Altered MicroRNA Processing in Heritable Pulmonary Arterial Hypertension

Abstract

Rationale: Heritable pulmonary arterial hypertension (HPAH) is primarily caused by mutations of the bone morphogenetic protein (BMP) type-II receptor (BMPR2). Recent identification of mutations in the downstream mediator Smad-8 (gene, SMAD9) was surprising, because loss of Smad-8 function in canonical BMP signaling is largely compensated by Smad-1 and -5. We therefore hypothesized that noncanonical pathways may play an important role in PAH. Objectives: To determine whether HPAH mutations disrupt noncanonical Smad-mediated microRNA (miR) processing. Methods: Expression of miR-21, miR-27a, and miR-100 was studied in pulmonary artery endothelial (PAEC) and pulmonary artery smooth muscle cells (PASMC) from explant lungs of patients with PAH. Measurements and Main Results: SMAD9 mutation completely abrogated miR induction, whereas canonical signaling was only reduced by one-third. miR-21 levels actually decreased, suggesting that residual canonical signaling uses up or degrades existing miR-21. BMPR2 mutations also led to loss of miR induction in two of three cases. HPAH cells proliferated faster than other PAH or controls. miR-21 and miR-27a each showed antiproliferative effects in PAEC and PASMC, and PAEC growth rate after BMP treatment correlated strongly with miR-21 fold-change. Overexpression of SMAD9 corrected miR processing and reversed the hyperproliferative phenotype. Conclusions: HPAH-associated mutations engender a primary defect in noncanonical miR processing, whereas canonical BMP signaling is partially maintained. Smad-8 is essential for this miR pathway and its loss was not complemented by Smad-1 and -5; this may represent the first nonredundant role for Smad-8. Induction of miR-21 and miR-27a may be a critical component of BMP-induced growth suppression, loss of which likely contributes to vascular cell proliferation in HPAH.Keywords: bone morphogenetic protein signaling; mutation; endothelium; smooth muscle Pulmonary arterial hypertension (PAH) is a sustained elevation of pulmonary artery pressure resulting from vascular remodeling and vessel narrowing that progressively obliterates the precapillary pulmonary arteries. It may be idiopathic (IPAH) or associated with an underlying condition, such as collagen vascular disease or congenital heart defect (APAH). After a recent reclassification, individuals with an identifiable genetic mutation or known family history are grouped together as heritable PAH (HPAH) (1). The primary genetic predisposition is a heterozygous mutation of the bone morphogenetic protein (BMP) receptor type-II (BMPR-II; gene symbol, BMPR2) (2-5). Recently, mutations affecting Smad-8 (gene symbol, SMAD9), a downstream mediator of BMP signaling, were also reported (6). Smad-8 is closely related to two other mediators, Smad-1 and -5, which are collectively known as the receptor Smads (R-Smads). In canonical BMP signaling, R-Smads are phosphorylated by the activated receptor complex, whereupon they associate with Smad-4 and translocate to the nucleus. Given this functional overlap betw...

Human Primary Lung Endothelial Cells in Culture

“…Additionally, all glassware and plastics used for cell culture were opened in a sterile work environment. This protocol is detailed to give investigators the ability to study more reliably the role of pulmonary endothelial cells in lung vascular diseases (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24).…”

Molecular Mechanisms of Pulmonary Arterial Remodeling

“…This group also demonstrated that the role of miR-204 and NFAT in PAH may be mediated by the DNA damage/ PARP-1 signaling pathway (59). Drake et al reported that heritable pulmonary arterial hypertension (HPAP)-associated mutation engender a primary defect in noncanonical miR processing, where bone morphogenetic protein (BMP) signaling is partially maintained (60). Smad-8 is responsible for this miR pathway.…”

Correction of Nonsense BMPR2 and SMAD9 Mutations by Ataluren in Pulmonary Arterial Hypertension

“…To date, over 200 distinct mutations have been identified in nearly 300 individuals, with nonsense point mutations accounting for approximately 29% overall (3,6). Less commonly, mutations are found in other BMP pathway genes: activin receptor-like kinase 1 (gene symbol, ACVRL1), endoglin (ENG), or Smad8 (gene symbol, SMAD9) (9)(10)(11)(12)(13)(14)(15)(16).…”

“…There are also several Smad4-independent functions, including post-transcriptional up-regulation of a subset of microRNAs (miRs) (17,18). Recently, we showed that, in cells from the explant lungs of patients with HPAH, mutations in BMPR2 or SMAD9 abrogate this BMP-mediated miR induction (16). Two of the affected miRs, miR-21 and miR-27a, are growth suppressive in pulmonary vascular cells.…”