|Fibroblast Growth Factor (FGF) Regulates Human Neuroectoderm Specification through ERK1/2-PARP-1 Pathway|
From Stem Cells
Current knowledge of the mechanisms of neural induction in mammals is not clear, but data from other model systems has allowed some details to be uncovered. Examinations on mammalian neural induction are mostly performed using in vitro models of neuroepithelial differentiation from embryonic stem cells (ESCs) and confirm the requirement of BMP inhibition and FGF activation in neural differentiation of mouse ESCs. Research from the group whose research is presented herein has found that phospho-SMAD1 was not altered in human ESCs undergoing neural differentiation in the presence of FGF signaling inhibitors, suggesting that FGFs regulate hESC neural differentiation independent of BMP/SMAD signaling (LaVaute et al). This led the group of Su-Chun Zhang, University of Wisconsin, Wisconsin ,USA to study in detail how FGF signalling regulates neuroectoderm specification of hESCs (Yoo et al) using a previously described efficient differentiation protocol for neuroepithelial (Pankratz et al).
Neural induction was measured by PAX6 and SOX2 expression, which was noted after day 6 of the protocol. Treatment of cultures at day 6 with an FGFR inhibitor (SU5402) blocked neural differentiation and this was associated with the inhibition of ERK1/2 phosphorylation and to a much lower extent AKT phosphorylation, but left phosphorylation of related kinases unaffected (p38 and JNK). Using an ERK1/2 specific inhibitor (U0126) between days 4 and 6 led to a decrease in PAX6+ cells (30% of cells) compared to control (80% of cells) as measured by FACS, while PAX6 and SOX2 RNA and protein was also decreased in the U0126 treated cells. Together, this suggests that FGF signals through ERK1/2 to mediate neural differentiation in hESCs.
Further analysis demonstrated that modulation of FGF-ERK did not affect SMAD activity, but did increase the activity of PARP1 while not affecting protein level. PARP1 is a chromatin-associated poly(ADP-ribosyl)transferase, which modifies various nuclear proteins by poly(ADP-ribosyl)ation (PAR) and detailed analysis showed that PAR activity corresponded well to expression of FGF and ERK1/2, and inhibition of FGF-ERK led to a drastic reduction in the PAR activity while not affecting protein levels. Direct inhibition of PARP1 by a small molecule inhibitor (PJ34) at day 4-6 of the differentiation protocol significantly reduced the PAX6+ neuroepithelial population, with loss of PAX6 and SOX2 RNA and protein. Constitutive knockdown of PARP1 in hESCs using lentivirally mediated shRNA expression did not affect the growth of hESCs and the expression of SOX2, OTX2, PAX6 and OCT4 were also not changed. Upon differentiation, all 4 markers were reduced at the protein and RNA level as compared to control suggesting the importance of PARP1 in neural differentiation.
As PAX6 is a transcriptional determinant of human neuroectoderm (Zhang et al), a possible FGF-ERK1/2-PARP1-mediated PAX6 regulatory mechanism was investigated. Differentiating hESC cultures at day 5 were treated with inhibitors for 2 hours and subsequent changes in PAX6 transcription were assayed and demonstrated that all 3 inhibitors suppressed PAX6 transcription. Subsequent ChIP analysis found that PARP1 was localized at the PAX6, but not SOX2, promoter and therefore may regulate PAX6 expression directly.
This study uncovers more of the regulatory mechanism in neuroectoderm specification, overall suggesting that FGFs regulate neuroectoderm specification of hESCs through the ERK1/2–PARP-1 pathway to control the transcription of PAX6.
LaVaute TM, Yoo YD, Pankratz MT et al.
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