Abstract
RationaleOur previous studies demonstrated that bone morphogenetic protein 4 (BMP4) mediated, elevated expression of canonical transient receptor potential (TRPC) largely accounts for the enhanced proliferation in pulmonary arterial smooth muscle cells (PASMCs). In the present study, we sought to determine the signaling pathway through which BMP4 up-regulates TRPC expression.MethodsWe employed recombinant human BMP4 (rhBMP4) to determine the effects of BMP4 on NADPH oxidase 4 (NOX4) and reactive oxygen species (ROS) production in rat distal PASMCs. We also designed small interfering RNA targeting NOX4 (siNOX4) and detected whether NOX4 knockdown affects rhBMP4-induced ROS, TRPC1 and 6 expression, cell proliferation and intracellular Ca2+ determination in PASMCs.ResultsIn rhBMP4 treated rat distal PASMCs, NOX4 expression was (226.73±11.13) %, and the mean ROS level was (123.65±1.62) % of that in untreated control cell. siNOX4 transfection significantly reduced rhBMP4-induced elevation of the mean ROS level in PASMCs. Moreover, siNOX4 transfection markedly reduced rhBMP4-induced elevation of TRPC1 and 6 proteins, basal [Ca2+]i and SOCE. Furthermore, compared with control group (0.21±0.001), the proliferation of rhBMP4 treated cells was significantly enhanced (0.41±0.001) (P<0.01). However, such increase was attenuated by knockdown of NOX4. Moreover, external ROS (H2O2 100 µM, 24 h) rescued the effects of NOX4 knockdown, which included the declining of TRPC1 and 6 expression, basal intracellular calcium concentration ([Ca2+]i) and store-operated calcium entry (SOCE), suggesting that NOX4 plays as an important mediator in BMP4-induced proliferation and intracellular calcium homeostasis.ConclusionThese results suggest that BMP4 may increase ROS level, enhance TRPC1 and 6 expression and proliferation by up-regulating NOX4 expression in PASMCs.
Highlights
Pulmonary hypertension (PH) is characterized by increased mean pulmonary arterial pressure . 25 mmHg
Cultured rat distal pulmonary arterial smooth muscle cells (PASMCs) were treated with recombinant human BMP4 (rhBMP4) or respective vehicle control
We found that rhBMP4 increased NADPH oxidase 4 (NOX4) protein expression and reactive oxygen species (ROS) mean level in rat distal PASMCs, and further investigated the role of NOX4 with a loss of function strategy using siRNA
Summary
Pulmonary hypertension (PH) is characterized by increased mean pulmonary arterial pressure (mPAP, at resting condition) . 25 mmHg. In PASMCs, BMP4 up-regulates TRPC1 and 6 expressions in rat pulmonary artery and PASMCs to increase [Ca2+]i and SOCE, further leads to increased proliferation, which leads to pulmonary small artery spasm contraction and remodeling, and eventually causes elevated pulmonary resistance and PH [6,15]. It still remains largely unclear how BMP4 induces TRPCs expression. We sought to wander: 1) whether BMP4, functions as a multiple faces’ factor, could influence ROS generation and NOX4 expression? 2) whether such induction controls the downstream TRPC expression and the intracellular calcium homeostasis? 3) whether these mechanisms fit into and explain the mechanisms through which BMP4-induced PASMCs proliferation and pulmonary vascular remodeling? This study aims to clarify the mechanism underlying BMP4 regulating calcium homeostasis and pulmonary vascular remodeling in PASMCs, to provide a theoretical basis for the subsequent development of drugs for the treatment
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