O1030: Decreased Camkkβ Signaling Leads to Down-regulation of PGC-1α Expression in Pulmonary Artery Endothelial Cells (PAECs) in Persistent Pulmonary Hypertension of the Newborn (PPHN)

Background: Persistent Pulmonary Hypertension of the Newborn (PPHN) is characterized by sustained elevation of pulmonary vascular resistance (PVR) and hypoxemia. Decreased mitochondrial function in pulmonary artery endothelial cells (PAECs) leads to impaired angiogenesis and elevated PVR in PPHN. We previously reported decreased levels of Peroxisome Proliferator-Activated Receptor Gamma Co-Activator-1α (PGC-1α), a key mitochondrial transcription factor, in PAECs in a fetal lamb model of PPHN. PGC-1α augments both nuclear- and mitochondrial-encoded genes when it is activated by phosphorylation at sites Thr-177 and Ser-538, and by deacetylation.

In the present study, we investigated the upstream mechanisms that lead to down-regulation of PGC-1α in PPHN PAECs. We hypothesized that decreased Ca2+/Calmodulin-dependent protein kinase kinase β (CaMKKβ), an upstream 5’-AMP-Activated Kinase (AMPK) activator, leads to reduced PGC-1α levels in PPHN. CaMKKβ can promote PGC-1α function via post-translational modifications by its client, AMPK, and transcriptional regulation by the CaMKIV/cAMP Response Element-Binding Protein (CREB)/Histone Deacetylases (HDACs)/Myocyte Enhancer Factor-2 (MEF-2) pathway, which may lead to both an increase in PGC-1α mRNA expression and increased deacetylation of PGC-1α by the HDAC proteins.

Methods: PAECs were isolated from fetal lambs with PPHN induced by prenatal ductus arteriosus constriction from 80% to term gestation and from matched controls. PAECs were grown to ≥70% confluence. Protein levels were assessed by immunoblotting, and phosphorylation and acetylation modifications were evaluated by immunoprecipitation. Data from control and PPHN groups were compared by T test with p< 0.05 considered as significant difference.

Results: PPHN lamb PAECs showed decreased CaMKKβ protein levels compared to controls (p=0.0371). Phos(Thr200)-CaMKIV (p=0.0267), CREB (p=0.0963), p(Ser133)-CREB (p=0.0182), HDACs 2 (p=0.0007) and 4 (p=0.0037), and MEF-2D (p=0.0391) protein levels were all significantly reduced in PPHN PAECs compared to controls. Acetylation of PGC-1α was increased in PPHN PAECs (p=0.0905), which was associated with decreased levels of HDACs 2 and 4.

Conclusion: Our data reveal new mechanisms for the reduction of PGC-1α, a key nodal point in mitochondrial biogenesis. The role of CaMKKβ downregulation in the decreased angiogenesis function of PAECs in PPHN requires further studies.