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. 2011 Dec 15;184(12):1400-8.
doi: 10.1164/rccm.201106-1130OC. Epub 2011 Sep 15.

Altered MicroRNA processing in heritable pulmonary arterial hypertension: an important role for Smad-8

Kylie M Drake  1 , Deborah Zygmunt, Lori Mavrakis, Phyllis Harbor, Lingli Wang, Suzy A Comhair, Serpil C Erzurum, Micheala A Aldred
Affiliations

Altered MicroRNA processing in heritable pulmonary arterial hypertension: an important role for Smad-8

Kylie M Drake et al. Am J Respir Crit Care Med. .

Abstract (VSports)

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 VSports手机版. .

Objectives: To determine whether HPAH mutations disrupt noncanonical Smad-mediated microRNA (miR) processing. V体育安卓版.

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. V体育ios版.

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 VSports最新版本. 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. V体育平台登录.

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Figures

Figure 1.
Figure 1.
Expression of miR-21 and its precursors in pulmonary artery endothelial cells (PAEC) (A) and pulmonary artery smooth muscle cells (PASMC) (B) after short-interfering RNA knockdown (KD) of BMPR2 or SMAD4. Bone morphogenetic protein (BMP) stimulation leads to an increase in pre- and mature–miR-21 in control and SMAD4-KD but not BMPR2-KD cells; ** P < 0.001. Basal level of mature miR-21 is significantly lower in BMPR2-KD PAEC than control cells; § P < 0.001. miR = microRNA.
Figure 2.
Figure 2.
RNA-immunoprecipitation (IP) of Flag-tagged Smad-8 in pulmonary artery endothelial cells. (A) Quantitative polymerase chain reaction demonstrates little or no primary microRNA (miR) bound to Smad-8 in vehicle-treated cells and significant induction of binding with bone morphogenetic protein (BMP)-9 treatment. Pre–miR-16 is not regulated by BMPs (see Figure E15) and showed no detectable binding to Smad-8. Control IPs with nonspecific IgG were negative for all miRs. No reverse transcriptase (RT) denotes a negative control with no RT, confirming there is no DNA contamination. (B) Western blot analysis with anti-Flag antibody confirms specificity of the pulldown and equal efficiency in vehicle and BMP9-treated samples.
Figure 3.
Figure 3.
Short-interfering RNA knockdown (KD) of any individual R-Smad (SMAD1, SMAD5, or SMAD9) leads to a paradoxical decrease in miR-21 levels after bone morphogenetic protein (BMP) stimulation; P ≤ 0.002. Similar effects are seen in pulmonary artery endothelial cells (PAEC) (A) and pulmonary artery smooth muscle cells (PASMC) (B). In contrast, expression of ID1, a downstream target of canonical BMP signaling, is still induced in both cell types; **P < 0.001. Basal level of mature miR-21 is significantly lower in Smad-KD PAEC than control cells; §P ≤; 0.006. miR = microRNA.
Figure 4.
Figure 4.
Timecourse of miR-21 expression in control (open symbols) and SMAD1-knockdown (KD) (solid symbols) pulmonary artery endothelial cells after bone morphogenetic protein-9 treatment at zero hours. Data are normalized to untreated cells at time zero. Primary miR-21 levels in both control and SMAD1-KD cells were essentially constant around 1; control cells are omitted for clarity. miR = microRNA.
Figure 5.
Figure 5.
Autoradiograph and quantitation of exogenous 32P-labeled miR-21 levels 24 hours after transfection, normalized to 5S ribosomal RNA to control for the amount of total RNA loaded. Densitometric ratios were then normalized to the untreated control in each experiment. Bone morphogenetic protein (BMP) stimulation leads to a decrease in miR-21 levels, which is blocked by SMAD4 short-interfering RNA knockdown (KD) but not SMAD1 or SMAD9, suggesting it is mediated by the canonical pathway. There is no decrease in cells treated with inhibitors of transcription (α-amanitin) or translation (puromycin or cyclohexamide). miR-27a levels are unchanged with BMP treatment. miR = microRNA.
Figure 6.
Figure 6.
Fold-change of microRNAs (miR) and ID1 in patient pulmonary artery endothelial cells (PAEC) when treated with bone morphogenetic protein (BMP)-9. Three of four patients with germline mutations show a significant decrease in miR-21 relative to basal levels (§ P < 0.01) and nonsignificant changes in miR-27a and miR-100, whereas ID1 is significantly increased (** P < 0.001) albeit with a smaller fold-change relative to control cells. The remaining patients showed significant induction of all miRs and ID1, with fold-changes that did not differ from control cells. APAH = associated pulmonary arterial hypertension; IPAH = idiopathic pulmonary arterial hypertension.
Figure 7.
Figure 7.
The growth rate of patient and control pulmonary artery endothelial cells (PAEC) stimulated with bone morphogenetic protein (BMP)-9 strongly correlates with the change in their miR-21 levels. miR = microRNA.
Figure 8.
Figure 8.
(A) Overexpression of SMAD9 normalizes the bone morphogenetic protein (BMP)–induced fold-change of all three microRNAs (miRs) in pulmonary artery endothelial cells (PAEC) with the SMAD9-R294X mutation; * P < 0.05 in analysis of variance post hoc pairwise test relative to fold-change in mock-treated control cells; ns = not significant. (B) Baseline proliferation rate and growth suppression in response to BMP9 are also completely corrected (post hoc P values vs. control at 72 h: R294X + GFP, P < 0.001; R294X + SMAD9, P = 0.138; R294X + SMAD9 + BMP9 vs. control + BMP9, P = 0.39). Mock-treated cells and control-GFP are omitted for clarity. GFP = green fluorescent protein.
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References

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