A64-04 Hypoxic Activation of P38 and Reduced Expression of MAPKAPK2 (MK2) in Pulmonary Arterial Smooth Muscle Cells (PASMCs)

Rationale Chronic hypoxia (CH) leads to pulmonary hypertension (PH) via sustained vasoconstriction and vascular remodeling, characterized by PASMC hyperproliferation, medial wall thickening, and muscle extension into distal arterioles. Prior studies showed CH increased PASMC proliferative and migratory capacity and resistance to apoptosis, processes dependent on activation of hypoxic inducible factor 1 (HIF-1). Pulmonary arteries from humans with PH and CH rats exhibited constitutive phosphorylation of p38 and this signaling was required for PASMC proliferation and vascular thickening. Prior research in cancer cells showed persistent phosphorylation of p38 and its downstream target, MK2, ultimately increased MK2 degradation and decreased total MK2 expression. Since MK2 plays a role as a nuclear chaperone of caspase-3, the final executioner of apoptosis, we sought to identify whether HIF-1 activation leads to activation of p38 and influences MK2 expression. Methods Intralobar pulmonary arteries were isolated from male Wistar rats by dissection. PASMCs were enzymatically isolated and grown in vitro. For experiments, PASMCs were growth arrested and subjected to 1) normoxia or hypoxia (1% O2) using a humidified modular incubator; 2) treatment with either vehicle or dimethyloxalylglycine (DMOG), a potent inhibitor of prolyl hydroxylases (PHDs) which leads to HIF-1 activation; or 3) treatment with an adenoviral construct containing constitutively active HIF-1. Immunoblot was performed to characterize expression of HIF-1, phosphorylated p38, phosphorylated MK2, and total MK2. RT-PCR was performed to determine expression of MK2 mRNA. Results Hypoxia induced phosphorylation of p38 and MK2 at 4 hours, followed by a decrease in total MK2 expression at 6 hours, which was sustained at 48 hours. Similarly, induction of HIF-1 with DMOG under normoxic conditions also induced phosphorylation of p38 and MK2 and decreased total MK2 expression. Forced expression of HIF-1 under normoxic conditions also resulted in decreased total MK2 expression. RT-PCR of PASMCs subjected to hypoxia or treated with DMOG showed no decrease in MK2 mRNA expression at 48 hours, suggesting this process is not due to direct transcriptional regulation by HIF-1. Conclusion Our work demonstrates induction of HIF-1 by multiple methods in PASMCs was associated with rapid phosphorylation of p38 and MK2 and that hypoxic activation of the p38-MK2 axis is associated with a subsequent decrease of total MK2 protein expression. Since MK2 plays an important role in mediating apoptosis by acting as a nuclear chaperone for caspase-3, our findings suggest a mechanism by which CH decreases total MK2 expression and its availability to facilitate apoptosis. This abstract is funded by: None