Satish Latha, Blair Harry C, Glading Angela, Wells Alan
Department of Pathology, 713 Scaife, University of Pittsburgh, Pittsburgh, PA 15261, USA.
Mol Cell Biol. 2005 Mar;25(5):1922-41. doi: 10.1128/MCB.25.5.1922-1941.2005.
Keratinocyte migration is critical to reepithelialization during wound repair. The motility response is promoted by growth factors, cytokines, and cytokines produced in the wound bed, including those that activate the epidermal growth factor (EGF) receptor. The Alu-Leu-Arg-negative CXC chemokine interferon-inducible protein 9 (IP-9; also known as CXCL11, I-TAC, beta-R1, and H-174) is produced by keratinocytes in response to injury. As keratinocytes also express the receptor, CXCR3, this prompted us to examine the role and molecular mechanism by which IP-9 regulates keratinocyte motility. Unexpectedly, as CXCR3 liganding blocks growth factor-induced motility in fibroblasts, IP-9 alone promoted motility in undifferentiated keratinocytes (37 +/- 6% of the level of the highly motogenic EGF) as determined in a two-dimensional in vitro wound healing assay. IP-9 even enhanced EGF-induced motility in undifferentiated keratinocytes (116 +/- 5%; P < 0.05 compared to EGF alone), suggesting two separate mechanisms of action. IP-9-increased motility and -decreased adhesiveness required the intracellular protease calpain. The increases in both motility and calpain activity by IP-9 were blocked by pharmacological and molecular inhibition of phospholipase C-beta3 and chelation of calcium, which prevented an intracellular calcium flux. Molecular downregulation or RNA interference-mediated depletion of mu-calpain (calpain 1) but not M-calpain (calpain 2) blocked IP-9-induced calpain activation and motility. In accord with elimination of IP-9-induced de-adhesion, RNA interference-mediated depletion of calpain 1 but not calpain 2 prevented cleavage of the focal adhesion component focal adhesion kinase and disassembly of vinculin aggregates. In comparison, EGF-induced motility of the same undifferentiated keratinocytes requires the previously described extracellular signal-regulated kinase to the M-calpain pathway. These data demonstrate that while both EGF- and IP-9-induced motility in keratinocytes requires calpain activity, the isoform of calpain triggered depends on the nature of the receptor for the particular ligand. Interestingly, physiological nonapoptotic calcium fluxes were capable of activating mu-calpain, implying that the calcium requirement of mu-calpain for activation is attained during cell signaling. This is also the first demonstration of differential activation of the two ubiquitous calpain isoforms in the same cell by different signals.
角质形成细胞迁移对于伤口修复过程中的再上皮化至关重要。伤口床中产生的生长因子、细胞因子及细胞因子可促进角质形成细胞的运动反应，其中包括那些激活表皮生长因子（EGF）受体的因子。Alu-Leu-Arg阴性CXC趋化因子干扰素诱导蛋白9（IP-9；也称为CXCL11、I-TAC、β-R1和H-174）由角质形成细胞在损伤后产生。由于角质形成细胞也表达受体CXCR3，这促使我们研究IP-9调节角质形成细胞运动的作用及分子机制。出乎意料的是，正如CXCR3配体阻断成纤维细胞中生长因子诱导的运动一样，在二维体外伤口愈合试验中测定，单独的IP-9可促进未分化角质形成细胞的运动（为高运动性EGF水平的37±6%）。IP-9甚至增强了未分化角质形成细胞中EGF诱导的运动（116±5%；与单独使用EGF相比，P<0.05），提示存在两种不同的作用机制。IP-9增加运动性和降低黏附性需要细胞内蛋白酶钙蛋白酶。IP-9引起的运动性增加和钙蛋白酶活性增加被磷脂酶C-β3的药理学和分子抑制以及钙螯合所阻断，这阻止了细胞内钙通量。通过分子下调或RNA干扰介导的μ-钙蛋白酶（钙蛋白酶1）而非M-钙蛋白酶（钙蛋白酶2）的缺失可阻断IP-9诱导的钙蛋白酶激活和运动。与消除IP-9诱导的去黏附一致，RNA干扰介导的钙蛋白酶1而非钙蛋白酶2的缺失可阻止黏着斑成分黏着斑激酶的裂解和纽蛋白聚集体的解体。相比之下，相同未分化角质形成细胞中EGF诱导的运动需要先前描述的细胞外信号调节激酶至M-钙蛋白酶途径。这些数据表明，虽然EGF和IP-9诱导的角质形成细胞运动都需要钙蛋白酶活性，但所触发的钙蛋白酶同工型取决于特定配体的受体性质。有趣的是，生理性非凋亡钙通量能够激活μ-钙蛋白酶，这意味着在细胞信号传导过程中达到了μ-钙蛋白酶激活所需的钙。这也是首次证明在同一细胞中两种普遍存在的钙蛋白酶同工型可被不同信号差异激活。
