BMC Genomics BioMed Central Open Access Research article Expression profiling and Ingenuity biological function analyses of interleukin-6- versus nerve growth factor-stimulated PC12 cells Dieter Kunz*†1, Gaby Walker†2, Marc Bedoucha3, Ulrich Certa4, Pia MärzWeiss2, Beatrice Dimitriades-Schmutz1 and Uwe Otten1 Address: 1Department of Biomedicine, Institute of Physiology, University of Basel, Pestalozzistrasse 25, CH-4056 Basel, Switzerland, 2Molecular Medicine Laboratories (MML), Hoffmann-La Roche Ltd., Grenzacherstrasse 2, CH-4002 Basel, Switzerland, 3Discovery Research (PRBD), Hoffmann-La Roche Ltd., Grenzacherstrasse 2, CH-4002 Basel, Switzerland and 4Non-Clinical Drug Safety (NCS), Hoffmann-La Roche Ltd., Grenzacherstrasse 2, CH-4002 Basel, Switzerland Email: Dieter Kunz* - dieter.kunz@unibas.ch; Gaby Walker - gaby.walker@roche.com; Marc Bedoucha - marc.bedoucha@roche.com; Ulrich Certa - ulrich.certa@roche.com; Pia März-Weiss - pia.maerz-weiss@roche.com; Beatrice DimitriadesSchmutz - beatrice.dimitriades@unibas.ch; Uwe Otten - uwe.otten@unibas.ch * Corresponding author †Equal contributors Published: 24 February 2009 BMC Genomics 2009, 10:90 doi:10.1186/1471-2164-10-90 Received: 4 December 2008 Accepted: 24 February 2009 This article is available from: http://www.biomedcentral.com/1471-2164/10/90 © 2009 Kunz et al; licensee 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. 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 growthassociated 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. Page 1 of 17 (page number not for citation purposes) BMC Genomics 2009, 10:90 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 (IL6R) 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 [5-7]. The transsignaling mechanism led to the development of a fusion protein in which IL-6 is covalently linked to s-IL6R 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 http://www.biomedcentral.com/1471-2164/10/90 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 IL6 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 Page 2 of 17 (page number not for citation purposes) BMC Genomics 2009, 10:90 -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). 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 -2fold 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). 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. 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, qRTPCR 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 IL6 (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 http://www.biomedcentral.com/1471-2164/10/90 cells, neither microarray nor qRT-PCR analyses revealed changes in RGE3B expression. 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. 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. 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 Page 3 of 17 (page number not for citation purposes) BMC Genomics 2009, 10:90 http://www.biomedcentral.com/1471-2164/10/90 Table 1: List of gene set regulated by IL-6 in PC12 cells Gene Cytokines chemokine ligand 13 chemokine ligand 10 Accession no. Fold change Subcellular location CXCL13 CXCL10 AF044196 U17035 43 7 Extracellular Space Extracellular Space CYP4F16 CP PADI3 ACSL1 TGM1 NOS2A OTC LOC374569 TREH KYNU NOS3 GATM GNAZ ST6GAL1 AKR1C1 MX1 ALDOC OAS1 PDIA2 RNMT POLA2 SRD5A1 ALAS2 GSTA3 UGT8 CDC42 CDO1 ST8SIA3 U39207 AF202115 D88034 D90109 M57263 U03699 M11266 AB009372 AF038043 U68168 AJ011115 U07971 U77485 M83143 BAA92883 P20591 X06984 Z18877 AAC50401 BAA82447 AJ245648 J05035 D86297 X78847 BC075069 U37720 M35266 X80502 424 191 142 102 93 58 43 37 35 25 21 14 14 14 12 9 3 3 3 3 -2 -2 -3 -3 -3 -4 -4 -5 Cytoplasm Extracellular Space Cytoplasm Cytoplasm Plasma Membrane Cytoplasm Cytoplasm Unknown Plasma Membrane Cytoplasm Cytoplasm Cytoplasm Plasma Membrane Cytoplasm Cytoplasm Nucleus Cytoplasm Cytoplasm Cytoplasm Nucleus Nucleus Cytoplasm Cytoplasm Cytoplasm Cytoplasm Cytoplasm Cytoplasm Cytoplasm G-protein coupled receptors adrenergic receptor, alpha-2B arginine vasopressin receptor 2 vasoactive intestinal peptide receptor 1 cholinergic receptor, muscarinic 3 cholinergic receptor, muscarinic 4 ADRA2B AVPR2 VIPR1 CHRM3 CHRM4 M32061 AAB87678 M86835 AB017656 M16409 26 5 -2 -3 -10 Plasma Membrane Plasma Membrane Plasma Membrane Plasma Membrane Plasma Membrane Growth factors regenerating islet-derived 3 alpha regenerating islet-derived 3 beta growth differentiation factor 15 platelet-derived growth factor alpha nudix-type motif 6 jagged 2 hepatocyte growth factor macrophage stimulating 1 REG3A REG3B GDF15 PDGFA NUDT6 JAG2 HGF MST1 L10229 S43715 AJ011970 M29464 AF188995 U70050 X84046 X95096 1084 672 80 69 22 5 4 -4 Extracellular Space Extracellular Space Extracellular Space Extracellular Space Extracellular Space Extracellular Space Extracellular Space Extracellular Space Ion channels glutamate receptor, ionotropic, delta 2 purinergic receptor P2X GRID2 P2RX2 U08256 Y10475 91 11 Plasma Membrane Plasma Membrane Kinases fyn-related kinase Janus kinase 2 phosphatidylinositol 4-kinase beta FRK JAK2 PI4KB U02888 U13396 D84667 122 120 2 Nucleus Cytoplasm Cytoplasm Enzymes cytochrome P450, 4f16 ceruloplasmin peptidyl arginine deiminase, type III acyl-CoA synthetase, member 1 transglutaminase 1 nitric oxide synthase 2A ornithine carbamoyltransferase Similar to Lysophospholipase trehalase kynureninase nitric oxide synthase 3 glycine amidinotransferase guanine nucleotide binding protein, alpha z ST6 galactosamide alpha-2,6-sialyltranferase 1 aldo-keto reductase, 1C1 myxovirus resistance 1 aldolase C 2',5'-oligoadenylate synthetase 1 protein disulfide isomerise, A2 RNA (guanine-7-) methyltransferase polymerase, alpha 2 steroid-5-alpha-reductase, alpha 1 aminolevulinate, delta-, synthase 2 glutathione S-transferase A3 UDP glycosyltransferase 8 cell division cycle 42 cysteine dioxygenase, type I ST8 alpha-2,8-sialyltransferase 3 Page 4 of 17 (page number not for citation purposes) BMC Genomics 2009, 10:90 http://www.biomedcentral.com/1471-2164/10/90 Table 1: List of gene set regulated by IL-6 in PC12 cells (Continued) pim-3 oncogene fer tyrosine kinase mitogen-activated protein kinase kinase 5 fibroblast growth factor receptor 1 activin receptor, type IIA PIM3 FER MAP2K5 FGFR1 ACVR2A AF086624 X13412 U37462 S54008 S48190 2 -2 -2 -3 -4 Unknown Cytoplasm Cytoplasm Plasma Membrane Plasma Membrane Nuclear receptors retinoic acid receptor alpha nuclear receptor, *C2 vitamin D receptor RARA NR3C2 VDR U15211 M36074 J03630 20 8 -4 Nucleus Nucleus Nucleus Peptidases complement component 1s caspase 1 proteasome subunit, alpha 1 kallikrein-related peptidase 8 C1S CASP1 PSMA1 KLK8 D88250 U14647 M29859 AJ005641 230 40 5 -5 Extracellular Space Cytoplasm Cytoplasm Extracellular Space Phosphatases pyruvate dehydrogenase phosphatase 2 protein tyrosine phosphatase receptor D PDP2 PTPRD AF062741 U57502 -4 -9 Cytoplasm Plasma Membrane Transcription regulators signal transducer and activator of transcription 1 Kruppel-like factor 6 HIV-1 Tat interacting protein HIV enhancer binding protein 2 upstream transcription factor 1 early growth response 1 interferon regulatory factor 1 signal transducer and activator of transcription 2 breast cancer 1 D site of albumin promoter binding protein nuclear factor I/B transcription elongation factor A 2 STAT1 KLF6 HTATIP HIVEP2 USF1 EGR1 IRF1 STAT2 BRCA1 DBP NFIB TCEA2 AF205604 AF072403 AAB18236 D37951 U41741 M18416 M34253 AF206162 U36475 J03179 Y07685 D12927 579 249 159 65 22 3 3 3 -2 -2 -2 -5 Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Transmembrane receptors oxidized low density lipoprotein receptor 1 histocompatibility 2, Q region locus 10 insulin-like growth factor 2 receptor Fc fragment of IgG receptor IIa (CD32) growth hormone receptor OLR1 H2-Q10 IGF2R FCGR2A GHR AB018097 M31018 NM_000876 M64368 Z83757 587 160 39 16 12 Plasma Membrane Plasma Membrane Plasma Membrane Plasma Membrane Plasma Membrane Transporters cadherin 17 solute carrier family 6, member 3 nucleoporin 153kDa solute carrier family 9, member 2 cadherin 17 lipocalin 2 syntaxin 4 secretory carrier membrane protein 2 solute carrier family 12, member 5 solute carrier family 30, member 2 syntaxin 5 CDH17 SLC6A3 NUP153 SLC9A2 CDH17 LCN2 STX4 SCAMP2 SLC12A5 SLC30A2 STX5 X78997 M80570 L06821 L11004 L46874 X13295 L20821 AF295405 U55816 U50927 U87971 273 90 83 32 13 9 3 2 -3 -5 -8 Plasma Membrane Plasma Membrane Nucleus Plasma Membrane Plasma Membrane Extracellular Space Plasma Membrane Cytoplasm Plasma Membrane Plasma Membrane Cytoplasm Others regenerating islet-derived 1 alpha TIMP metallopeptidase inhibitor 1 calcitonin-related polypeptide beta fibrinogen gamma chain trans-golgi network protein 2 LIM and senescent cell antigen-like domains 1 alpha-2-HS-glycoprotein REG1A TIMP1 CALCB FGG TGOLN2 LIMS1 AHSG J05722 L31883 M11596 J00734 X53565 AAA20086 M29758 796 210 195 164 113 94 80 Extracellular Space Extracellular Space Extracellular Space Extracellular Space Cytoplasm Plasma Membrane Extracellular Space Page 5 of 17 (page number not for citation purposes) BMC Genomics 2009, 10:90 http://www.biomedcentral.com/1471-2164/10/90 Table 1: List of gene set regulated by IL-6 in PC12 cells (Continued) ribosomal protein L3-like collagen, type IV, alpha 5 parvalbumin YTH domain containing 1 growth hormone releasing hormone annexin A1 collagen, type XII, alpha 1 regenerating islet-derived 3 gamma adenylate cyclase activating polypeptide 1 heat shock protein 90 kDa, alpha B 1 luteinizing hormone beta galectin 5 myocilin prolactin family 8a81 troponin C type 2 ribosomal protein L18a fibrinogen beta chain tropomyosin 3 tubulin, beta extracellular proteinase inhibitor growth associated protein 43 galectin 9 tubulin, alpha 4a BCL2-like 11 integrin alpha 7 syndecan 2 zinc finger protein 260 filamin C metallothionein 3 arginine vasopressin fasciculation and elongation protein zeta 1 crystallin, alpha B neurofascin RPL3L COL4A5 LOC4951 YTHDC1 GHRH ANXA1 COL12A1 REG3G ADCYAP1 HSP90AB1 LHB LGALS5 MYOC PRL8A8 TNNC2 RPL18A FGB TPM3 TUBB EXPI GAP43 LGALS9 TUBA4A BCL2L11 ITGA7 SDC2 ZNF260 FLNC MT3 AVP FEZ1 CRYAB NFASC AAC50777 AB041350 J02705 AF144731 Z34092 M19967 U57362 L20869 S83513 S45392 U25653 L36862 AF093567 AB000107 J05598 X14181 U05675 X72859 AB011679 X13309 M16736 U72741 M13444 AF136927 X65036 M81687 U56862 AF119148 S65838 M25646 U63740 U04320 U81036 60 59 58 39 31 29 26 24 20 20 17 8 8 8 8 7 6 4 4 3 3 3 3 2 -2 -2 -2 -3 -3 -4 -4 -6 -7 Unknown Extracellular Space Unknown Cytoplasm Extracellular Space Plasma Membrane Extracellular Space Extracellular Space Extracellular Space Cytoplasm Extracellular Space Extracellular Space Cytoplasm Extracellular Space Unknown Cytoplasm Extracellular Space Cytoplasm Cytoplasm Extracellular Space Plasma Membrane Extracellular Space Cytoplasm Cytoplasm Plasma Membrane Plasma Membrane Nucleus Cytoplasm Cytoplasm Extracellular Space Cytoplasm Nucleus Plasma Membrane Gene description names, gene symbols are from IPA Tool; accession numbers are from GenBank Table 2: List of gene set regulated by NGF in PC12 cells Gene Enzymes rat senescence marker protein 2A gene myosin, heavy chain 3 lecithin-cholesterol acyltransferase UDP glucuronosyltransferase 2, polypeptide A1 contactin 4 phosphodiesterase 4B, gulonolactone (L-) oxidase superoxide dismutase 3 fibronectin 1 acetylcholinesterase tryptophan hydroxylase 1 aldo-keto reductase family 1, member C1 guanine nucleotide binding protein, alpha z aminoadipate aminotransferase phospholipase D2 N-deacetylase/N-sulfotransferase 1 phosphate cytidylyltransferase 2 peptidylprolyl isomerase A Rab geranylgeranyltransferase alpha glutathione S-transferase A3 cytochrome P450, 4F4 Accession no. SMP2A MYH3 LCAT UGT2A1 CNTN4 PDE4B GULO SOD3 FN1 ACHE TPH1 AKR1C1 GNAZ AADAT PLD2 NDST1 PCYT2 PPIA RABGGTA GSTA3 CYP4F4 X63410 K03468 X54096 X57565 U35371 J04563 J03536 Z24721 X15906 S50879 X53501 BAA92883 U77485 Z50144 D88672 M92042 AF080568 M19533 L10415 X78847 U39206 Fold change 303 133 101 63 44 37 34 28 28 28 24 10 9 5 4 3 2 -2 -2 -3 -3 Subcellular location Cytoplasm Cytoplasm Extracellular Space Cytoplasm Plasma Membrane Cytoplasm Cytoplasm Extracellular Space Plasma Membrane Plasma Membrane Cytoplasm Cytoplasm Plasma Membrane Cytoplasm Cytoplasm Cytoplasm Cytoplasm Cytoplasm Unknown Cytoplasm Cytoplasm Page 6 of 17 (page number not for citation purposes) BMC Genomics 2009, 10:90 http://www.biomedcentral.com/1471-2164/10/90 Table 2: List of gene set regulated by NGF in PC12 cells (Continued) 3-hydroxyanthranilate 3,4-dioxygenase stearoyl-Coenzyme A desaturase 2 aldo-keto reductase family 1, member C3 myxovirus resistance 2 serine dehydratase HAAO SCD2 AKR1C3 MX2 SDS D28339 AB032243 L32601 X52711 M38617 -3 -4 -6 -10 -11 Cytoplasm Cytoplasm Cytoplasm Nucleus Cytoplasm G-protein coupled receptors calcitonin/calcitonin-related polypeptide alpha angiotensin II receptor 1 cholinergic receptor, muscarinic 3 parathyroid hormone receptor 1 CALCA AGTR1 CHRM3 PTHR1 V01228 NM_009585 AB017656 M77184 136 50 -2 -3 Plasma Membrane Plasma Membrane Plasma Membrane Plasma Membrane Growth factors growth differentiation factor 15 transforming growth factor beta 1 brain-derived neurotrophic factor neuregulin 1 GDF15 TGFB1 BDNF NRG1 AJ011970 X52498 X67108 U02324 131 101 89 -3 Extracellular Space Extracellular Space Extracellular Space Extracellular Space Ion channels calcium channel, voltage-dependent, beta 2 glutamate receptor, ionotropic, delta 2 sodium channel, voltage-gated, type II, beta potassium inwardly-rectifying channel J4 solute carrier family 9 member 3 purinergic receptor P2X, ligand-gated ion channel 2 sodium channel, voltage-gated, type I, alpha purinergic receptor P2X-like 1 gamma-aminobutyric acid A receptor gamma 2 CACNB2 GRID2 SCN2B KCNJ4 SLC9A3 P2RX2 SCN1A P2RXL1 GABRG2 M80545 U08256 U37147 X87635 M85300 Y10475 M22253 X92070 X56313 90 78 73 51 40 13 12 -2 -19 Plasma Membrane Plasma Membrane Plasma Membrane Plasma Membrane Plasma Membrane Plasma Membrane Plasma Membrane Plasma Membrane Plasma Membrane Kinases G protein-coupled receptor kinase 5 protein kinase, cGMP-dependent, type II mitogen-activated protein kinase kinase kinase kinase 1 calcium/calmodulin-dependent serine protein kinase discs, large homolog 1 phosphatidylinositol 4-kinase beta discoidin domain receptor family member 1 non-metastatic cells 6 GRK5 PRKG2 MAP4K1 CASK DLG1 PI4KB DDR1 NME6 NM_005308 Z36276 Y09010 U47110 U14950 D84667 L26525 AF051943 131 68 25 3 3 3 -8 -14 Plasma Membrane Cytoplasm Cytoplasm Plasma Membrane Plasma Membrane Cytoplasm Plasma Membrane Extracellular Space Peptidases carboxypeptidase A3 ADAM metallopeptidase domain 17 Proteasome subunit alpha 1 protein disulfide isomerase family A member 3 caspase 1 CPA3 ADAM17 PSMA1 PDIA3 CASP1 U67914 AJ012603 M29859 D63378 U14647 5 4 3 2 -5 Extracellular Space Plasma Membrane Cytoplasm Cytoplasm Cytoplasm Phosphatases dual specificity phosphatase 6 protein phosphatase 1 subunit 1A protein tyrosine phosphataser type 11 DUSP6 PPP1R1A PTPN11 U42627 AJ276593 U09307 53 18 -2 Cytoplasm Cytoplasm Cytoplasm Transcription regulators jun dimerization protein 2 cAMP responsive element modulator JDP2 CREM U53449 Z15158 -2 -4 Nucleus Nucleus Transmembrane receptors cholinergic receptor, nicotinic, beta 1 CHRNB1 X74833 39 Plasma Membrane Transporters solute carrier family 1 member 1 solute carrier family 22, member 3 gap junction protein, beta 2 solute carrier family 1, member 3 solute carrier family 22, member 6 vacuolar protein sorting 33 homolog B solute carrier family 30, member 1 syntaxin 4 murinoglobulin 1 ATPase, Cu++ transporting, beta polypeptide SLC1A1 SLC22A3 GJB2 SLC1A3 SLC22A6 VPS33B SLC30A1 STX4 MUG1 ATP7B U21104 AF055286 X51615 S59158 AF008221 U35245 U17133 L20821 J03552 AF120492 238 95 55 6 6 4 3 2 -2 -6 Plasma Membrane Plasma Membrane Plasma Membrane Plasma Membrane Plasma Membrane Cytoplasm Plasma Membrane Plasma Membrane Extracellular Space Cytoplasm Page 7 of 17 (page number not for citation purposes) BMC Genomics 2009, 10:90 http://www.biomedcentral.com/1471-2164/10/90 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 glutathione S-transferase A3 aldo-keto reductase family 1, member C1 GNAZ GSTA3 AKR1C1 14 -3 12 9 -3 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 purinergic receptor P2X, ligand-gated ion channel GRID2 P2RX2 91 11 78 13 Kinases phosphatidylinositol 4-kinase beta PI4KB 2 3 Peptidases caspase 1 proteasome subunit alpha 1 CASP1 PSMA1 40 5 -5 3 STX4 3 2 113 94 94 26 3 106 75 3 28 3 Transporters syntaxin 4 Others trans-golgi network protein 2 LIM and senescent cell antigen-like domains 1 fibrinogen gamma chain collagen, type XII, alpha 1 extracellular proteinase inhibitor TGOLN2 LIMS1 FGG COL12A1 EXPI Gene description names, gene symbols are from IPA Tool 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 REG3Bexpression 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 IL6/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 Page 8 of 17 (page number not for citation purposes) BMC Genomics 2009, 10:90 GeneChip Changes in Figure IL-6-stimulated 1in expression PC12ofcells GAP-43determined and REG3B by qRT-PCR mRNAversus levels 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. cellular adhesion and cellular polarisation [30-32]. 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 http://www.biomedcentral.com/1471-2164/10/90 [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 upregulated 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 [46-51]. 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]. Page 9 of 17 (page number not for citation purposes) BMC Genomics 2009, 10:90 http://www.biomedcentral.com/1471-2164/10/90 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 [61-64] 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]. Figure Changes sus NGF-stimulated 2in expression PC12 of GAP-43cells mRNA levels in IL-6- verChanges in expression of GAP-43- mRNA levels in IL6- versus NGF-stimulated PC12 cells. qRT-PCR analyses were performed as described in the Methods section. 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 195fold, 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. 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 [56-60]. The data herein are confirmative to our previous study in which we 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 IL6. 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 upregulates major components of the ECM including collagen, type XI, alpha1 (COL11A1), COL12A1, fibronectin1 (FN1) as well as fibrillin2 (FN2) (Table 2). Page 10 of 17 (page number not for citation purposes) BMC Genomics 2009, 10:90 http://www.biomedcentral.com/1471-2164/10/90 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 IL-6-regulated genes Cancer Cellular Growth and Proliferation Cell Death Cell-to-Cell Signalling and Interaction Tissue Development Cellular Movement Cellular Development Small Molecule Biochemistry ... Nervous system development and function NGF-regulated genes Cellular growth and proliferation Cell-to-cell signalling and interaction Molecular transport Cancer Cellular movement Cell death Neurological diseases Nervous system development and function Number of genes Significance (p-value) 61 54 47 46 45 39 38 37 2.98 × 10-6 to 5.16 × 10-3 1.14 × 10-6 to 5.16 × 10-3 4.54 × 10-6 to 5.16 × 10-3 2.26 × 10-7 to 5.16 × 10-3 2.26 × 10-7 to 5.15 × 10-3 9.19 × 10-6 to 5.16 × 10-3 8.56 × 10-6 to 4.85 × 10-3 1.32 × 10-5 to 4.47 × 10-3 24 2.83 × 10-5 to 3.77 × 10-3 37 31 30 30 29 29 29 29 7.86 × 10-5 to 8.88 × 10-3 1.03 × 10-4 to 7.43 × 10-3 8.89 × 10-6 to 8.70 × 10-3 1.03 × 10-4 to 7.43 × 10-3 2.41 × 10-5 to 8.70 × 10-3 2.73 × 10-5 to 8.77 × 10-3 1.07 × 10-4 to 8.70 × 10-3 1.60 × 10-4 to 8.70 × 10-3 p-values are from IPA Tool 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 combi- nation 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. Page 11 of 17 (page number not for citation purposes) BMC Genomics 2009, 10:90 http://www.biomedcentral.com/1471-2164/10/90 Table 5: Ingenuity biological function analyses of IL-6-versus NGF-regulated genes in PC12 cells (selected) Category Sub-Category or Function annotation Cellular Growth and Proliferation Growth of cells Proliferation of cells Cellular Movement Cell movement Chemotaxis Cell-To-Cell Signaling and Interaction Adhesion of cells Cell Signaling Quantity of calcium Production of nitric oxide Flux of calcium Cell surface receptor linked signal transduction Small Molecule Biochemistry Quantity of cyclic AMP IL-6 regulated genes in PC12 cells p-value Molecules NGF-regulated genes in PC12 cells p-value Molecules 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 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 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 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 4.05 × 10-4 ANXA1, CDC42, CXCL10, CXCL13, FCGR2A, FER, FGFR1, GNAZ, HGF, IGF2R, LGALS9, NOS3, VIPR1 3.82 × 10-3 AGTR1, AKR1C3, BDNF, CALCA, CD44, CHRM3, DDR1, FN1, GDF15, GRK5, NPPC, NRG1, PPIA, PTPN11, TAC1, TGFB1 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 6.29x10-5 AGTR1, BDNF, CALCA, CD44, FN1, GNAZ, NAP1L4, PDE4B, PPIA, PTPN11, SCN2B, TAC1, TGFB1 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 3.25 × 10-3 ADCYAP1, AVP, CHRM3, CXCL10, CXCL13, FCGR2A, GHRH, HGF, NOS3, NOS2A, VDR 1.33 × 10-3 IRF1, JAK2, MST1, NOS3, NOS2A, STAT1 8.89x10-6 AGTR1, BDNF, CALCA, CHRM3, FN1, GRK5, NPPC, PLD2, PPIA, PTHR1, PTPN11, SELP, TAC1, TGFB1 - 1.67 × 10-3 ADCYAP1, ANXA1, AVP, CHRM3, CXCL10, CXCL13, FCGR2A, P2RX2 1.45 × 10-3 ACVR2A, ANXA1, CDC42, CXCL10, FCGR2A, FGFR1, ITGA7, JAK2, KLF6, LIMS1, PDGFA, PTPRD, STAT1 1.00 × 10-5 ADCYAP1, AVP, CHRM4, CXCL10, GAP43, GHRH, GNAZ, NOS3, VIPR1 Production of cyclic 2.17 × 10-4 ADCYAP1, AVP, GHRH, GNAZ, NOS3, AMP NOS2A, VIPR1 Accumulation of cyclic 1.21 × 10-3 ADCYAP1, AVP, AVPR2, CHRM3, GHRH, AMP VIPR1 Formation of cyclic AMP 1.28 × 10-4 ADCYAP1, AVP, AVPR2, GHRH, GANZ Release of Ca2+ 9.82 × 10-5 ANXA1, AVP, CHRM3, FCGR2A, FGB, FGG Quantity of cholesterol - 2.20x10-3 - CALCA, CHRM3, FN1, NPPC, P2RX2, PPIA, TGFB1 - 6.03x10-3 BDNF, CALCA, GNAZ, NPPC, PTHR1 4.35 × 10-4 CALCA, CHRM3, GRK5, PTHR1, TAC1, TGFB1 7.26 × 10-4 CALCA, GNAZ, PTHR1, TAC1 2.85 × 10-3 ATP7B, BDNF, CALCA, GULO, LCAT Page 12 of 17 (page number not for citation purposes) BMC Genomics 2009, 10:90 http://www.biomedcentral.com/1471-2164/10/90 Table 5: Ingenuity biological function analyses of IL-6-versus NGF-regulated genes in PC12 cells (selected) (Continued) 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 8.02 × 10-3 ADCYAP1, CDC42, GAP43, HGF, TPM3 3.60 × 10-3 ADCYAP1, BCL2L11, GDF15, HGF, RARA, REG3A 6.57 × 10-3 GRID2, NFASC 3.14 × 10-2 GAP43 1.25 × 10-2 HGF - - - - 1.25 × 10-2 EGR1 - - - - 1.60 × 10-4 BDNF, CHRM3, CHRNB1, NRG1, PPP1R1A - - - - 2.88 × 10-4 BDNF, CACNB2, CHRM3, CHRNB1, GABRG2, P2RX2, SCN2B, SLC1A1, SLC1A3 4.79 × 10-4 BDNF, CNTN4, GRID2, NRG1, PDIA3, UBB - - 7.73 × 10-4 7.73 × 10-4 7.73 × 10-4 8.92 × 10-4 development of neurites - - migration of nervous tissue cell lines proliferation of nervous tissue cell lines - - CALCA, TAC1 BDNF, CD44 ACHE, BDNF BDNF, GDF15, NRG1, PDIA3, SLC1A3, TGFB1 2.83 × 10-3 ACHE, BDNF, GRID2, NRG1, PDIA3, PTPN11 3.38 × 10-3 NRG1, TGFB1 - - 6.67 × 10-3 NPPC, TGFB1 Nervous system development and function growth of neurites survival of neurons development of synapse fasciculation of axons complexity of dendritic trees long-term potentiation of dentate gyrus neurological process of synapse synaptic transmission neurological process of axons, neurites activation of nerves binding of neurites size of cell body survival of neurons -, no subcategories found in IPA Tool; p-values and gene symbols are from IPA Tool 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 downregulation 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 Page 13 of 17 (page number not for citation purposes) BMC Genomics 2009, 10:90 http://www.biomedcentral.com/1471-2164/10/90 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]) Gene family Gene abbreviation IL-6-stimulated PC12 cells 9 hours 96 hours Pituitary adenylate cyclase activating polypeptide ADCYAP1 + + BCL2-like protein BCL2L11 + n.c. Chemokine Ligands CXCL1 + + Cytochrome P450 proteins CYP1B1 + Gene abbreviation 24 hours ADCYAP1 + BCL2L11 + CXCL10 CXCL13 + + CYP4F16 + + Early growth response EGR1 + n.c. EGR1 + Glutathione S-transferase GSTA3 + n.c. GSTA3 - Heat shock proteins HSP27B1 + n.c. HSP90B1 + + JAK2 + Janus kinase JAK2 Kruppel-like factors KLF4 KLF9 + + n.c. n.c. KLF6 + Nuclear factors NFIA + n.c. NFIB + Nuclear receptors NR4A3 NR4A2 NR4A1 + + + n.c. n.c. n.c. NR3C2 + ST8SIA3 ST6GAL1 + SLC6A3 + SLC12A5 - SLC30A2 - TUBB + TIMP1 + Sialytransferases ST8SIA1 ST6GAL1 Solute carrier proteins SLC1A3 SLC2A1 SLC2A3 + + + + + n.c. + + + SLC7A1 SLC7A3 + + + SLC18A2 + + SLC24A2 Tubulins TUBA1 Tissue Inhibitor of metalloproteinase TIMP1 + - + n.c. + +, upregulated -, downregulated; n.c., not changed from control cultures; gene symbols are from IPA Tool Page 14 of 17 (page number not for citation purposes) BMC Genomics 2009, 10:90 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 A3'; rat S12 reverse: 5'-CCT TGG CCT GAG ATT CTT TGC3'; 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. http://www.biomedcentral.com/1471-2164/10/90 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. Authors' contributions DK and GW generated the microarray data and drafted the manuscript. UC provided the microarray facility. MB performed the statistical analyses of the microarrays. BD and PM performed the cell-culture of PC12 cells. DK and UO provided support, direction and oversight of the experiments and revised the final manuscript. UO holds the SNF grant. 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). References 1. 2. 3. 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 IPAprogram 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. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. Taga T, Kishimoto T: gp 130 and the interleukin-6 family of cytokines. Annu Rev Immunol 1997, 15:797-819. 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