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Gata4 and Sp1 regulate expression of the erythropoietin receptor in cardiomyocytes.

ABSTRACT: Experimental studies indicate significant cardioprotective effects of recombinant erythropoietin (Epo) by binding to the Epo receptor (EpoR) and by inducing various molecular mechanisms, including activation of Gata4, a transcription factor that induces anti-apoptotic genes. However, specific molecular mechanisms of EpoR regulation in cardiomyocytes are unknown. We identified a 774 bp regulatory domain in the EpoR 5' flanking region by reporter gene assays in murine HL-1 cardiomyocytes. The binding sites for Gata and Sp transcription factors both significantly contributed to EpoR promoter activity. DNA-binding studies (EMSA and ChIP assays) identified Gata4 and Sp1 as EpoR promoter-binding proteins in HL1 cardiomyocytes. Although Sp1 alone stimulates EpoR only slightly, forced expression of Gata4 significantly induced EpoR mRNA expression. In addition, knockdown of Gata4 (but also of Sp1) resulted in a significant decrease of EpoR transcript levels in HL-1 cardiomyocytes. Cumulative in vitro data suggest that function of the Sp1 site is essential for the Gata4-mediated transcription. In vivo, analysis of transgenic mice expressing an inducible small-hairpin RNA against Gata4 confirmed suppression of EpoR expression in the heart. Treating mice with high-dose doxorubicin not only resulted in Gata4 protein depletion, but also down-regulated EpoR, followed by up-regulation of EpoR transcripts when Gata4 levels recovered. In conclusion, we identified Gata4 as novel regulator of EpoR transcription in cardiomyocytes. In models of cardiac injury, down-regulation of Gata4 or Sp1 may limit the accessibility of the EpoR for binding of erythropoiesis-stimulating agents (ESA). Thereby our data underline the essential role of Gata4 in mediating cardioprotective effects.

SUBMITTER: Salisch Sv

PROVIDER: S-EPMC3918051 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Gata4 and Sp1 regulate expression of the erythropoietin receptor in cardiomyocytes.

Salisch Sandy von Sv Klar Martin M Thurisch Boris B Bungert Jörg J Dame Christof C

Journal of cellular and molecular medicine 20110901 9

Experimental studies indicate significant cardioprotective effects of recombinant erythropoietin (Epo) by binding to the Epo receptor (EpoR) and by inducing various molecular mechanisms, including activation of Gata4, a transcription factor that induces anti-apoptotic genes. However, specific molecular mechanisms of EpoR regulation in cardiomyocytes are unknown. We identified a 774 bp regulatory domain in the EpoR 5' flanking region by reporter gene assays in murine HL-1 cardiomyocytes. The bind ...[more]

PMID: 21029371

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Project description:Recent studies indicate that mammals, including humans, maintain some capacity to renew cardiomyocytes throughout postnatal life. Yet, there is little or no significant cardiac muscle regeneration after an injury such as acute myocardial infarction. By contrast, zebrafish efficiently regenerate lost cardiac muscle, providing a model for understanding how natural heart regeneration may be blocked or enhanced. In the absence of lineage-tracing technology applicable to adult zebrafish, the cellular origins of newly regenerated cardiac muscle have remained unclear. Using new genetic fate-mapping approaches, here we identify a population of cardiomyocytes that become activated after resection of the ventricular apex and contribute prominently to cardiac muscle regeneration. Through the use of a transgenic reporter strain, we found that cardiomyocytes throughout the subepicardial ventricular layer trigger expression of the embryonic cardiogenesis gene gata4 within a week of trauma, before expression localizes to proliferating cardiomyocytes surrounding and within the injury site. Cre-recombinase-based lineage-tracing of cells expressing gata4 before evident regeneration, or of cells expressing the contractile gene cmlc2 before injury, each labelled most cardiac muscle in the ensuing regenerate. By optical voltage mapping of surface myocardium in whole ventricles, we found that electrical conduction is re-established between existing and regenerated cardiomyocytes between 2 and 4 weeks post-injury. After injury and prolonged fibroblast growth factor receptor inhibition to arrest cardiac regeneration and enable scar formation, experimental release of the signalling block led to gata4 expression and morphological improvement of the injured ventricular wall without loss of scar tissue. Our results indicate that electrically coupled cardiac muscle regenerates after resection injury, primarily through activation and expansion of cardiomyocyte populations. These findings have implications for promoting regeneration of the injured human heart.

Project description:BackgroundThe capsaicin receptor, transient receptor potential vanilloid type -1 (TRPV1) directs complex roles in signal transduction including the detection of noxious stimuli arising from cellular injury and inflammation. Under pathophysiologic conditions, TRPV1 mRNA and receptor protein expression are elevated in dorsal root ganglion (DRG) neurons for weeks to months and is associated with hyperalgesia. Building on our previous isolation of a promoter system for the rat TRPV1 gene, we investigated the proximal TRPV1 P2-promoter by first identifying candidate Sp1-like transcription factors bound in vivo to the P2-promoter using chromatin immunoprecipitation (ChIP) assay. We then performed deletion analysis of GC-box binding sites, and quantified promoter activity under conditions of Sp1 / Sp4 over-expression versus inhibition/knockdown. mRNA encoding Sp1, Sp4 and TRPV1 were quantified by qRT-PCR under conditions of Sp1/Sp4 over-expression or siRNA mediated knockdown in cultured DRG neurons.ResultsUsing ChIP analysis of DRG tissue, we demonstrated that Sp1 and Sp4 are bound to the candidate GC-box site region within the endogenous TRPV1 P2-promoter. Deletion of GC-box "a" or "a + b" within the P2- promoter resulted in a complete loss of transcriptional activity indicating that GC-box "a" was the critical site for promoter activation. Co-transfection of Sp1 increased P2-promoter activity in cultured DRG neurons whereas mithramycin-a, an inhibitor of Sp1-like function, dose dependently blocked NGF and Sp1-dependent promoter activity in PC12 cells. Co-transfection of siRNA directed against Sp1 or Sp4 decreased promoter activity in DRG neurons and NGF treated PC12 cells. Finally, electroporation of Sp1 or Sp4 cDNA into cultures of DRG neurons directed an increase in Sp1/Sp4 mRNA and importantly an increase in TRPV1 mRNA. Conversely, combined si-RNA directed knockdown of Sp1/Sp4 resulted in a decrease in TRPV1 mRNA.ConclusionBased on these studies, we now propose a model of TRPV1 expression that is dependent on Sp1-like transcription factors with Sp4 playing a predominant role in activating TRPV1 RNA transcription in DRG neurons. Given that increases of TRPV1 expression have been implicated in a wide range of pathophysiologic states including persistent painful conditions, blockade of Sp1-like transcription factors represents a novel direction in therapeutic strategies.

Project description:The intestinal epithelium performs vital roles in organ function by absorbing nutrients and providing a protective barrier. The zinc-finger containing transcription factors GATA4 and GATA6 regulate enterocyte gene expression and control regional epithelial cell identity in the adult intestinal epithelium. Although GATA4 and GATA6 are expressed in the developing intestine, loss of either factor alone during the period of epithelial morphogenesis and cytodifferentiation fails to disrupt these processes. Therefore, we tested the hypothesis that GATA4 and GATA6 function redundantly to control these aspects of intestinal development. We used Villin-Cre, which deletes specifically in the intestinal epithelium during the period of villus development and epithelial cytodifferentiation, to generate Gata4Gata6 double conditional knockout embryos. Mice lacking GATA4 and GATA6 in the intestinal epithelium died within 24h of birth. At E18.5, intestinal villus architecture and epithelial cell populations were altered. Enterocytes were lost, and goblet cells were increased. Proliferation was also increased in GATA4-GATA6 deficient intestinal epithelium. Although villus morphology appeared normal at E16.5, the first time at which both Gata4 and Gata6 were efficiently reduced, changes in expression of markers of enterocytes, goblet cells, and proliferative cells were detected. Moreover, goblet cell number was increased at E16.5. Expression of the Notch ligand Dll1 and the Notch target Olfm4 were reduced in mutant tissue indicating decreased Notch signaling. Finally, we found that GATA4 occupies chromatin near the Dll1 transcription start site suggesting direct regulation of Dll1 by GATA4. We demonstrate that GATA4 and GATA6 play an essential role in maintaining proper intestinal epithelial structure and in regulating intestinal epithelial cytodifferentiation. Our data highlight a novel role for GATA factors in fine tuning Notch signaling during intestinal epithelial development to repress goblet cell differentiation.

Dataset Information

Gata4 and Sp1 regulate expression of the erythropoietin receptor in cardiomyocytes.

Publications

Gata4 and Sp1 regulate expression of the erythropoietin receptor in cardiomyocytes.

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