Medical and Research Services, Ralph H. Johnson Veterans Affairs Medical Center, Medical University of South Carolina, Charleston, SC 29425-6290, USA.
Mol Pharmacol. 2010 Jul;78(1):126-34. doi: 10.1124/mol.110.064840. Epub 2010 Apr 12.
We have shown previously that the vasoactive peptide bradykinin (BK) stimulates proliferation of a cultured murine cell model of the inner medullary collecting duct (mIMCD-3 cells) via transactivation of epidermal growth factor receptor (EGFR) by a mechanism that involves matrix metalloproteinases (collagenase-2 and -3). Because collagenases lack an integral membrane domain, we hypothesized that receptors for extracellular matrix proteins, integrins, may play a role in BK-induced signaling by targeting collagenases to the membrane, thus forming a functional signaling complex. BK-induced phosphorylation of extracellular signal-regulated protein kinase (ERK) in mIMCD-3 cells was reduced by approximately 65% by synthetic peptides containing an Arg-Gly-Asp sequence, supporting roles for integrins in BK-induced signaling. Neutralizing antibody against alpha5beta1 integrin partially (approximately 60%) blocked BK-induced ERK activation but did not affect EGF-induced ERK activation. Silencing of alpha5 and beta1 expression by transfecting cells with small interfering RNAs (siRNA) significantly decreased BK-induced ERK activation (approximately 80%) and EGFR phosphorylation (approximately 50%). This effect was even more pronounced in cells that were cotransfected with siRNAs directed against both collagenases and alpha5beta1 integrin. On the basis of our results, we suggested that integrin alpha5beta1 is involved in BK-induced signaling in mIMCD-3 cells. Using immunoprecipitation/Western blotting, we demonstrated association of BK B(2) receptor with alpha5beta1 integrin upon BK treatment. Furthermore, BK induced association of alpha5beta1 integrin with EGFR. These data provide the first evidence that specific integrins are involved in BK B(2) receptor-induced signaling in kidney cells, and ultimately might lead to development of new strategies for treatment of renal tubulointerstitial fibrosis.
我们之前已经表明，血管活性肽缓激肽（BK）通过涉及基质金属蛋白酶（胶原酶-2 和 -3）的表皮生长因子受体（EGFR）的反式激活，刺激培养的小鼠内髓集合管细胞模型（mIMCD-3 细胞）的增殖。由于胶原酶缺乏完整的膜结构域，我们假设细胞外基质蛋白受体整合素可能通过将胶原酶靶向膜，从而形成功能性信号复合物，在 BK 诱导的信号转导中发挥作用。在 mIMCD-3 细胞中，含有精氨酸-甘氨酸-天冬氨酸序列的合成肽使 BK 诱导的细胞外信号调节蛋白激酶（ERK）磷酸化减少约 65%，支持整合素在 BK 诱导的信号转导中的作用。针对 alpha5beta1 整合素的中和抗体部分（约 60%）阻断 BK 诱导的 ERK 激活，但不影响 EGF 诱导的 ERK 激活。用小干扰 RNA（siRNA）转染细胞沉默 alpha5 和 beta1 表达可显著降低 BK 诱导的 ERK 激活（约 80%）和 EGFR 磷酸化（约 50%）。在共转染针对胶原酶和 alpha5beta1 整合素的 siRNA 的细胞中，这种效应更为明显。根据我们的结果，我们认为整合素 alpha5beta1 参与 mIMCD-3 细胞中的 BK 诱导信号转导。通过免疫沉淀/Western 印迹，我们证明了 BK 处理后 BK B（2）受体与 alpha5beta1 整合素的结合。此外，BK 诱导 alpha5beta1 整合素与 EGFR 的结合。这些数据首次证明了特定的整合素参与肾脏细胞中 BK B（2）受体诱导的信号转导，并最终可能导致开发治疗肾间质纤维化的新策略。
