{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2024,4,8]],"date-time":"2024-04-08T16:00:36Z","timestamp":1712592036856},"reference-count":46,"publisher":"Wiley","issue":"3","license":[{"start":{"date-parts":[[2013,9,1]],"date-time":"2013-09-01T00:00:00Z","timestamp":1377993600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"},{"start":{"date-parts":[[2013,9,1]],"date-time":"2013-09-01T00:00:00Z","timestamp":1377993600000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/journals.sagepub.com\/page\/policies\/text-and-data-mining-license"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R01 HL\u2010087176","R03 HL\u2010114601"],"award-info":[{"award-number":["R01 HL\u2010087176","R03 HL\u2010114601"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.sagepub.com"],"crossmark-restriction":true},"short-container-title":["Pulm. circ."],"published-print":{"date-parts":[[2013,9]]},"abstract":"<jats:p>The natural history of familial pulmonary arterial hypertension (PAH) typically involves mutations in and\/or haploinsuffciency of BMPR2 (gene for bone morphogenetic protein receptor type 2) but with low penetrance (10%\u201315%), delayed onset (in the third or fourth decade), and a gender bias (two\u2010 to fourfold more prevalent in postpubertal women). Thus, investigators have sought an understanding of \u201csecond\u2010hit\u201d modalities that might affect BMPR2 anterograde trafficking and\/or function. Indeed, vascular lung lesions in PAH have been reported to contain enlarged \u201cvacuolated\u201d endothelial and smooth muscle cells with dilated endoplasmic reticulum (ER) cisternae, increased ER structural protein reticulon 4 (also called Nogo\u2010B), and enlarged and fragmented Golgi apparatus. We recently replicated this cellular phenotype in primary human pulmonary arterial endothelial cells and human pulmonary arterial smooth muscle cells in culture by acute knockdown of the estradiol 17\u03b2 (E2)\u2013responsive proteins signal transducer and activator of transcription 5a (STAT5a) and STAT5b using small interfering RNAs (siRNAs). We have now investigated whether functional haploinsufficiences of these molecules, alone or in combination with other modalities, might interfere with anterograde membrane trafficking using (<jats:italic>a<\/jats:italic>) the quantitative tsO45VSV\u2010G\u2010GFP trafficking assay and (<jats:italic>b<\/jats:italic>) assays for cell\u2010surface localization of Flag\u2010tagged BMPR2\u00a0molecules. The G glycoprotein of the vesicular stomatitis virus (VSV\u2010G) trafficking assay was validated in EA.hy926 endothelial cells by showing that cells exposed to monocrotaline pyrrole displayed reduced anterograde trafficking. Thereafter, the combinatorial knockdowns of STAT5a, STAT5b, BMPR2, and\/or endothelial nitric oxide synthase as well as exposure to E2 or 2\u2010methoxyestradiol were observed to significantly inhibit VSV\u2010G trafficking. These combinations also led to intracellular trapping of wild\u2010type Flag\u2010tagged BMPR2. Overexpression of the PAH disease\u2013derived F14 and KDF mutants of BMPR2, which were trapped in the ER\/Golgi, also inhibited VSV\u2010G trafficking in trans. Moreover, probenecid, a chemical chaperone in clinical use today, partially restored cell\u2010surface localization of the KDF but not the F14\u00a0mutant. These data identify several combinatorial modalities that inhibit VSV\u2010G anterograde trafficking and cause mislocalization of BMPR2. These modalities merit consideration in defining aspects of the late\u2010developing and gender\u2010biased natural history of human PAH.<\/jats:p>","DOI":"10.1086\/674336","type":"journal-article","created":{"date-parts":[[2014,3,7]],"date-time":"2014-03-07T14:17:42Z","timestamp":1394201862000},"page":"533-550","update-policy":"http:\/\/dx.doi.org\/10.1177\/sage-journals-update-policy","source":"Crossref","is-referenced-by-count":9,"title":["Subcellular Mechanisms in Pulmonary Arterial Hypertension: Combinatorial Modalities that Inhibit Anterograde Trafficking and Cause Bone Morphogenetic Protein Receptor Type 2\u00a0Mislocalization"],"prefix":"10.1002","volume":"3","author":[{"given":"Yang\u2010Ming","family":"Yang","sequence":"first","affiliation":[{"name":"Department of Cell Biology and Anatomy New York Medical College Valhalla New York USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kirk B.","family":"Lane","sequence":"additional","affiliation":[{"name":"Department of Medicine Vanderbilt University School of Medicine Nashville Tennessee USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Pravin B.","family":"Sehgal","sequence":"additional","affiliation":[{"name":"Department of Cell Biology and Anatomy New York Medical College Valhalla New York USA"},{"name":"Department of Medicine New York Medical College Valhalla New York 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