Extracellular Calpains Increase Tubular Epithelial Cell Mobility

Abstract

Calpains are intracellular Ca2؉ -dependent cysteine proteases that are released in the extracellular milieu by tubular epithelial cells following renal ischemia. Here we show that externalized calpains increase epithelial cell mobility and thus are critical for tubule repair. In vitro, exposure of human tubular epithelial cells (HK-2 cells) to -calpain limited their adhesion to extracellular matrix and increased their mobility. Calpains acted primarily by promoting the cleavage of fibronectin, thus preventing fibronectin binding to the integrin ␣ v ␤ 3 . Analyzing downstream integrin effects, we found that the cyclic AMP-dependent protein kinase A pathway was activated in response to ␣ v ␤ 3 disengagement and was essential for calpain-mediated increase in HK-2 cell mobility. In a murine model of ischemic acute renal failure, injection of a fragment of calpastatin, which specifically blocked calpain activity in extracellular milieu, markedly delayed tubule repair, increasing functional and histological lesions after 24 and 48 h of reperfusion. These findings suggest that externalized calpains are critical for tubule repair process in acute renal failure.Calpains are intracellular Ca 2ϩ -dependent cysteine proteases (1). The major isozymes, calpain 1 or -calpain and calpain 2 or m-calpain, are distributed ubiquitously and activated in vitro by micromolar and millimolar concentrations of Ca 2ϩ , respectively. They are heterodimers composed of a ϳ80-kDa catalytic subunit (encoded by CAPN1 and CAPN2 for -and m-calpain, respectively) and a common ϳ30-kDa regulatory subunit (encoded by CAPN4). Binding of Ca 2ϩ to -or m-calpain induces the release of constraints imposed by domain interactions and results in a two-stage activation process: first, the release of ϳ30-kDa regulatory subunit; and second, the rearrangement of the active site cleft in ϳ80-kDa catalytic subunit (2). Calpain activity is tightly controlled by calpastatin, a specific endogenous inhibitor, which contains four equivalent inhibitory domains (1). By conducting limited proteolysis of intracellular substrates, calpain activity has been shown to be critical for a great diversity of cellular responses. They include rearrangement of cytoskeletal linkages to the plasma membrane during cell adhesion and mobility, modification of molecules in signal transduction pathways, degradation of enzymes controlling the cell cycle, and activation of proteolytic cascades leading to cell apoptosis or necrosis (1,3,4).Recently, several groups showed that calpains may be released from cells into the extracellular environment and thus may have an extracellular role. Like other intracellular enzymes, calpains may indeed leak out from injured and dying cells such as hepatocytes exposed to toxic chemicals (5). The release of intracellular calpains from blood mononuclear cells (6), osteoblasts (7), chondrocytes (8), and parathyroid cells (9) is not due to cell death, but rather to a nonclassical pathway of secretion, which involves in certain cells the shedding of membrane vesi...

Calpains Contribute to Vascular Repair in Rapidly Progressive Form of Glomerulonephritis: Potential Role of Their Externalization

“…Our previous studies indicated that extracellular calpains play a role in tubular epithelium regeneration after renal ischemia/reperfusion via the detachment of FN fragments from the extracellular matrix, 31 consistent with findings of other groups. 32 Thus, to identify molecular mechanisms whereby extracellular calpains speed up endothelium regeneration, we evaluated the ability of calpains to hydrolyze proteins of extracellular matrix in 1D SDS-PAGE experiments (Supplemental Figure VII).…”

“…Previous studies have suggested a role for protease-induced cleavage products of extracellular matrix proteins in vascular development and repair. 36 Because calpains are known to cleave FN, 31,32 we focused our studies on this protein. Exposure of native human FN to calpains generated various fragments encompassing the C-terminal heparin-II domains 9 to 10 and 13 to 14 which bind the ␣ 5 ␤ 1 integrin and VEGF, respectively.…”

Cigarette smoke impairs airway epithelial barrier function and cell–cell contact recovery

“…In addition to its effects on cell-cell contacts, EGFR activity can affect cell-matrix contacts by induction of calpain activity and cleavage of fibronectin and/or focal adhesion component talin [32][33][34]. In our setting, the modest CSE-induced increase in high-frequency capacitance reflected effects on cell-matrix contacts, and the presence of AG1478 abrogated these effects.…”

Calpains Released by T Lymphocytes Cleave TLR2 To Control IL-17 Expression

“…For instance, externalized calpains activate anti-inflammatory cytokines (TGF-b) (13) and inactivate proinflammatory proteins (chemerins) (14). In addition, we demonstrated that extracellular calpains participate in both epithelium regeneration in a model of acute kidney injury (15) and angiogenesis in models of kidney inflammation (10). The opposite activities of intra-and extracellular calpains raise questions regarding the mechanisms of calpain secretion.…”