Figure 2. BRCC3 overexpression ameliorates the BMP pathway in PASMCs.
A and B, RNAs extracted from PASMCs transfected with Ad-Null or Ad-BRCC3 (n=3, each condition) were subjected to RNA-seq analyses. A, GO enrichment analysis using DAVID for the top upregulated and downregulated genes (fold change in expression >1.5 or < –1.5; P<0.05) in PASMCs transfected with Ad-Null or Ad-BRCC3 and plotted as –log (P value) [–Log10(P)]. B, Heatmaps showing differentially expressed genes involved in BMP (9 differentially expressed genes) and TGF-β signaling pathways (9 differentially expressed genes), positive regulation of cell migration (34 differentially expressed genes), and SMC proliferation and apoptosis (57 differentially expressed genes) in PASMCs transfected with Ad-Null vs Ad-BRCC3. C, PASMCs were transfected with Ad-Null or Ad-BRCC3 for 24 hours, then exposed to hypoxia (1% O2) or normoxia for an additional 48 hours. Western blot analysis of protein levels of BMP2, BMPR2, ALK2, pSmad1/5, Smad1, PPARγ, p53, Id1, BRCC3, and β-actin. D, PASMCs were transfected with Ad-Null, Ad-BRCC3, or Ad-BRCC3 shRNA for 24 hours, then cultured under hypoxia for 48 hours and treated with vehicle or BMP2 (25 ng/mL) for 2 hours before harvesting. Western blot analysis of protein levels of pSmad1/5, Smad1, PPARγ, Id1, BRCC3, and β-actin. Data in C and D are mean±SEM from 4 to 6 independent experiments. Normally or nonnormally distributed data were analyzed by 1-way ANOVA (C) and 2-way ANOVA (D) or the Kruskal-Wallis test between multiple groups. Data in C, *P<0.05 vs Nor+Ad-Null, #P<0.05 vs Hyp+Ad-Null. Data in D, *P<0.05 vs Ad-Null in vehicle (Veh) group; #P<0.05 vs Ad-Null in BMP2 group. ALK2 indicates activin receptor-like kinase-2; BMP, bone morphogenetic protein; BMPR2, bone morphogenetic protein type 2 receptor; BRCC3, BRCA1/BRCA2-containing complex subunit 3; GO, Gene Ontology; PASMC, pulmonary arterial smooth muscle cell; RNA-seq, RNA sequencing; SMC, smooth muscle cell; and TGF-β, transforming growth factor-β.