3, PRNP, SH2B3, SKIL, SMAD7, SPRED1, SPRED2, SPRY2, TNFRSF12A, ZFP36
ATF3, CCND1, CDKN1A, CLCF1, CXCR4, DUSP5, EGR1, EGR2, EGR3, FASLG, FOS, FOSL1, FOSL2, FST, HES1, HRH1, IL1B, JUNB, KLF10, LIF, MAFF, MCL1, MYC, NAB2, NR4A1, NR4A2, NR4A3, PRNP, SH2B3, SKIL, SMAD7, SPRED1, SPRED2, SPRY2, TNFRSF12A, ZFP36
Maturation of cells
CCND1, CDKN1A, EGR1, FASLG, FOS, HES1, IL1B, LFNG, LIF, MYC, NR4A2, PRNP
4. CELL MORPHOLOGY
ADM, ATF3, CCND1, CDKN1A, EGR1, EZR, FOS, FOSL1, GDF15, GEM, JUNB, KLF2, LIF, MYC, PLK3, SERPINE1
CCND1, CDKN1A, KLF2, MYC
5. CELLULAR FUNCTION AND MAINTAINANCE
T cell development
CCND1, CDKN1A, CXCR4, EGR1, EGR2, EGR3, EZR, FASLG, FOS, IER3, IL1B, JUNB, KLF2, KLF10, LFNG, LIF, LTB, MYC, NR4A1, PRNP, SH2D2A, SMAD7
Cell death of T lymphocytes
CDKN1A, CXCR4, EGR1, EGR3, EZR, FASLG, FOS, IER3, IL1B, KLF2, MYC, NR4A1, SH2D2A, SMAD7
6. GENE EXPRESSION
ATF3, CCND1, CDKN1A, CSRNP1, DUSP4, EGR1, EGR2, EGR4, ETV5, FASLG, FOS, FOSB, FOSL1, FOSL2, FST, HES1, IL1B, JUNB, KLF2, KLF10, LIF, MAFF, MYC, NAB2, NOC2L, NR4A1, NR4A2, NR4A3, PBX2, PER2, RYBP, SGK1, SH2B3, SH2D2A, SKIL, SMAD7, ST18, ZFP36
CCND1, EGR2, FOS, FOSB, FOSL1, FOSL2, KLF2, LIF, MYC, NR4A1, NR4A2
FOS, FOSB, HES1, KLF10, MYC, NR4A2
Furthermore, 32 genes, including c-MYC, EGR1, EGR2, HES1, and KLF2 of 58 genes that were downmodulated by imatinib and upregulated upon stimulation with NGF are involved in survival and proliferation, suggesting that NGF/TrkA signaling may take over the survival and/or mitogenic signal in the imatinib treated HMC-1(V560G c-Kit) cells using these genes.
Novel target genes, KLF2, and SMAD7 which were induced by NGF-TrkA signaling are involved in anti apoptosis signal in hematopoietic cell system
Furthermore, to assess the degree of apoptosis, sister culture cells were stained by an in situ cell death detection kit (Roche, Mannheim, Germany) for terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). In agreement with data obtained from caspase 3 cleavage, TUNEL positive cells appeared within 6 h after imatinib treatment in both KLF2 specific siRNA and control siRNA treated cells. However, numbers of TUNEL positive cells increased significantly faster in KLF2 siRNA treated cells than in control siRNA transfected cells 6 (p = 0.008), 9 (p = 0.009) and 15 h (p = 0.0005) after imatinib treatment (Figure 5C).
Since KLF2 specific siRNA transfectants still grow in the presence of NGF and imatinib, additional survival signals may be mediated by NGF treatment. However, our data strongly suggest that KLF2 is involved in an anti-apoptosis signal.
Cell differentiation and self-renewal are paralleled by a timely, ordered expression of a set of cytokines, growth factors and corresponding receptors. Many members of receptor tyrosine kinase family have emerged as key regulators of these critical cellular processes [4, 29, 30]. Humans have 58 known receptor tyrosine kinases, which fall into 20 subfamilies. Despite differences in structure, many of tyrosine kinases signal through the same pathways to typically enhance proliferation and prolong viability. These pathways include activation of the Ras/Raf/Erk, STATs and PI3K. These facts raised the question of whether each receptor tyrosine kinase is associated with a similar signaling potential, regulated by different expression patterns in different cell types, or whether each tyrosine kinase exhibits a unique signaling pathway.
It has previously been shown that TrkA and c-Kit are co-expressed in mast cells and hematopoietic CD34 positive cells. The treatment of CD34 positive cells with NGF showed the synergistic effects with the SCF treatment on colony formation. For mast cell culture in vitro, bone marrow cells are cultivated for 4-6 weeks in the presence of SCF, interleukin (IL) 3 and IL4 . We examined whether mouse primary mast cells can survive in the presence of NGF, or NGF and IL3/IL4 in the absence of SCF. Under these conditions mouse mast cells did not survive in the absence of SCF. These data suggest that NGF does not assume the role of SCF in normal mast cells. According to PANTHER analysis [32, 33], the difference of gene upregulation of cytokines, growth factors, and their receptors between SCF and NGF stimulation is significant, suggesting that upregulation of cytokines and their receptors play a role in survival of normal mast cells. In agreement with these data, few genes encoding cytokines/their receptors in PC12 cells were upregulated 24 h after NGF treatment , suggesting that NGF poorly induces cytokine and growth factor genes in different cell types.
It has been shown that STAT5 is required for c-Kit mediated mast cell survival and differentiation . Although NGF does not induce tyrosine phosphorylation of STATs, HMC-1(V560G c-Kit) cells survive by NGF stimulation without c-Kit signaling. Thereby our array data provide novel candidate genes, KLF2, SMAD7, PBX2, and HOXB8 which are induced by NGF/TrkA activation in hematopoietic cells, and have not been reported as NGF target genes in the PC12 cell system [7–14]. On the other hand, another known target gene of NGF treatment in PC12 cells, wingless-related MMTV integration site 7B (Wnt7b)  was not upregulated by NGF treatment in HMC-1 (V560G c-Kit) cells, suggesting that Wnt7b may be a specific target gene for NGF signaling in neuronal cells. These data indicate that most NGF upregulated genes were common, but some of them may be cell-type specific. However, we cannot presently rule out the possibility that the difference of upregulated genes is due to differences between human (HMC-1) and rat (PC12) cells.
Interestingly, KLF2, SMAD7, PBX2, and HOXB8 are suggested to be involved in self-renewal or in anti differentiation signal of stem cells or hematopoietic stem cells [26, 34–37]. We show here that KLF2 modulates imatinib-mediate apoptosis. Along the same line, it has been shown that KLF2-deficient T cells had a spontaneously activated phenotype and died rapidly from Fas-ligand-induced apoptosis , and induction of KLF2 expression corresponded with long-term T cell survival , suggesting that KLF2 plays a role in T cell survival. Furthermore, KLF2-/- embryos have a significantly increased number of primitive erythroid cells undergoing apoptotic cell death. These data suggest that the upregulation of the KLF2 gene induced by the stimulation with NGF plays a role in the survival signal in imatinib treated HMC-1(V560G c-Kit) cells.
We compared the signaling of two structurally and functionally diverse receptor tyrosine kinases, c-Kit and TrkA, in hematopoietic cells. The c-Kit activation induces cytokines and their receptors, but TrkA does not, suggesting that the part of the signal pathways induced by the two receptors is different. However, TrkA is able to induce common novel downstream targets such as KLF2 and SMAD7 which has not been reported in the neuronal system, indicating that NGF induces genes which are involved in stem cell maintenance similar to c-Kit signaling in hematopoietic cells. Furthermore, upregulation of KLF2 may be involved in NGF mediated survival of imatinib treated cells.
HMC-1(V560G c-Kit)  were grown in RPMI1640 medium supplemented with 10-20% (v/v) fetal calf serum (FCS). The presence of V560G mutation and the absence of 816 mutation in c-Kit was confirmed by sequencing.
HMC-1(V560G c-Kit) cells were grown in medium containing 10% FCS in the presence of 5 μM imatinib (kindly provided by Novartis, Basel, Switzerland) and/or 100 ng/ml human recombinant NGF (PeproTech Inc., Rocky Hill, NJ). Cells were counted in a Neubauer chamber using 0.1% Trypan Blue (Sigma-Aldrich Chemie GmbH, Steinheim, Germany).
To assess the degree of apoptosis, an in situ cell death detection kit (Roche, Mannheim, Germany) was used for terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining.
Growth factor stimulation, and RNA-isolation
Cells were serum starved for 17 h, then treated with d im ethyl s ulfo xide (DMSO) or 5 μM imatinib for 4 hours prior to stimulation with 100 ng/ml mouse recombinant SCF (PeproTech Inc.) or NGF, respectively. After 30 or 120 min the stimulation was stopped in ice-cold PBS. RNA was isolated from growth factor treated or untreated HMC-1(V560G c-Kit) cells using RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Residual DNA contamination was removed with DNAseI (Invitrogen GmbH, Darmstadt, Germany) according to the manufacturer's recommendations, and the RNA was again purified with RNeasy Mini kit (Qiagen).
The "Whole Human Genome Microarray" (G4112F, ID 014850, Agilent Technologies) used in this study contained 45015 oligonucleotide probes covering the entire human transcriptome. cRNA-synthesis was performed with the "Low RNA Input Linear Amplification Kit PLUS, Two-Color" (#5188-5340, Agilent Technologies) as directed by the manufacturer. cRNA fragmentation, hybridization and washing steps were also performed exactly as recommended by the manufacturer "Two-Color Microarray-Based Gene Expression Analysis Protocol V5.5" (see http://www.agilent.com for details) except that 4 μg of each labeled cRNA were used for hybridization. Slides were scanned on the Agilent Micro Array Scanner G2505 B at two different PMT settings, namely 100% (default setting) and 5%, to increase the dynamic range of the measurements. Data extraction and normalization were performed with the "Feature Extraction Software V126.96.36.199" by using the recommended default extraction protocol file: GE2-v5_95_Feb07.xml. Only probes with allocated gene symbols and arithmetic mean intensity >50 for both channels were considered for further analysis. Genes with p value ≤ 0.0001 and fold induction ratio of ≥ 2 were considered significantly induced.
The complete microarray data have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO series accession number GSE28045.
Functional and gene ontology analysis
Categorization of genes according to protein class was done using PANTHER (Protein Analysis Through Evolutionary Relationships) Classification Systems . For each protein class, PANTHER calculates the number of genes identified in that category in both the list of differentially regulated genes and a reference list containing all the probe sets present on the chip and compares these results using the binomial test to determine if there are more genes than expected in the differentially regulated list . Over-representation is defined by p < 0.05. Functional Analysis identifying the biological functions that were most significant to the data set were carried out using Ingenuity Pathways Analysis (IPA) (Ingenuity Systems, Mountain View, CA). Right-tailed Fisher's exact test was used to calculate a p-value determining the probability that each biological function and/or disease assigned to that data set is due to chance alone.
RT-PCR and qRT-PCR
Reverse transcription was carried out using oligo dT primers and the Omniscript reverse transcriptase kit (Qiagen) following the instructions provided. PCRs were set up according to the following profile: an initial denaturation step of 94°C for 2 min, repeating cycles of 94°C for 30 seconds, annealing temperature given for each primer pair for 1 minute, and 72°C for 1 minute. Following primer pairs were used: human EGR-1 (NM_001964) forward primer: 5'-CAGCAGTCCCATTTACTCAG-3', reverse primer: 5'-GACTGGTAGCTGGTATTG-3' (annealing temperature 56°C, product size 345 bp); human KLF2 (NM_016270) forward primer: 5'-CTACACCAAGAGTTCGCATCTG-3', reverse primer: 5'-CCGTGTGCTTTCGGTAGTG-3'(annealing temperature 57°C, product size 137 bp); human SMAD7 (NM_005904) forward primer: 5'-ACTCCAGATACCCGATGGATTT-3', reverse primer: 5'-CCTCCCAGTATGCCACCAC-3' (annealing temperature 57°C, product size 174 bp); human beta-actin (ACTB, NM_001101) forward primer: 5'-CCCAAGGCCAACCGCGAGAAGAT-3', reverse primer: 5'-GTCCCGGCCAGCCAGGTCCAG-3' (annealing temperature 66°C, product size 219 bp). Separation of the DNA fragments was carried out on 2% (w/v) agarose gels, stained with ethidium bromide (2 μg/ml) and photographed under UV light. Quantitative (TaqMan) RT-PCR was performed as previously described . TaqMan probes (Applied Biosystems, assay-IDs as follows: Hs00152928_m1(EGR1); Hs00357891_s1(JUNB); Hs00170630_m1(FOS); Hs00153408_m1(MYC); Hs00360439_g1(KLF2); Hs99999905_m1(GAPDH); Hs99999908_m1(GUSB)) were used with TaqMan® Fast Universal PCR Master Mix (2×) (Applied Biosystems).
Transfection, RNA interference and immunoblotting
SiRNA against human LKLF (KLF2) and control siRNA was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). 4 × 106 HMC-1(V560G c-Kit) cells were transfected with 200 pmol of siRNA using Amaxa® Cell Line Nucleofector Kit L with program T-020 in an Amaxa® Nucleofector® II device according to the manufacturer's instructions. Two days after transfection, cells were treated with imatinib (5 μM) for up to 15 h. During imatinib treatment, aliquots were prepared for analysis by TUNEL staining or immunoblot.
For immunoblot analysis, whole cell lysates were prepared using 1 × SDS buffer (80 mM Tris/HCl, pH 6.8, 2% sodium dodecyl sulfate (SDS), 10% glycerol, 5% beta-mercaptoethanol, 0.01% bromphenole blue). Then, cell lysates were analyzed for cleavage fragments of caspase 3 by immunoblot analysis using a polyclonal antibody against cleaved caspase-3 (Asp175) (Cell Signaling Technology, Beverly, MA) or GAPDH (Santa Cruz Biotechnology) as described previously . Knockdown of KLF2 was verified by semi-quantitative RT-PCR and quantitative analysis was performed using TINA2.0 software (Raytest Isotopenmessgeraete GmbH, Straubenhardt, Germany).
We thank Sabine Klebba-Färber for technical assistance and Bruce Boschek for critically reading the manuscript. P.D. was supported by the MD PhD program of the Medizinische Hochschule Hannover (MHH), A. K. was supported by the Madeleine Schickedanz-Kinderkrebsstiftung, Wiedeking-Stiftung, Habilitationsstipendium (MHH) and the HiLF program (MHH). The research was supported by Sonderforschungsbereich 566 (B2, Z2), and by the Leistungsorientierte Mittelvergabe (LOM) of MHH with Frauen-Faktor. The publication of this article was funded by the program "Open Access Publizieren" of Deutsche Forschungsgemeinschaft.
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