Epidermal Growth Factor Receptor Activation of Calpain Is Required for Fibroblast Motility and Occurs via an ERK/MAP Kinase Signaling Pathway

Abstract

To become migratory, cells must reorganize their connections to the substratum, and during locomotion they must break rear attachments. The molecular and biochemical mechanisms underlying these biophysical processes are unknown. Recent studies have implicated both extracellular signal-regulated kinase/mitogen-activated protein (ERK/MAP) kinase and calpain (EC 3.4.22.17) in these processes, but it is uncertain whether these are two distinct pathways acting on different modes of motility. We report that cell deadhesion involved in epidermal growth factor (EGF) receptor-mediated fibroblast motility requires activation of M-calpain downstream of ERK/MAP kinase signaling. NR6 fibroblasts expressing full-length wild type epidermal growth factor receptor required both calpain and ERK activation, as demonstrated by pharmacological inhibitors (calpeptin and calpain inhibitor I and PD98059, respectively) for EGF-induced deadhesion and motility. EGF induced rapid activation of calpain that was preventable by molecular inhibition of the Ras-Raf-MEK but not phospholipase C␥ signaling pathway, and calpain was stimulated by transfection of constitutively active MEK. Enhanced calpain activity was not mirrored by increased calpain protein levels or decreased levels of its endogenous inhibitor calpastatin. The link between ERK/MAP kinase signaling and cell motility required the M-isoform of calpain (calpain II), as determined by specific antisense-mediated down-regulation. These data promote a previously undescribed signaling pathway of ERK/MAP kinases activating calpain to destabilize cell-substratum adhesions in response to EGF stimulation.Cell motility is a central process involved in many physiological events including tumor invasion, embryonic development, and wound healing. Cell motility has been found to be a very complex biophysical process involving multiple factors working together to accomplish concerted movement. To begin to understand the molecular bases of motility, fibroblast cell motility has been deconstructed into four separate events: extension of the lammelipod, formation of new focal adhesions at the leading edge, breaking of adhesions at the trailing edge, and translocation of the cell mass (1). Failure of any one of these steps is sufficient to prevent cell motility (2, 3).These motility events are regulated by both cell intrinsic and extrinsic properties (4, 5). Among the latter are regulatory inputs from growth factor receptors and the strength and nature of cell-substratum interactions. Previously, it had been demonstrated that motility of adherent cells occurs in a biphasic relationship to adhesiveness of the surface (6, 7). Recently, we have shown that this also holds for growth factor-induced motility, in that epidermal growth factor (EGF) 1 receptor-mediated fibroblast motility is noted only at intermediate levels of cell adhesiveness to substrata (8, 9). Thus, signals that regulate adhesiveness to the substratum are of high importance, since the rear deadhesion of the cell from its substrate can be ...

Podosome-type adhesions and focal adhesions, so alike yet so different

“…Indeed, Zaidel-Bar , 2007 et al recent studies have demonstrated that the calpain family has a regulatory function in cell motility, partly through the capacity to down regulate integrin-mediated adhesion complexes ( ; ; ; ). In that Glading , 2000et al Dourdin , 2001et al Bhatt , 2002et al Glading , 2002 context, talin seems to be one of major target of calpain leading to a rate-limiting step critical for FA disassembly ( ). It Franco , 2004 et al has also been shown that the expression of a calpain-resistent cortactin impaired cell migration and increased transient membrane…”

Scaffold mediated regulation of MAPK signaling and cytoskeletal dynamics: A perspective

“…Calpain cleavage of talin here is a rate limiting step in disassembly of other focal adhesion components including zyxin, paxillin and vinculin [190]. Recent studies have demonstrated that growth factor stimulation of calpain activity and cellular deadhesion is dependent upon ERK [191,192]. Calpain is directly phosphorylated by ERK in response to EGF stimulation and this increases calpain's proteolytic activity [96].…”

“…Indeed, calpain may be one of the "relaxing" factors targeted to focal adhesions by microtubules for focal adhesion disassembly [140]. Src [200], FAK and MEKK1 [193] appear to regulate calpain activity in part through ERK phosphorylation of the protease [96,192]. FAK null cells exhibit less stable microtubules [201], lower calpain activity [193], decreased ERK signaling [198,202,203] and reduced migratory potential due to defects in focal adhesion disassembly [86], consistent with the view that signaling from FAK to ERK may alter migratory responses through ERK stimulated calpain proteolysis of focal adhesion components and actin remodeling, and microtubule dynamics.…”

Ceramide triggers an NF-κB-dependent survival pathway through calpain

“…28 Therefore, we utilized an Figure 6 Capn4 silencing and overexpression of calpastatin correlate with increased p50 and p105 protein levels in vivo. Panel a: Capn4 siRNA or control siRNA were transfected in the indicated cell lines.…”

“…Binding of ligand to EGFR was shown to lead to activation of milli-calpain subsequent to ERK/MAP kinase signalling. 28,29 Endoplasmic reticulum (ER) stress-inducing agents such as thapsigargin, ionomycin and ceramides are widely used as triggers of calpain activity 27 to elucidate the molecular events underlying calpain-involved signalling.…”