PITX2 Isoform-specific Regulation of Atrial Natriuretic Factor Expression: SYNERGISM AND REPRESSION WITH Nkx2.5

Mrudula Ganga,Herbert M Espinoza,Carol J Cox,Lisa Morton,Tord A Hjalt,Youngsook Lee,Brad A Amendt

doi:10.1074/jbc.m210163200

Abstract

PITX2 and Nkx2.5 are two of the earliest known transcriptional markers of vertebrate heart development. Pitx2-/- mice present with severe cardiac malformations and embryonic lethality, demonstrating a role for PITX2 in heart development. However, little is known about the downstream targets of PITX2 in cardiogenesis. We report here that the atrial natriuretic factor (ANF) promoter is a target of PITX2. PITX2A, PITX2B, and PITX2C isoforms differentially activate the ANF promoter. However, only PITX2C can synergistically activate the ANF promoter in the presence of Nkx2.5. We further demonstrate that the procollagen lysyl hydroxylase (PLOD1) promoter is regulated by Nkx2.5. Mechanistically, PITX2C and Nkx2.5 synergistically regulate ANF and PLOD1 expression through binding to their respective DNA elements. Surprisingly, PITX2A activation of the ANF and PLOD1 promoters is repressed by co-transfection of Nkx2.5 in the C3H10T1/2 embryonic fibroblast cell line. Pitx2a and Pitx2c are endogenously expressed in C3H10T1/2 cells, and these cells express factors that differentially regulate PITX2 isoform activities. We provide a new mechanism for the regulation of heart development by PITX2 isoforms through the regulation of ANF and PLOD1 gene expression and Nkx2.5 transcriptional activity.

Highlights

PITX2 and Nkx2.5 are two of the earliest known transcriptional markers of vertebrate heart development
Cell-specific Repression of PITX2A Activity by Nkx2.5 Requires Both Factors to Bind the Promoter—We have shown that PITX2A and PITX2C do not interact with Nkx2.5 in a functional assay using the thymidine kinase (TK)-Bic and TK-NKE promoters in CHO cells
Pitx2 is co-expressed with the transcription factors Nkx2.5 and GATA-4 in the heart (23,26 –28,30)

Summary

Introduction

PITX2 and Nkx2.5 are two of the earliest known transcriptional markers of vertebrate heart development. Pitx2a and Pitx2c are endogenously expressed in C3H10T1/2 cells, and these cells express factors that differentially regulate PITX2 isoform activities. We provide a new mechanism for the regulation of heart development by PITX2 isoforms through the regulation of ANF and PLOD1 gene expression and Nkx2.5 transcriptional activity. Asymmetric expression of Pitx was first detected in stage 7 embryos on the left-sided lateral mesoderm [11]. More importantly to this report, it has recently been shown that differential expression of Pitx isoforms is a key mechanism for heart development [13,14,15,16]. Pitx appears to be a key gene for determination of leftness in heart formation, consistent with the fact that cardiac defects are among the associated defects of Axenfeld-Rieger syndrome. The PLOD genes encode enzymes that hydroxylate lysines in collagens; the hydroxylysine residues act as attachment sites for carbohydrate units and provide stability to in-

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