Vascular smooth muscle cells (VSMCs) 3 are highly plastic cells that undergo phenotype modulation in response to physiological and pathological cues (1). In response to vascular injury or growth factors, such as platelet-derived growth factor (PDGF) (2), VSMCs dedifferentiate and adopt a highly migratory, proliferative phenotype known as a "synthetic" phenotype that is required for vascular injury repair or during angiogenesis (1). However, prolonged or deregulated dedifferentiation can cause occlusion of the vasculature and contributes to development of vascular proliferative disorders, such as atherosclerosis, restenosis following angioplasty, as well as both systemic and pulmonary hypertension (1). Unlike PDGF, the TGF-␤ family of growth factors, including TGF-␤ and BMP4, promote a less migratory and proliferative phenotype known as the "contractile" phenotype (3). Contractile VSMC phenotype is characterized by alterations in the gene expression profile of VSMC. In particular, high expression of VSMC-specific genes, such as smooth muscle ␣-actin (SMA), calponin1 (CNN), and SM22␣ (SM22) are associated with the contractile VSMC phenotype. Transcription of contractile genes is regulated by SRF through a DNA sequence motif known as the CArG box (CC(A/T) 6 GG), which is present in the promoters of VSMC-specific genes (1). A coactivator of SRF, Myocd, interacts with SRF and activates VSMC expression of contractile genes (4 -6). Similarly, the Myocd-related transcription factor (MRTF) family of proteins, MRTF-A and MRTF-B, are also involved in the transcriptional regulation of contractile gene markers as coactivators of SRF (7,8). Myocd is constitutively localized to the nucleus and its activity is regulated primarily at the level of expression. Conversely, MRTFs are sequestered in the cytoplasm through interaction with monomeric G-actin (9, 10). In response to BMP4 or other stimuli, Rho signaling promotes actin polymerization and MRTF translocation into the nucleus where MRTFs associate with SRF (3), resulting in the activation of contractile gene transcription. Unlike BMP4, TGF-␤ does not activate Rho signal-* This work was supported, in whole or in part, by National Institutes of Health 3 The abbreviations used are: VSMC, vascular smooth muscle cell; TGF-{␤}, transforming growth factor-{␤}; PDGF, platelet-derived growth factor; PAI-1, plasminogen activator inhibitor-1; PDCD4, programed cell death 4; BMP, bone morphogenetic protein; MRTF, Myocd-related transcription factor; PASMC, pulmonary artery smooth muscle cell; ESC, embryonic stem cell; SRF, serum response factor; Myocd, myocardin; miRNA, microRNA; pri-miRNA; primary miRNA; SBE, Smad-binding element; rAoSMC, rat aortic SMC; qRT, quantitative reverse transcriptase.

Summary Ingested dsRNAs trigger RNA interference (RNAi) in many invertebrates including the nematode Caenorhabditis elegans. Here we show that the C. elegans apical intestinal membrane protein SID-2 is required in C. elegans for the import of ingested dsRNA and, when expressed in Drosophila S2 cells, SID-2 enables the uptake of dsRNAs. SID-2-dependent dsRNA transport requires an acidic extracellular environment and is selective for dsRNAs with at least 50 base pairs. Through structure-function analysis, we identify several SID-2 regions required for this activity including three extracellular, positively-charged, histidines. Finally, we find that SID-2-dependent transport is inhibited by drugs that interfere with vesicle transport. Therefore, we propose that environmental dsRNAs are imported from the acidic intestinal lumen by SID-2 via endocytosis and are released from internalized vesicles in a secondary step mediated by the dsRNA-channel SID-1. Similar multistep mechanisms may underlie the widespread observations of environmental RNAi.

SUMMARY Double-stranded RNA (dsRNA) is a common by-product of viral infections and acts as a potent trigger of anti-viral immunity. In the nematode C. elegans, sid-1 encodes a dsRNA transporter that is highly conserved throughout animal evolution, but the physiological role of SID-1 and its orthologs remains unclear. Here, we show that the mammalian SID-1 ortholog, SIDT2, is required to transport internalized extracellular dsRNA from endocytic compartments into the cytoplasm for immune activation. Sidt2 deficient mice exposed to extracellular dsRNA, encephalomyocarditis virus (EMCV) and herpes simplex virus 1 (HSV-1) show impaired production of anti-viral cytokines and – in the case of EMCV and HSV-1 – reduced survival. Thus, SIDT2 has retained the dsRNA transport activity of its C. elegans ortholog, and this transport is important for antiviral immunity.

Differentiation of lung vascular smooth muscle cells (vSMCs) is tightly regulated during development or in response to challenges in a vessel specific manner. Aberrant vSMCs specifically associated with distal pulmonary arteries have been implicated in the pathogenesis of respiratory diseases, such as pulmonary arterial hypertension (PAH), a progressive and fatal disease, with no effective treatment. Therefore, it is highly relevant to understand the underlying mechanisms of lung vSMC differentiation. miRNAs are known to play critical roles in vSMC maturation and function of systemic vessels; however, little is known regarding the role of miRNAs in lung vSMCs. Here, we report that miR-29 family members are the most abundant miRNAs in adult mouse lungs. Moreover, high levels of miR-29 expression are selectively associated with vSMCs of distal vessels in both mouse and human lungs. Furthermore, we have shown that disruption of miR-29 in vivo leads to immature/synthetic vSMC phenotype specifically associated with distal lung vasculature, at least partially due to the derepression of KLF4, components of the PDGF pathway and ECM-related genes associated with synthetic phenotype. Moreover, we found that expression of FBXO32 in vSMCs is significantly upregulated in the distal vasculature of miR-29 null lungs. This indicates a potential important role of miR-29 in smooth muscle cell function by regulating FBXO32 and SMC protein degradation. These results are strongly supported by findings of a cell autonomous role of endogenous miR-29 in promoting SMC differentiation in vitro. Together, our findings suggested a vessel specific role of miR-29 in vSMC differentiation and function by targeting several key negative regulators.

Background:The BMP signaling pathway modulates the expression of protein coding genes and non-coding RNAs. Results: BMP4-Smad pathway represses the transcription of miR-302ϳ367 cluster and depresses the expression of the type II BMP receptor. Conclusion: BMP4 treatment facilitates the BMP signaling pathway by down-regulation of miR-302. Significance: Autoregulatory mechanism of control of BMP signaling pathway via miRNA and its target.