IntroductionWe previously showed that patient-derived induced pluripotent stem cells (hiPSCs) differentiated into hepatocytes is a relevant model to study the impact of PCSK9 gain-of-function (GOF) ...and loss-of-function (LOF) mutations on cholesterol metabolism regulation. The hiPSCs hepatic differentiation process reproduces the major steps of mammalian liver development. Upon PCSK9 gene expression analysis, we discovered that PCSK9 expression is tightly regulated during hiPSCs differentiation, with the highest expression level in undifferentiated hiPSCs.HypothesisUndifferentiated hiPSCs and their early differentiation will provide a novel approach to highlight new PCSK9 functions.MethodsControl hiPSCs or carrying the S127R GOF or R104C/V114A LOF mutations, and hiPSCs silenced for PCSK9 expression with the use of a shRNA, were analyzed in undifferentiated hiPSCs (gene expression and protein) and during their endoderm (hepatocytes) and mesoderm (cardiomyocytes) differentiation (gene expression). The impacts of LOF mutations and shRNA-mediated PCSK9 expression inhibition on hiSPCs proliferation were also evaluated. Finally, differential PCSK9 expression during early mouse embryonic development has been elucidated by immunofluorescent staining.ResultsWe detected a differential expression of several genes of a cell signaling pathway involved in hiPSCs pluripotency and differentiation. The impact on protein effectors has been verified and we identified a new potential intracellular target of PCSK9. While we did not observe an effect during endoderm differentiation of hiPSCs, we detected an abnormal expression of the endodermal transcription factor FOXA2 during the mesoderm differentiation whenever PCSK9 activity or expressions were modified. Finally, our study showed that the R104C/V114A PCSK9 LOF mutations or PCSK9 silencing with shRNA significantly reduced hiPSCs proliferation. PCSK9 expression during early mouse development and throughout hiPSCs hepatic and cardiac differentiation will be discussed.ConclusionsWe described for the first time the expression of PCSK9 in pluripotent stem cells, and the use of patient-specific hiPSCs allowed us to highlight a new PCSK9 function in developmental processes.
Loss of the nuclear hormone receptor hepatocyte nuclear factor 4alpha (HNF4alpha) in hepatocytes results in a complex pleiotropic phenotype that includes a block in hepatocyte differentiation and a ...severe disruption to liver function. Recent analyses have shown that hepatic gene expression is severely affected by the absence of HNF4alpha, with expression of 567 genes reduced by > or =2.5-fold (P < or = 0.05) in Hnf4alpha(-/-) fetal livers. Although many of these genes are direct targets, HNF4alpha has also been shown to regulate expression of other liver transcription factors, and this raises the possibility that the dependence on HNF4alpha for normal expression of some genes may be indirect. We postulated that the identification of transcription factors whose expression is regulated by HNF4alpha might reveal roles for HNF4alpha in controlling hepatic functions that were not previously appreciated. Here we identify cyclic adenosine monophosphate responsive element binding protein H (CrebH) as a transcription factor whose messenger RNA can be identified in both the embryonic mouse liver and adult mouse liver and whose expression is dependent on HNF4alpha. Analyses of genomic DNA revealed an HNF4alpha binding site upstream of the CrebH coding sequence that was occupied by HNF4alpha in fetal livers and facilitated transcriptional activation of a reporter gene in transient transfection analyses. Although CrebH is highly expressed during hepatogenesis, CrebH(-/-) mice were viable and healthy and displayed no overt defects in liver formation. However, upon treatment with tunicamycin, which induces an endoplasmic reticulum (ER)-stress response, CrebH(-/-) mice displayed reduced expression of acute phase response proteins.
These data implicate HNF4alpha in having a role in controlling the acute phase response of the liver induced by ER stress by regulating expression of CrebH.
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