SIDT2 Transports Extracellular dsRNA into the Cytoplasm for Innate Immune Recognition.

Double-stranded RNA (dsRNA) is a common by-product of viral infections and acts as a potent trigger of antiviral 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 antiviral 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.

Uptake of extracellular double-stranded RNA by SID-2.

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 that, 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.

Disruption of miR-29 Leads to Aberrant Differentiation of Smooth Muscle Cells Selectively Associated with Distal Lung Vasculature.

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.

Inhibition of microRNA-302 (miR-302) by bone morphogenetic protein 4 (BMP4) facilitates the BMP signaling pathway.

The signaling pathway mediated by BMPs plays an essential role during development as well as the maintenance of homeostasis in adult. Aberrant activation or inactivation of BMP signaling can lead to developmental defects and various human disorders. To fine-tune its activity, BMP signaling is regulated both positively and negatively by extrinsic and intrinsic regulatory factors that modulate binding of ligand to the receptors, and the activity of receptors and their dedicated signal transducers, the Smad proteins. Upon BMP binding to the receptor complex, Smad proteins translocate to the nucleus and modulate gene expression transcriptionally by directly associating with the promoter region of target genes, or post-transcriptionally through modulation of microRNA (miRNA) synthesis. In this study, we demonstrate that BMP signaling down-regulates transcription of the miRNA-302∼367 gene cluster. We show that the type II BMP receptor (BMPRII) is a novel target of miR-302. Upon overexpression, miR-302 targets a partially complementary sequence localized in the 3'-untranslated region (UTR) of BMPRII transcripts and leads to destabilization of the transcripts and down-regulation of BMP signaling. We propose that the negative regulatory loop of BMP4-miR-302-BMPRII is a potential mechanism for the maintenance and fine-tuning of the BMP signaling pathway in various systems.

down-regulation of Kruppel-like factor-4 (KLF4) by microRNA-143/145 is critical for modulation of vascular smooth muscle cell phenotype by transforming growth factor-beta and bone morphogenetic protein 4.

In the postnatal vasculature, fully differentiated and quiescent vascular smooth muscle cells (VSMCs) in a "contractile" phenotype are required for the normal regulation of vascular tone. The transforming growth factor-ß (TGF-ß) superfamily of growth factors (TGF-ßs and bone morphogenetic proteins (BMPs)) are potent inducers of contractile phenotype and mediate (i) induction of contractile genes, and (ii) inhibition of VSMC growth and migration. Transcription of contractile genes is positively regulated by a regulatory DNA element called a CArG box. The CArG box is activated by the binding of serum response factor and its coactivators, myocardin (Myocd) or Myocd-related transcription factors (MRTFs). Krüppel-like factor-4 (KLF4) is known to inhibit activation of the CArG box. However, the potential role of KLF4 in the contractile activities of TGF-ß or BMP has not been explored. Here, we demonstrate that TGF-ß and BMP4 rapidly down-regulate KLF4 through induction of microRNA-143 (miR-143) and miR-145, which leads to a reduction of KLF4 transcripts and decreased KLF4 protein expression. Inhibition of miR-145 prevents down-regulation of KLF4 and activation of contractile genes by TGF-ß or BMP4, suggesting that modulation of KLF4 is a prerequisite for induction of contractile genes by TGF-ß and BMP4. Interestingly, both TGF-ß and BMP4 activate transcription of the miR-143/145 gene cluster through the CArG box, however, TGF-ß mediates this effect through induction of Myocd expression, whereas BMP4 utilizes nuclear translocation of MRTF-A. Thus, this study sheds light on both the similarities and the differences of TGF-ß and BMP4 signaling in the regulation of KLF4 and contractile genes.

SID-1 Domains Important for dsRNA Import in Caenorhabditis elegans.

In the nematode Caenorhabditis elegans, RNA interference (RNAi) triggered by double-stranded RNA (dsRNA) spreads systemically to cause gene silencing throughout the organism and its progeny. We confirm that Caenorhabditis nematode SID-1 orthologs have dsRNA transport activity and demonstrate that the SID-1 paralog CHUP-1 does not transport dsRNA. Sequence comparison of these similar proteins, in conjunction with analysis of loss-of-function missense alleles, identifies several conserved 2-7 amino acid microdomains within the extracellular domain (ECD) that are important for dsRNA transport. Among these missense alleles, we identify and characterize a sid-1 allele, qt95, which causes tissue-specific silencing defects most easily explained as a systemic RNAi export defect. However, we conclude from genetic and biochemical analyses that sid-1(qt95) disrupts only import, and speculate that the apparent export defect is caused by the cumulative effect of sequentially impaired dsRNA import steps. Thus, consistent with previous studies, we fail to detect a requirement for sid-1 in dsRNA export, but demonstrate for the first time that SID-1 functions in the intestine to support environmental RNAi (eRNAi).

The amiloride derivative phenamil attenuates pulmonary vascular remodeling by activating NFAT and the bone morphogenetic protein signaling pathway.

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.