Bone Morphogenetic Protein4 Regulates Cytoplasmic Calcium In Distal Pulmonary Artery Smooth Muscle Cells Of Rat

OBJECTIVE:To investigate that machenism of [Ca2+] i change in peripheral PASMCs exposed to BMP4 and role of BMPs antagonists in pathogenesis of hypoxic pulmonary arterie hypertension.METHODS:1 Determine whether there was [Ca2+] i chang in distal PASMCs exposed to BMP4 (50 ng/ml)Distal PASMCs were treated with BMP4 (50 ng/ml). Ca²⁺ in PASMCs was measured using fluorescent microscopy and Ca²⁺sensitive dye-Fura-2.2 Signaling Pathway between BMP4 and TRPC1,6 in distal pulmonary artery smooth muscle cells.1) BMP4 induced expression of TRPC1, 6 mRNA and ProteinThe PASMCs were treated with BMP4 (50 ng/ml) or vehicle for up to 60 hours. TRPC1, 6 mRNA extracted from treated cells were amplified by a standard PCR protocol using IQ SYBR Green supermix Real-Time PCR. Western Blotting was performed for identification of TRPC1, 6 proteins extracted from cells treated with BMP4 BMP4 (50 ng/ml).2) Determine which signaling pathway was involved in BMP4-induced TRPC1, 6 expression up-regulatin.BMP4/Smad1/5/8 and ERK1/2, p38MAPK were examined in distal PASMCs treated with BMP4 using Western Blots. (Different time-point treatment) The PASMCs were pre-treated with inhibitor of ERK1/2 (PD098059 10μM), ERK1/2 pathway was detected using Western Blot. The PASMCs were pre-treated with inhibitor of p38MAPK (SB2035805μM), P38MAPK pathway was detected using Western Blot.3) Determine whether a kind of the inhibitors decreased [Ca2+]i in PASMCs exposed to BMP4The PASMCs exposed to BMP4 were pre-treated (30 min) with inhibitors of ERK1/2 (PD098059 10μM), p38MAPK (SB203580 5μM).Ca²⁺in PASMCs was measured using fluorescent microscopy and Ca²⁺sensitive dye-Fura-2. 3 Role of BMPs antagonists in pathogenesis of hypoxic pulmonary hypertension1) Determine whether there are some BMPs antagonists in lung tissue of mouse and how many kinds are there, so BMPs antagonists were established by polymerase chain reaction (PCR) and confirmed by gene sequencing.2) Identificat whether BMPs antagonists are depended-BMP4, and whether they are relative with hypoxia.They were detected by Real-time fluorescence quantitative PCR, the samples were lung tissue isolated from BMP4+/+ or BMP4+/- mice. (Wild-type or HET).RESULTS:1 [Ca2+] i in peripheral PASMCs treated with BMP4 was increased. 2 BMP4 induces expression of TRPC1, 6 mRNA and Protein1) Incubation of peripheral PASMCs with BMP-4 (50ng/mL) led to phosphorylation of Smad1/5/8. Reprobing of blots for total Smad5 confirmed equal loading. Rapid phosphorylation of Smad1/5/8 was seen after 15 minutes of stimulation with BMP4.2) Incubation of peripheral PASMCs with BMP-4 (50 ng/mL) led to phosphorylation of p38MAPK and ERK1/2. Reprobing of blots for total p38MAPK and ERK1/2 confirmed equal loading. BMP4 stimulation led to rapid (15 min) transient activation of P38MAPK and ERK1/2, which returned to baseline by 30minuts, then phosphorylation, was evident at 4 hour. 3) The effect of MAPK inhibitors was examined at the 15-min time point. SB203580 and PD09859 can inhibit special signaling pathway compared with BMP4 alone.4) Immunoblotting analysis showed that three different concentrations of BMPRâ…¡siRNA were silented BMPRâ…¡protein expression almost 100% compared to no-targeting siRNA as a negative control. To confirm equal loading, blots were incubated withβ-actin antibody. In next experiments, 50nM concentration of BMPRâ…¡siRNA was used.5) Real-time PCR demonstrated that induction of TRPC1 mRNA in response to BMP4 (50ng/ml) was decreased by the p38MAPK inhibitor, SB203580 or the ERK1/2 inhibitor, PD98059.6) Real-time PCR demonstrted that induction of TRPC6 mRNA in response to BMP4 (50ng/ml) was reduced by the p38MAPK inhibitor SB203580 or the ERK1/2 inhibitor, PD980509.7) The PASMCs transfected with no-targeting control siRNA showed no effect on BMP4-mediated P-Smad1/5/8 induction. However, with BMPRâ…¡siRNA, P-Smad1/5/8 was reduced comparing with the no-targeting control level.8) In the PASMCs the pre-treated (30 min) with inhibitor of ERK1/2 (PD098059 10μM ), p38MAPK (SB203580 5μm), expressions of TRPC1, 6 protein were decreased.9) The increase of [Ca2+] i induced by BMP4 was attenuated by SB or PD.3 Eight BMPs antagonists were identificated by polymerase chain reaction (PCR) in lung tissue of mouse and confirmed by gene sequencingThe samples from lung tissue isolated from BMP4+/+ or BMP4+/- mice were detected by Real-time fluorescence quantitative PCR, and they were relative with hypoxia, most of the antagnists mRNA were increased after hypoxic 1 day and reduced with hypoxic time, which returned to basline by hypoxic 21 days. CONCLUSIONS:1. [Ca²⁺] i in peripheral PASMCs treated with BMP4 was increased; TRPC1, 6 are an important target of BMP signaling in PASMCs. Inhibition of p38MAPK and ERK1/2 parthway will decrease expression of TRPC1, 6 and represent a novel theory about PAH.2. There were eight BMP antagonists in lung tissue of mouse. Most of BMPs antagonists are depended-hypoxia.