Cell-specific Activation of the Atrial Natriuretic Factor Promoter by PITX2 and MEF2A

Abstract

The PITX2 homeodomain protein is mutated in patients with Axenfeld-Rieger syndrome and is involved in the development of multiple organ systems, including the heart. We have examined the interaction of PITX2 isoforms with myocyte-enhancing factor 2A (MEF2A), which is a known regulator of cardiac development. A direct interaction between PITX2a and MEF2A was demonstrated using yeast two-hybrid and GST pulldown assays. To study the functional significance of this interaction, we used the atrial natriuretic factor (ANF) promoter. Coexpression of MEF2A and PITX2a or Pitx2c resulted in a strong synergistic activation of the ANF promoter in LS8 oral epithelial cells but not in other cell lines (NIH/3T3, Chinese hamster ovary, or C2C12). The synergism was dependent on promoter context, because it required MEF2 binding sites and was not seen with two other PITX2 target promoters. DNA binding by MEF2A was required but not sufficient for synergism. Upstream activators of p38 MAP kinases, MKK3 and MKK6, increased PITX2a and Pitx2c activity to yield up to 90-fold activation of the ANF promoter in LS8 cells. Because Axenfeld-Rieger syndrome is autosomal dominant and affects development of the oral epithelium, we tested one of the known PITX2 mutants. The PITX2a-K88E mutant protein suppressed wild type PITX2a synergism with MEF2A. These results demonstrate a promoter-and cell-specific functional interaction between PITX2 and MEF2A and suggest the possibility of coordinate control by these factors in the oral epithelium.The PITX2 gene was cloned based on its linkage in AxenfeldRieger syndrome (ARS) 1 (1). ARS is an autosomal dominant human disorder characterized by ocular anterior chamber anomalies causing glaucoma in Ͼ50% of affected individuals as well as dental hypoplasia, mild craniofacial dysmorphism, and umbilical stump abnormalities (1-3). Other features associated with this syndrome include abnormal cardiac, limb, and pituitary development. PITX2 is a member of the bicoid/paired-like homeodomain family (3, 4). There are three isoforms, PITX2a, b, c, that differ only in their amino-terminal regions. The Pitx2 knock-out mouse is lethal at embryonic day 15 because of cardiac abnormalities and shows defects in cardiac positioning, atrial septation, and outflow tract development (4). Pitx2 is a proliferation target in the Wnt/Dvl pathway, which, when disrupted, leads to cardiac outflow tract abnormalities (5). Pitx2 has also been shown to be a downstream target in left-right asymmetry pathways and organogenesis (6 -9). It is expressed asymmetrically on the left side of the lateral plate mesoderm and derivative organs, including the heart and the gut.We have used the yeast two-hybrid assay to analyze candidate PITX2 interacting partners. With respect to the role of PITX2 in heart development, we were intrigued by the recent findings that PITX2 could activate the promoter of the atrial natriuretic factor (ANF) gene (10). ANF plays a key role in electrolyte and fluid homeostasis in the cardiovascular system by causing natriure...

Molecular Biology of the Natriuretic Peptide System

“…26 GATAindependent interactions (eg, between dHAND and MEF-2c and between PITX2 and MEF2A) on the ANP gene promoter have also been documented. 27,28 Consistent with the in vitro observations, cardiac-specific deletion of GATA4 results in diminished ANP gene expression and impairment in the hypertrophic response. 29 The rat ANP gene promoter is also regulated by the homeodomain protein Nkx2-5 19,30 and the T box factor Tbx5, 31 both of which have binding sites in the proximal 5Ј flanking sequence of the ANP gene.…”

Smad4-Irf6 genetic interaction and TGFβ-mediated IRF6 signaling cascade are crucial for palatal fusion in mice

“…1J; supplementary material Table S1). The MEF2 transcription factor is regulated by the p38 MAPK pathway (Han and Molkentin, 2000;Toro et al, 2004). We found a potential MEF2 recognition site in the Irf6 genomic region (Fig.…”

Pitx2c overexpression promotes cell proliferation and arrests differentiation in myoblasts

“…Recent in vitro studies have shown that Pitx2c and Nkx2.5 (a cardiac-specific transcription factors) synergistically regulate the Nppa (atrial natriuretic factor, ANF) promoter. Cotransfection experiments have revealed a role of p38 MAP kinases in up-regulating Pitx2c activation of the ANF promoter in the presence of MEF2A, providing a molecular mechanism for Pitx2c regulation of heart development (Ganga et al, 2003;Toro et al, 2004). Of interest, it has been reported that Pitx2 is a target gene in the Wnt/Dvl2/beta-catenin pathway and operates in specific cell types to control proliferation by regulating expression of the growth control genes cyclin D1, cyclin D2, and c-Myc (Kioussi et al, 2002;Baek et al, 2003).…”