FGF2 Signaling in Mouse Embryonic Fibroblasts Is Crucial for ... - MDC

Relative expression level54321+ FGF2 3 d– FGF2 3 d+ FGF2 5 d– FGF2 5 dRelative expression level654321+ FGF2 3 d– FGF2 3 d+ FGF2 5 d– FGF2 5 da0mcpt8plfgrem1il6inhbbtncgzmdtgfbimmp3Genesserpine 1fgf7tnsf11saa3cxcl5cxcl14ccl2b0plfGenesbmp4Fig. 4. Validation of the global gene expression analysis with realtimePCR. a FGF2-dependent gene regulation of the secreted moleculesobserved in the microarray analysis was validated by realtimePCR in the presence or absence of FGF2 for 3 or 5 days (asindicated). b As in a . Omission of FGF2 in the MEF culture wasleading to decreased expression of proliferin, whereas expressionof bmp4 was increased around 2-fold. RNA expression was analy z e d a s d e s c r i b e d i n f i g u re 1b. d = D ay s .Validation of the FGF2-Dependent Gene Regulationin MEFsTo validate the expression of FGF2-regulated genesidentified in the microarray study with PMEF-N, we performedreal-time PCR analyses with CF1 MEFs that areroutinely used to culture H9 hESCs ( fig. 4 a). The expressionlevels for granzyme E, which was identified in themicroarray analysis, were not analyzed, since it is highlyhomologous to granzyme D. MEFs were grown for 3 and5 days in stem cell medium with or without FGF2. Mostof the identified genes were regulated by FGF2 as predictedby the microarray analysis. The strongest downregulationafter withdrawal of FGF2 was observed in thegene expression level of mast cell protease 8 (mcpt8) andproliferin. The genes for inhibin B and fgf7 showed onlya modest downregulation in the absence of FGF2 by 50and 30%, respectively. Five of the genes, that is, mcpt8,proliferin, gremlin 1, tenascin and tnsf11, showed significantlyhigher expression levels (around 3-fold) after 5days in FGF2 medium compared to 3 days. This suggeststhat regulation of these genes is further induced when theMEFs adapt to the culture conditions in stem cell medium.However, 4 of the genes studied, that is, saa3 andgenes for chemokine ligands cxcl5, cxcl14 and ccl2, werenot significantly downregulated in the absence of FGF2under these conditions.Among the 17 identified genes, those for the moleculesInhibin B (Activin B), Gremlin 1 and FGF7 (KGF) areof special interest, since they regulate pathways whichhave already been shown to be involved to keep hESCs inthe undifferentiated state.Inhibin B, as a homodimer, forms the ligand ActivinB and activates the transcription factors Smad2/3. Wehave shown previously that Smad2/3 are indeed activated[Besser, 2004] and their activation is required to maintainhESCs undifferentiated [Amit et al., 2004; Beattie et al.,2005; James et al., 2005]. Interestingly, Beattie et al. [2005]described that addition of FGF7 in feeder-free conditionsin the presence of Activin A and nicotinamide had astrong proliferative effect on hESCs. It was argued thatwhile Activin A maintained the pluripotent state underthose conditions, FGF7 was required for the proliferationof the cells, while nicotinamide was required to blockapoptosis [Beattie et al., 2005].Gremlin 1 is an inhibitor of BMP signaling [Hsu et al.,1998] and we have shown that this pathway is inhibitedin the undifferentiated state [Besser, 2004; James et al.,2005]. In line with these observations, it has been describedthat activation of BMP signaling in the undifferentiatedstate leads to the differentiation of hESCs intotrophoblasts [Xu et al., 2002]. Suppression of BMP signalingis required to maintain the pluripotent state [Xu et al.,2005]. This appears to be especially important whenhESCs are maintained in a pluripotent state with MEF-CM supplemented with KSR, since this supplement containsBMP activity [Xu et al., 2005]. It has also been suggestedthat MEFs produce BMPs during the conditioningphase and that the expression of bmp4 in the MEF cultureMaintenance of Pluripotency by MEFsRequires FGF2 SignalingCells Tissues Organs 2008;188:52–61 59

aESCM + FGF2ESCM – FGF2U0126DMSO1 2 3 4p-ERK1/2ERK1/2-TubulinRelative expression levelb3.02.52.01.51.00.50+ FGF2– FGF2U0126DMSOmcpt8plfil6mmp3grem1GenesinhBgzmdtgficxcl14Fig. 5. Influence of MEK inhibitor on the expression of the FGF2-regulated genes in MEFs. a Western blot analysis of MEFs grownwith FGF2 (+ FGF2) and treated with a specific MEK inhibitor(U0126), DMSO or cells grown without FGF2 (–FGF2). Twentymicrograms of total protein from whole cell extracts were probedw it h a nt i b o d ie s a g a i n s t pho s pho -E R K1/2 , E R K a nd -tubulin (asloading control). Phosphorylation of ERK1/2 was decreased in theabsence of FGF2. U0126 was able to block phosphorylation ofERK1/2 almost completely, whereas the solvent control withDMSO showed no effect. b Real-time PCR analysis of MEFs culturedunder the indicated conditions showed a downregulation ofthe target genes dependent on FGF2 treatment. U0126 treatmentin the presence of FGF2 showed only an effect on some of theFGF2 target genes, that is, mcpt8, plf, il6, mmp3, grem1 andtgf i. DMSO solvent control had no effect.is downregulated by FGF2 [Greber et al., 2007]. To testthis possibility, we determined the mRNA level for bmp4in the treated MEFs ( fig. 4 b). Indeed, while the mRNAlevels for proliferin decreased in the absence of FGF2, theexpression level for bmp4 was increased around 2-foldafter 3 days in culture without FGF2. After 5 days culturingwith FGF2, we observed a further decrease in bmp4expression as opposed to some of the FGF2-induced geneswhere an increased expression was observed.Effect of the MEK Inhibitor U0126 on FGF2-RegulatedGenesTo investigate how FGF2 signaling is promoting the expressionof the target genes, we analyzed the activationstate of Akt and ERK signaling [Eswarakumar et al., 2005].No difference in the phosphorylation of Akt in MEFstreated with or without FGF2 was observed in Westernblot analysis using a phosphospecific Akt antibody, suggestingthat FGF2 does not regulate Akt activity underthese conditions (data not shown). The phosphorylation ofERK1/2, on the other hand, changed depending on FGF2treatment ( fig. 5 a). We therefore used the specific inhibitorof MEK1/2 signaling (U0126) [Favata et al., 1998] to inhibitthe activation of ERKs. The inhibitor U0126 was ableto block the phosphorylation of ERK1/2 almost completely,even stronger than observed by withdrawal of FGF2. Asa control, treatment with DMSO showed no effects of thephosphorylation pattern ( fig. 5 a). Based on these results,we analyzed the expression of the FGF2-regulated genesby real-time PCR. As shown before in figure 3 a, the levelfor mcpt8, proliferin, inhibin B, gremlin 1 and fgf7 decreasedin the absence of FGF2. Note that this experimentwas carried out over 3 days to avoid toxic effects of the inhibitor( fig. 5 b). Interestingly, U0126 treatment showed aneffect on some of the FGF2 target genes (mcpt8, plf, il6,mmp3, grem1 and tgf i) but not on others (inh B andgzmd). In addition, decreased gene expression by FGF2withdrawal was in all cases more pronounced than the decreasein the presence of U0126. The drop in phospho-ERK levels was stronger in the presence of U0126 than bywithdrawal of FGF2. Thus, these results suggest that FGF2signaling acts in part independently of the Ras/ERK pathwayin MEFs ( fig. 5 b). Further experiments are necessaryto determine how FGF2 elicits the regulation of targetgenes in MEFs under these conditions.S u m m a r yThis study shows that FGF2 signaling is required forMEFs to produce secreted factors supporting the undifferentiatedgrowth of hESCs. No effect of the modulation60Cells Tissues Organs 2008;188:52–61Diecke/Quiroga-Negreira/Redmer/Besser

of FGF2 signaling directly in the hESCs was observed.Based on these observations, we identified 17 genes regulatedby FGF2 by a microarray analysis, of which 13 wereconfirmed by real-time PCR in MEFs from the CF1mouse strain. Among these genes we identified factors,that is Inhibin B, Gremlin 1 and FGF7, that have beenpreviously implicated in the maintenance of hESC pluripotency.The results presented in this study will be importantfor the development of culture protocols forhESCs replacing the undefined CM from MEFs by mediacompositions with controlled recombinant factors free ofcontaminating animal material.AcknowledgementsWe would like to thank Ali H. Brivanlou for his support duringthe microarray analysis in his laboratory at Rockefeller Universityand Agnés Viale at the genomic core facility of MSKCC forher support in this analysis. 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S. Diecke et al. Maintenance of Pluripotency by MEFs requires FGF2 signalingSupplementary MaterialSequences of upstream and downstream primers for genes analyzed by realtimeRT-PCR (h – human, m – mouse) are listed below including the length ofthe expected product in bases pairs (bp; Length).Gene Forward Primer Reverse Primer Lengthlefty-A (h) 5’-gggaattgggatacctggat-3’ 5’-ctaaatatgcacgggcaagg-3’ 206 bpoct4 (h) 5’-gaaggatgtggtccgagtgt-3’ 5’-gtgacagagacagggggaaa-3’ 243 bpnanog (h) 5’-accagaactgtgttctcttccacc-3’ 5’-ggttgctccaggttgaattgttcc-3’ 333 bp-actin (h) 5’-tggcaccacaccttctacaatgagc-3’ 5’-gcacagcttctccttaatgtcacgc-3’ 400 bpnodal (h) 5’-agacatcatccgcagcctaca-3’ 5’-gacctgggacaaagtgacagtgaa -3’ 268 bpmsx2 (h) 5’-aaatctggttccagaaccgaaggg-3’ 5’-atgggaagcacaggtctatggaacg-3’ 187 bpinhbb (m) 5’-tcttcatcgactttcggctcatcg-3’ 5’-caaagtagagcatggacatggagc-3’ 234 bpmcpt8 (m) 5’-atgacatcatgctcctgaagctgg-3’ 5’-caaagtagagcatggacatggagc-3’ 225 bpgzmd (m) 5’-aacgtttccgacactacactgagacc-3’ 5’-ttcatgttcctgcttatccacggc-3’ 208 bpsaa3 (m) 5’-atcttgatcctgggagttgacagc-3’ 5’-atgtcccgtgaacttctgaacagc-3’ 237 bpil6 (m) 5’-tctggtcttctggagtaccatagc-3’ 5’-attggatggtcttggtccttagcc-3’ 232 bpplf (m) 5’-tgttgccttatttcccatgtgtgc-3’ 5’-aagagctgaatagtgtgtgagcctgg-3’ 250 bptnfsf11 (m) 5’-atcgctctgttcctgtactttcgagc-3’ 5’-atgtgttcgagttccttctgcacg-3’ 206 bpcxcl14 (m) 5’-tccaagtgtaagtgttccaggaaggg-3’ 5’-ttgatgaagcgtttggtgctctgc-3’ 188 bpmmp3 (m) 5’-aagggtggatgctgtctttgaagc-3’ 5’-gccatagcacatgctgaacaaagc-3’ 230 bpgrem1 (m) 5’-taagtcaaagcgggcacattcagc-3’ 5’-tgccttctagcaaaggctgtttcc-3’ 214 bptnc (m) 5’-aagactgcaaagagcaaaggtgcc-3’ 5’-acacacattgtccttggttgctgc-3’ 150 bpcxcl5 (m) 5’-tggcatttctgttgctgttcacgc-3’ 5’-tgcagggatcacctccaaattagc-3’ 152 bpfgf7 (m) 5’-aatgacatgagtccggagcaaacg-3’ 5’-attgccacaattccaactgccacg-3’ 232 bpserpine1 (m) 5’-tgcctgacatgtttagtgcaaccc-3’ 5’-tgtgccgaaccacaaagagaaagg-3’ 210 bptgfbi (m) 5’-tgagcaacgtcaacatcgaactgc-3’ 5’-accttgtcaatgagatgcaccacg-3’ 226 bpccl2 (m) 5’-ttgtcaccaagctcaagagagagg-3’ 5’-tacagaagtgctgaggtggtgtgg-3’ 224 bpbmp4 (m) 5’-agaagaataagaactgccgtcgcc-3’ 5’-atggcatggttggttgagttgagg-3’ 160 bp-24-