Yamada Kiyomi, Green Karen G, Samarel Allen M, Saffitz Jeffrey E
Department of Pathology, Center for Cardiovascular Research, Washington University School of Medicine, St Louis, MO, USA.
Circ Res. 2005 Aug 19;97(4):346-53. doi: 10.1161/01.RES.0000178788.76568.8a. Epub 2005 Jul 21.
To define mechanisms regulating expression of cell-cell junction proteins, we have developed an in vitro system in which neonatal rat ventricular myocytes were subjected to pulsatile stretch. Previously, we showed that expression of the gap junction protein, connexin (Cx) 43, is increased by approximately 2-fold after 1 hour of stretch, and this response is mediated by stretch-induced secretion of vascular endothelial growth factor (VEGF). Here, we report that the mechanical junction proteins plakoglobin, desmoplakin, and N-cadherin are also upregulated by pulsatile stretch but by a mechanism independent of VEGF or other secreted chemical signals. Stretch-induced upregulation of mechanical junction proteins was blocked by anti-beta1 and anti-beta3 integrin antibodies. Transfection of cells with adenovirus expressing GFP-FRNK, a dominant-negative inhibitor of focal adhesion kinase (FAK)-dependent signaling, blocked stretch-induced upregulation of Cx43 and mechanical junction proteins but did not block the ability of exogenous VEGF to upregulate Cx43 expression. Conditioned medium removed from uninfected cells after stretch increased Cx43 expression when added to nonstretched cells, and this effect was blocked by anti-VEGF antibodies, but stretch-conditioned medium from GFP-FRNK cells had no effect on Cx43 expression. The src kinase inhibitor 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine blocked stretch-induced upregulation of mechanical junction proteins but not Cx43. Thus, stretch upregulates expression of both electrical and mechanical junction proteins via integrin-dependent activation of FAK. Stretch-induced upregulation of Cx43 expression is mediated by FAK-dependent secretion of VEGF. In contrast, stretch-induced upregulation of adhesion junction proteins involves intracellular mechanotransduction pathways initiated via integrin signaling and acting downstream of src kinase.
为了确定调节细胞间连接蛋白表达的机制，我们开发了一种体外系统，在该系统中对新生大鼠心室肌细胞施加脉动拉伸。此前，我们发现，在拉伸1小时后，间隙连接蛋白连接蛋白（Cx）43的表达增加了约2倍，并且这种反应是由拉伸诱导的血管内皮生长因子（VEGF）分泌介导的。在此，我们报告，机械连接蛋白桥粒斑珠蛋白、桥粒芯蛋白和N-钙黏蛋白也会因脉动拉伸而上调，但通过一种独立于VEGF或其他分泌化学信号的机制。抗β1和抗β3整合素抗体可阻断拉伸诱导的机械连接蛋白上调。用表达GFP-FRNK（一种粘着斑激酶（FAK）依赖性信号传导的显性负性抑制剂）的腺病毒转染细胞，可阻断拉伸诱导的Cx43和机械连接蛋白上调，但不阻断外源性VEGF上调Cx43表达的能力。拉伸后从未感染细胞中去除的条件培养基添加到未拉伸细胞中时会增加Cx43表达，且这种效应被抗VEGF抗体阻断，但来自GFP-FRNK细胞的拉伸条件培养基对Cx43表达没有影响。src激酶抑制剂4-氨基-5-（4-氯苯基）-7-（叔丁基）吡唑并[3,4-d]嘧啶可阻断拉伸诱导的机械连接蛋白上调，但不影响Cx43。因此，拉伸通过整合素依赖性激活FAK上调电连接蛋白和机械连接蛋白的表达。拉伸诱导的Cx43表达上调由FAK依赖性VEGF分泌介导。相比之下，拉伸诱导的黏附连接蛋白上调涉及通过整合素信号传导启动并作用于src激酶下游的细胞内机械转导途径。
