Abeyrathna Prasanna, Kovacs Laszlo, Han Weihong, Su Yunchao
Department of Pharmacology & Toxicology, Medical College of Georgia, Augusta University, Augusta, Georgia;
Department of Pharmacology & Toxicology, Medical College of Georgia, Augusta University, Augusta, Georgia; Department of Medicine, Medical College of Georgia, Augusta University, Augusta, Georgia; Vascular Biology Center, Medical College of Georgia, Augusta University, Augusta, Georgia; and Research Service, Charlie Norwood Veterans Affairs Medical Center, Augusta, Georgia
Am J Physiol Cell Physiol. 2016 Jul 1;311(1):C24-34. doi: 10.1152/ajpcell.00295.2015. Epub 2016 Apr 20.
Calpain is a family of calcium-dependent nonlysosomal neutral cysteine endopeptidases. Akt is a serine/threonine kinase that belongs to AGC kinases and plays important roles in cell survival, growth, proliferation, angiogenesis, and cell metabolism. Both calpain and Akt are the downstream signaling molecules of platelet-derived growth factor (PDGF) and mediate PDGF-induced collagen synthesis and proliferation of pulmonary artery smooth muscle cells (PASMCs) in pulmonary vascular remodeling. We found that inhibitions of calpain-2 by using calpain inhibitor MDL28170 and calpain-2 small interfering RNA attenuated Akt phosphorylations at serine-473 (S473) and threonine-308 (T308), as well as collagen synthesis and cell proliferation of PASMCs induced by PDGF. Overexpression of calpain-2 in PASMCs induced dramatic increases in Akt phosphorylations at S473 and T308. Moreover, knockout of calpain attenuated Akt phosphorylations at S473 and T308 in smooth muscle of pulmonary arterioles of mice with chronic hypoxic pulmonary hypertension. The cell-permeable-specific transforming growth factor (TGF)-β receptor inhibitor SB431542 attenuated Akt phosphorylations at both S473 and T308 induced by PDGF and by overexpressed calpain-2 in PASMCs. Furthermore, SB-431452 and knocking down activin receptor-like kinase-5 significantly reduced PDGF-induced collagen synthesis and cell proliferation of PASMCs. Nevertheless, neutralizing extracellular TGF-β1 using a cell-impermeable TGF-β1 neutralizing antibody did not affect PDGF-induced Akt phosphorylations at S473 and T308. Furthermore, inhibition of mammalian target of rapamycin complex 2 (mTORC2) by knocking down its component protein Rictor prevented Akt phosphorylations at S473 and T308 induced by PDGF and by overexpressed calpain-2. These data provide first evidence supporting that calpain-2 upregulates PDGF-induced Akt phosphorylation in pulmonary vascular remodeling via an intracrine TGF-β1/mTORC2 mechanism.
钙蛋白酶是一类依赖钙的非溶酶体中性半胱氨酸内肽酶。Akt是一种丝氨酸/苏氨酸激酶，属于AGC激酶家族，在细胞存活、生长、增殖、血管生成和细胞代谢中发挥重要作用。钙蛋白酶和Akt均为血小板衍生生长因子（PDGF）的下游信号分子，在肺血管重塑过程中介导PDGF诱导的肺动脉平滑肌细胞（PASMCs）胶原蛋白合成和增殖。我们发现，使用钙蛋白酶抑制剂MDL28170和钙蛋白酶-2小干扰RNA抑制钙蛋白酶-2可减弱丝氨酸-473（S473）和苏氨酸-308（T308）位点的Akt磷酸化，以及PDGF诱导的PASMCs胶原蛋白合成和细胞增殖。在PASMCs中过表达钙蛋白酶-2可显著增加S473和T308位点的Akt磷酸化。此外，在慢性低氧性肺动脉高压小鼠的肺小动脉平滑肌中敲除钙蛋白酶可减弱S473和T308位点的Akt磷酸化。细胞可渗透的特异性转化生长因子（TGF）-β受体抑制剂SB431542可减弱PDGF和PASMCs中过表达的钙蛋白酶-2诱导的S473和T308位点的Akt磷酸化。此外，SB-431452和敲低激活素受体样激酶-5可显著降低PDGF诱导的PASMCs胶原蛋白合成和细胞增殖。然而，使用细胞不可渗透的TGF-β1中和抗体中和细胞外TGF-β1并不影响PDGF诱导的S473和T308位点的Akt磷酸化。此外，通过敲低其组成蛋白Rictor抑制哺乳动物雷帕霉素靶蛋白复合物2（mTORC2）可阻止PDGF和过表达的钙蛋白酶-2诱导的S473和T308位点的Akt磷酸化。这些数据首次提供了证据，支持钙蛋白酶-2在肺血管重塑过程中通过自分泌TGF-β1/mTORC2机制上调PDGF诱导的Akt磷酸化。
