Summary The signal transducers of the Transforming Growth Factor β (TGFβ)/Bone Morphogenetic Protein (BMP), the Smads, promote the expression of a subset of miRNAs by facilitating the cleavage reaction by Drosha. The mechanism that limits Smad-mediated processing to a selective group of miRNAs remained hitherto unexplored. In this study, we expand the number of TGFβ/BMP-regulated miRNAs (T/B-miRs) to 20. Interestingly, a majority of T/B-miRs contain a consensus sequence (R-SBE) within the stem region of the primary transcripts of T/B-miRs (pri-T/B-miRs). Here, we demonstrate that Smads directly bind the R-SBE. Mutation of the R-SBE abrogates TGFβ/BMP-induced recruitment of Smads, Drosha, and DGCR8 to pri-T/B-miRs, and impairs their processing, while introduction of R-SBE to unregulated pri-miRNAs is sufficient to recruit Smads and allow regulation by TGFβ/BMP. Thus, Smads are multifunctional proteins which modulate gene expression transcriptionally through DNA binding, and post-transcriptionally by pri-miRNA binding and regulation of miRNA processing.

The platelet-derived growth factor (PDGF) signaling pathway is a critical regulator of animal development and homeostasis. Activation of the PDGF pathway leads to neointimal proliferative responses to artery injury; it promotes a switch of vascular smooth muscle cells (vSMC) to a less contractile phenotype by inhibiting the SMC-specific gene expression and increasing the rate of proliferation and migration. The molecular mechanism for these pleiotropic effects of PDGFs has not been fully described. Here, we identify the microRNA-221 (miR-221), a small noncoding RNA, as a modulator of the phenotypic change of vSMCs in response to PDGF signaling. We demonstrate that miR-221 is transcriptionally induced upon PDGF treatment in primary vSMCs, leading to down-regulation of the targets c-Kit and p27Kip1. Down-regulation of p27Kip1 by miR-221 is critical for PDGF-mediated induction of cell proliferation. Additionally, decreased c-Kit causes inhibition of SMC-specific contractile gene transcription by reducing the expression of Myocardin (Myocd), a potent SMC-specific nuclear coactivator. Our study demonstrates that PDGF signaling, by modulating the expression of miR-221, regulates two critical determinants of the vSMC phenotype; they are SMC gene expression and cell proliferation.Unlike many terminally differentiated cells, smooth muscle cells (SMCs) 3 can switch between differentiated and dedifferentiated phenotypes in response to changes in the local environment (2). In response to vascular injury, quiescent contractile vSMCs reduce the expression of SMC-specific genes such as ␣-smooth muscle actin (SMA), smooth muscle calponin (CNN), SM22␣ (SM22), and smooth muscle myosin heavy chain, increase proliferation, and synthesize collagens and matrix metalloproteinases (2, 9). Although this phenotype switch is believed to be essential for repair of vascular injury, deregulation of this process also plays a role in the pathogenesis of various human diseases, including atherosclerosis, hypertension, asthma, and cancer. Therefore, a complete understanding of the molecular mechanisms of vSMC phenotype regulation is essential for treatment or prevention of vascular disorders.PDGFs potently mediate the vSMC phenotype switch from a differentiated, contractile state to a dedifferentiated, synthetic state by repressing SMC marker gene expression as well as promoting vSMC proliferation and migration (2, 10). Consistent with the effect of PDGF on vSMC, up-regulation of molecules in the PDGF signaling pathway is found in various cardiovascular disorders and vascular injuries, including atherosclerosis and restenosis (1). PDGF released from platelets and endothelial cells at sites of vascular injury is thought to be a contributing factor to atherosclerosis (11). Inhibition of PDGF signaling by the PDGF receptor (PDGFR) kinase inhibitor imatinib mesylate (Gleevec) demonstrated a major protective effect on atherosclerosis development (1). It is of note that hyperactivation of the PDGFR pathway has been implicated in the pathogenesis of id...

In response to vascular injury, smooth muscle cells (SMCs) 2 exhibit a phenotypic change characterized by loss of contractility and abnormal proliferation, migration, and matrix secretion. This "synthetic" phenotype plays an active role in repair of the vascular damage. Upon resolution of the injury, local environmental signals within the vessel prompt SMCs to reacquire their "contractile" phenotype. SMC phenotype modulation contributes to the pathogenesis of numerous cardiovascular disorders, including PAH, post-angioplasty restenosis and atherosclerosis (1). Idiopathic PAH is a rare disease typically fatal because of right ventricular failure. PAH is characterized by elevated pulmonary vascular resistance and pulmonary arterial pressure with increased muscularization of small arteries, thickening or fibrosis of the intima, and the presence of plexiform lesions (2, 3). Proliferation and dedifferentiation of SMCs appear to be the major events in the formation of lesions found in pulmonary arteries of patients with PAH (2, 3). Several lines of evidence implicate the BMP pathway in the etiology of PAH. First, heterozygous mutations in the gene encoding BMPRII, the type II subunit of the BMP receptor, were identified in patients with both familial and sporadic PAH (4, 5). Furthermore, the presence of BMPRII mutations in PAH patients curtails the available therapeutic options, as the patients affected are unlikely to demonstrate vasoreactivity and respond to longterm therapy with calcium channel blockers (6, 7). Second, loss of expression of either the type I (8) or the type II subunit (9, 10) of the BMP receptor has been observed in sporadic or secondary forms of PAH. Third, transgenic mice expressing a catalytically inactive BMPRII mutant in SMCs are predisposed to develop PAH in response to different stimuli (11,12). Fourth, experimental induction of PAH in rodents via hypoxia or monocrotaline injection correlates with a down-regulation of either the type I or the type II BMP receptor and with a general repression of BMP signaling (13-15). Despite these overwhelming correlations, the etiological mechanism of action of the BMP signaling pathway in PAH remains unclear.BMPs represent the largest group in the transforming growth factor ␤ (TGF␤) superfamily of growth factors (16 -18). During embryonic development, the BMP pathway plays multiple essential roles in the induction of ventral mesoderm, cardiac myogenesis, and vasculogenesis (19). Targeted inactivation of the BMP signal transducers Smad1 and Smad5 display a severe vascular phenotype (20). However, the effects of BMPs on adult VSMCs are not completely understood. It has been reported that BMPs inhibit proliferation and induce apoptosis in serumstarved pulmonary artery smooth muscle cells (PASMC) (10,21). It is also known that BMP7 stimulates the maintenance of the SMC phenotype in aortic SMCs, while BMP2 and BMP4 appear to have opposing effects on expression of SMC-specific genes in . These results suggest that the effects of BMP signaling on VSMCs are compl...

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