GATA-6 mediates human bladder smooth muscle differentiation: involvement of a novel enhancer element in regulating alpha-smooth muscle actin gene expression.
Hollow organs exposed to pathological stimuli undergo phenotypic modulation characterized by altered expression of smooth muscle contractile proteins and loss of normal function. The molecular mechanisms that regulate smooth muscle differentiation, especially in organs other than the vasculature, are poorly understood. In this study, we describe a role for the GATA-6 transcription factor in regulation of human bladder smooth muscle differentiation. Knockdown of endogenous GATA-6 in primary human bladder smooth muscle cells (pBSMC) led to decreased mRNA levels of the differentiation markers alpha-smooth muscle actin (alpha-SMA), calponin, and smooth muscle myosin heavy chain. Similar effects were obtained following downregulation of GATA-6 by forskolin-induced elevation of intracellular cAMP levels. Forskolin treatment of pBSMC abolished recruitment of GATA-6 to the alpha-SMA promoter in vivo and reduced activity of human alpha-SMA promoter-directed gene expression by >60%. This inhibitory effect was rescued by enforced expression of wild-type GATA-6 but not by a zinc-finger-deleted mutant, GATA-6-DeltaZF, which lacks DNA-binding ability. In silico analysis of a region of the human alpha-SMA promoter, described previously as a transcriptional enhancer, identified a putative GATA-binding site at position -919/-913. Point mutation of this site in SMA-Luc abrogated GATA-6-induced activation of promoter activity. Together, these results provide the first evidence for a functional role for GATA-6 in regulation of bladder smooth muscle differentiation. In addition, these findings demonstrate that GATA-6 regulates human alpha-SMA expression via a novel regulatory cis element in the alpha-SMA promoter-enhancer.
Kanematsu
Akihiro
A
Urological Diseases Research Center, John F. Enders Research Laboratories, Rm. 1077, 300 Longwood Ave., Boston, MA 02115, USA. rosalyn.adam@childrens.harvard.edu
Ramachandran
Aruna
A
Adam
Rosalyn M
RM
D000199:Actins / Q000235:genetics*; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D015536:Down-Regulation / Q000502:physiology; D004742:Enhancer Elements, Genetic / Q000502:physiology*; D050993:GATA6 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D009130:Muscle, Smooth / Q000166:cytology*; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology; D001743:Urinary Bladder / Q000166:cytology*
D000199:Actins; D050993:GATA6 Transcription Factor; D012333:RNA, Messenger; D000242:Cyclic AMP