[Show abstract][Hide abstract] ABSTRACT: Pulmonary artery hypertension (PAH) patients exhibit elevated levels of inflammatory cytokines and infiltration of inflammatory cells in the lung. Concurrently, mutations of bmpr2, the gene encoding the type II receptor of bone morphogenetic proteins (BMP), are found in ∼75% of patients with familial PAH, but a possible nexus between increased inflammation and diminished BMP signaling has hitherto remained elusive. We previously showed that BMP4 triggers nuclear localization of the Myocardin-related transcription factor A (MRTF-A) in human pulmonary artery smooth muscle cells (PASMC), resulting in the induction of contractile proteins. Here we report the BMPR2-dependent repression of a set of inflammatory mediators in response to BMP4 stimulation of PASMC. Forced expression of MRTF-A precisely emulates the anti-inflammatory effect of BMP4, while MRTF-A depletion precludes BMP4-mediated cytokine inhibition. BMP4 and MRTF-A block signaling through NF-κB, the keystone of most pathways leading to inflammatory responses, at the level of chromatin recruitment and promoter activation. Moreover, MRTF-A physically interacts with RelA/p65, the NF-κB subunit endowed with a transcription activation domain. Interestingly, the MRTF-A-NF-κB interaction is mutually antagonistic: stimulation of NF-κB signaling by TNFα, as well as p65 overexpression, hinders MRTF-A activity and the expression of contractile genes. Thus, a molecular inhibitory pathway linking BMP4 signaling, activation of MRTF-A, and inhibition of NF-κB provides insights into the etiology of PAH and a potential focus of therapeutic intervention.
[Show abstract][Hide abstract] ABSTRACT: The bone morphogenetic protein 4 (BMP4) signaling pathway plays a critical role in the promotion and maintenance of the contractile phenotype in vascular smooth muscle cell (vSMC). Misexpression or inactivating mutations of the BMP receptor gene can lead to dedifferentiation of vSMC characterized by increased migration and proliferation that is linked to vascular proliferative disorders. Previously we demonstrated that vSMCs increase microRNA-21 (miR-21) biogenesis upon BMP4 treatment, which induces contractile gene expression by targeting programmed cell death 4 (PDCD4). To identify novel targets of miR-21 that are critical for induction of the contractile phenotype by BMP4, biotinylated miR-21 was expressed in vSMCs followed by an affinity purification of mRNAs associated with miR-21. Nearly all members of the dedicator of cytokinesis (DOCK) 180-related protein superfamily were identified as targets of miR-21. Down-regulation of DOCK4, -5, and -7 by miR-21 inhibited cell migration and promoted cytoskeletal organization by modulating an activity of small GTPase. Thus, this study uncovers a regulatory mechanism of the vSMC phenotype by the BMP4-miR-21 axis through DOCK family proteins.
[Show abstract][Hide abstract] ABSTRACT: Pulmonary artery hypertension (PAH) is characterized by elevated pulmonary artery resistance and increased medial thickness due to deregulation of vascular remodeling. Inactivating mutations of the BMPRII gene, which encodes a receptor for bone morphogenetic proteins (BMPs), are identified in ∼60% of familial PAH (FPAH) and ∼30% of idiopathic PAH (IPAH) patients. It has been hypothesized that constitutive reduction in BMP signal by BMPRII mutations may cause abnormal vascular remodeling by promoting dedifferentiation of vascular smooth muscle cells (vSMCs). Here, we demonstrate that infusion of the amiloride analog phenamil during chronic-hypoxia treatment in rat attenuates development of PAH and vascular remodeling. Phenamil induces Tribbles homolog 3 (Trb3), a positive modulator of the BMP pathway that acts by stabilizing the Smad family signal transducers. Through induction of Trb3, phenamil promotes the differentiated, contractile vSMC phenotype characterized by elevated expression of contractile genes and reduced cell growth and migration. Phenamil activates the Trb3 gene transcription via activation of the calcium-calcineurin-nuclear factor of activated T cell (NFAT) pathway. These results indicate that constitutive elevation of Trb3 by phenamil is a potential therapy for IPAH and FPAH.
[Show abstract][Hide abstract] ABSTRACT: The signal transducers of the transforming growth factor beta (TGFbeta)/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 TGFbeta/BMP-regulated miRNAs (T/B-miRs) to 20. Of interest, 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 TGFbeta/BMP-induced recruitment of Smads, Drosha, and DGCR8 to pri-T/B-miRs and impairs their processing, whereas introduction of R-SBE to unregulated pri-miRNAs is sufficient to recruit Smads and to allow regulation by TGFbeta/BMP. Thus, Smads are multifunctional proteins that modulate gene expression transcriptionally through DNA binding and posttranscriptionally through pri-miRNA binding and regulation of miRNA processing.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Vascular smooth muscle cells (VSMCs), unlike other muscle cells, do not terminally differentiate. In response to injury, VSMCs change phenotype, proliferate, and migrate as part of the repair process. Dysregulation of this plasticity program contributes to the pathogenesis of several vascular disorders, such as atherosclerosis, restenosis, and hypertension. The discovery of mutations in the gene encoding BMPRII, the type II subunit of the receptor for bone morphogenetic proteins (BMPs), in patients with pulmonary arterial hypertension (PAH) provided an indication that BMP signaling may affect the homeostasis of VSMCs and their phenotype modulation. Here we report that BMP signaling potently induces SMC-specific genes in pluripotent cells and prevents dedifferentiation of arterial SMCs. The BMP-induced phenotype switch requires intact RhoA/ROCK signaling but is not blocked by inhibitors of the TGFbeta and PI3K/Akt pathways. Furthermore, nuclear localization and recruitment of the myocardin-related transcription factors (MRTF-A and MRTF-B) to a smooth muscle alpha-actin promoter is observed in response to BMP treatment. Thus, BMP signaling modulates VSMC phenotype via cross-talk with the RhoA/MRTFs pathway, and may contribute to the development of the pathological characteristics observed in patients with PAH and other obliterative vascular diseases.
[Show abstract][Hide abstract] ABSTRACT: Bone morphogenetic protein (BMP) signaling regulates many different biological processes, including cell growth, differentiation, and embryogenesis. BMPs bind to heterogeneous complexes of transmembrane serine/threonine (Ser/Thr) kinase receptors known as the BMP type I and II receptors (BMPRI and BMPRII). BMPRII phosphorylates and activates the BMPRI kinase, which in turn activates the Smad proteins. The cytoplasmic region of BMPRII contains a "tail" domain (BMPRII-TD) with no enzymatic activity or known regulatory function. The discovery of mutations associated with idiopathic pulmonary artery hypertension mapping to BMPRII-TD underscores its importance. Here, we report that Tribbles-like protein 3 (Trb3) is a novel BMPRII-TD-interacting protein. Upon BMP stimulation, Trb3 dissociates from BMPRII-TD and triggers degradation of Smad ubiquitin regulatory factor 1 (Smurf1), which results in the stabilization of BMP receptor-regulated Smads and potentiation of the Smad pathway. Downregulation of Trb3 inhibits BMP-mediated cellular responses, including osteoblast differentiation of C2C12 cells and maintenance of the smooth muscle phenotype of pulmonary artery smooth muscle cells. Thus, Trb3 is a critical component of a novel mechanism for regulation of the BMP pathway by BMPRII.
[Show abstract][Hide abstract] ABSTRACT: Germ line mutations in the bone morphogenetic protein (BMP) receptor type II (BMPRII) gene have been found in >50% of familial idiopathic pulmonary arterial hypertension (IPAH) patients and in 30% of sporadic cases of IPAH. Mutations of BMPRII occur in the extracellular ligand-binding domain, in the cytoplasmic serine/threonine kinase domain, or in the long carboxy terminus domain of unknown function. In this study, we demonstrate that BMPs promote apoptotic cell death in normal human pulmonary artery smooth muscle cells (PASMCs) by activation of caspases-3, -8, and -9, cytochrome c release, and downregulation of Bcl-2. Normal PASMCs expressing a kinase domain mutant or a carboxy-terminal domain deletion mutant of BMPRII identified in IPAH patients are resistant to BMP-mediated apoptosis. This dominant-negative effect may act in heterozygous patients and lead to the development of the pulmonary vascular medial hypertrophy found in IPAH patients. Our study also demonstrates an essential role of the carboxy terminus domain of BMPRII in the activation of the apoptotic signaling cascade.