Kruppel-like factor 4 (KLF4/GKLF) is a target of bone morphogenetic proteins and transforming growth factor β1 in the regulation of vascular smooth muscle cell phenotype

Vascular smooth muscle cell (VSMC) differentiation and phenotypic modulation is characterized by changes in mRNA expression for smooth muscle (SM) marker contractile proteins such as alpha-SM actin and SM22alpha. Transforming growth factor beta1 (TGF-beta1) is a potent VSMC differentiation factor; however, it is not known if other TGF-beta-superfamily members, in particular the bone morphogenetic proteins (BMPs), modulate VSMC phenotype. Here we demonstrate that a large subset of TGF-beta-superfamily members and their type I receptors are differentially co-expressed as VSMC phenotype changes during fetal/neonatal development and that BMP2, -4, and -6 reciprocally regulate SM-marker mRNA and protein expression in vitro. BMP2 and BMP6 decrease expression of the SM markers alpha-SM actin, SM22alpha, and calponin in rat VSMCs, whereas BMP4 increases their expression. The effects of BMP-2, -4, and -6 on SM marker gene transcription are mediated through a consensus TGF-beta-controlling element, the TCE, which is common to regulatory regions of SM-marker genes. Moreover, co-treatment experiments revealed that BMP-2, -4, and -6 each inhibit TGF-beta1-modulated increases in SM22a reporter gene activity. Regardless of whether they positively or negatively regulate SM marker expression, TGF-beta1 and BMP-2, -4, and -6 all induced binding of the Kruppel-like transcription factor, GKLF/KLF4, to the TGF-beta control element. Induction of KLF4 was confirmed by immunocytochemistry and Western blotting, which revealed that a lower molecular weight KLF4 protein is induced after treatment with TGF-beta-superfamily members. Taken together, our results demonstrate that multiple members of the TGF-beta superfamily act in concert to modulate VSMC phenotype.