Overactivation of Ca2+/Calmodulin‐Dependent Protein Kinase IV and IIδ Contributes to Enhancing Pulmonary Arterial Smooth Muscle Cell Proliferation in Patients with Idiopathic Pulmonary Arterial Hypertension

Abstract

IntroductionA rise in cytosolic free Ca2+ concentration ([Ca2+]cyt) in pulmonary arterial smooth muscle cells (PASMCs) causes pulmonary vasoconstriction and increases PASMC proliferation, leading to concentric arterial wall thickening. Ca2+/Calmodulin‐Dependent Protein Kinases (CaMK) are a family of serine/threonine kinases that are activated by an increase in [Ca2+]cyt. Akt is another serine/threonine protein kinase that has been observed to have increased activity due to a rise in [Ca2+]cyt, and its overexpression has been implicated in excessive cell proliferation. CaMKIV has been shown to cause phosphorylation of Akt while CaMKIIδ has been shown to cause increases in vascular remodeling and vascular smooth muscle cell proliferation. STIM2, a Ca2+ sensor in sarcoplasmic reticulum (SR), has been suggested to mediate increases in the resting [Ca2+]cyt in PASMCs from patients with idiopathic pulmonary arterial hypertension (IPAH). PDGF‐R is a tyrosine kinase receptor that, when activated by PDGF, can cause cell proliferation. Our hypothesis is that overactivation of CaMKIV and CaMKIIδ increases activity of Akt, causing upregulation of STIM2 and PDGF‐R, which stimulate PASMC proliferation in patients with IPAH.MethodsPASMCs from both healthy subjects and patients with IPAH were used for this study. Measurement of protein levels was done using conventional western blot protocol. Knockdown of specific proteins was done using siRNA. Measurement of cell proliferation was done by measuring EdU incorporation rate, an indicator of DNA synthesis, and total cell number after 48 hours.ResultsIn this study, we found that the protein level of CaMKIV is significantly increased in PASMCs from patients with IPAH (1.00±0.588 vs 11.4±1.29, n=3 normal subjects and 5 patients, p=0.001). Knockdown of CaMKIV in IPAH PASMCs with CaMK IV specific siRNA significantly decreased the protein level of STIM2, PDGF‐R‐α and PDGF‐R‐β, and also significantly decreased cell proliferation, which is indicated by the decreased EdU incorporation rate and total cell number in IPAH PASMCs. Knockdown of CaMKIIδ significantly decreased phosphorylation of Akt, EdU incorporation rate and total cell number. In addition, knockdown of Akt resulted in a decrease in STIM2 and PDGF‐R‐β.ConclusionTogether, these data indicate that overactivation of CaMKIV and CaMKIIδ in IPAH PASMCs, due to upregulation of CaMKIV, CaMKIIδ and/or increased resting [Ca2+]cyt, contributes to enhancing PASMCs proliferation by increasing activation of Akt, causing upregulation of STIM2 and PDGF‐R. CaMKIV and CaMKIIδ activation‐induced upregulation of STIM2 will further raise the resting [Ca2+]cyt and subsequently activate CaMKIV and CaMKIIδ, forming a positive feedback loop. The overactivation of CaMKIV and CaMKIIδ may be an important mechanism involved in sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling in patients with IPAH.Support or Funding InformationThis work was supported in part by the grants from the National Heart, Lung and Blood Institute of the National Institutes of Health (HL135807 and HL125208).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.