Interferon-Inducible Protein 9 (CXCL11)-Induced Cell Motility in Keratinocytes Requires Calcium Flux-Dependent Activation of μ-Calpain

Regulated cell motility is critical to wound healing (25, 35). During repair, tissue deposition and remodeling by the immigrant fibroblasts and keratinocytes result in the regeneration of an intact skin barrier and functional organ. The cells from the remaining epidermal and dermal layers must proliferate and migrate to repopulate the nascent wound. The basal keratinocytes undergo a transition that enables such repopulation while the provisional matrix is invaded by fibroblasts as the first step in regenerating the future dermal layer. The numerous growth factors present throughout repair, including high levels of epidermal growth factor (EGF) receptor (EGFR) ligands such as HB-EGF and transforming growth factor alpha, are thought to promote these mitogenic and motogenic responses (26, 35, 45, 50, 60). However, the process of cell repopulation is limited late in the process of healing to prevent fibroplasia and excess matrix deposition. Late in the repair process, members of the Alu-Leu-Arg (ELR)-negative family of CXC chemokines appear (13, 42). It has been proposed that the migration of fibroblasts and keratinocytes is controlled by the waves of these growth factors and chemokines produced throughout wound repair (9, 35, 42, 44, 60). This would include signals to promote as well as inhibit cell migration. Active cell locomotion requires the coordination of a number of cellular processes that should be common among cell types (18, 28). Thus, as numerous external signals can modulate cell motility, two key questions are which biochemical pathway is actuated to promote migration during regeneration and whether these differ between cell types. Any such signaling pathway needs to affect key biophysical processes. During cell migration, tail de-adhesion may be rate-limiting; in experimental models, failure to detach limits cell motility (23, 44). Activation of calpain (EC 3.4.22.17), an intracellular limited protease, is required for integrin-mediated tail de-adhesion on moderately and highly adhesive substrata (30, 37) and for growth factor-induced motility (17, 43). This intracellular protease is a key switch, as calpain inhibitors convert EGFR-mediated signals from cell motility to matrix contractility (1). Thus, calpain activators appear to shift a wide range of cells to motility-permissive adhesion regimens, while inhibitory signals for calpain block productive locomotion. As such, we proposed that keratinocyte motility was dependent on calpain activity. This requirement for calpain activity provides a target for regulating cell motility (18). Confounding any analysis, two calpain isoforms with seemingly identical target specificities are present in practically all cells (48). In vitro, calpain 1 (μ-calpain) is activated at nearly micromolar concentrations of calcium; calpain 2 (M-calpain) requires millimolar calcium levels. While calcium fluxes have been postulated to regulate μ-calpain, this has yet to be demonstrated conclusively in living cells (5), and the signaling cascade that triggers this isoform during cell locomotion remain undefined (30). Furthermore, the physiologically relevant activators of M-calpain are unknown since intracellular calcium levels fail to reach the nearly millimolar concentrations required in vitro (22). Still, we know that plasma membrane-localized M-calpain is activated subsequent to growth factor signaling by direct extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAP kinase) phosphorylation (12, 16, 17, 44). However, what signals and respective intracellular signaling pathways operate during reepithelialization by keratinocytes remains an open question (42). In an initial exploration (43, 44), we reported that ELR-negative CXC chemokines, present during the process of wound repair (13, 42), can block fibroblast motility by preventing activation of M-calpain and subsequent de-adhesion, demonstrating that this might be a physiologically operative pathway. Of this family of chemokines, interferon-inducible protein 10 (IP-10) appears to be produced by the neovasculature deep in the dermis (13). A related ELR-negative CXC chemokine, IP-9, also called beta-R1 (40), H174 (24), and I-TAC (10), is produced by basal keratinocytes in response to immune-mediated injuries (52). Previously, we demonstrated that IP-9 is a wound response factor (42). These ELR-negative CXC chemokines were originally found as modulators of cells of the hematopoietic lineage, but chemokine receptors have been found on endothelial and epithelial cells (36, 38, 44, 47, 63). The ELR-negative members of the CXC family of chemokines, all of which bind to a common CXCR3 receptor (2, 14, 27), inhibit endothelial cell proliferation, migration (21, 31, 51), and fibroblast migration (44). These appear to act dominantly over promitogenic and promotility chemokines and growth factors (43, 44). IP-9 is a wound response factor situated in the right place to limit fibroblast repopulation and promote the remodeling phase (42). However, with IP-9 being produced by keratinocytes in or near the wound bed, the situation for the reepithelializing keratinocytes is of great interest; would this chemokine, which serves to “mature” the dermis, also have the collateral effect of slowing reepithelialization? Here we report that keratinocyte motility is promoted by both EGF and IP-9 through their activation of calpains. Furthermore, IP-9 does not block EGF-induced motility in keratinocytes, opposite to its effects on fibroblasts. Keratinocyte motility induced by EGF requires the pathway previously described in fibroblasts, which culminates in activation of the calpain 2 isoform, M-calpain (17). In undifferentiated keratinocytes, IP-9 also activated calpain, though it was the μ-calpain isoform and not the M-calpain isoform that was both triggered and required. This chemokine activation occurred through a phospholipase Cβ (PLCβ)-mediated calcium flux in distinction to the ERK MAP kinase signaling cascade that growth factors utilize to activate M-calpain (17). These two pathways converge at cell-diminished cell adhesion to substratum as mirrored in vinculin aggregate disassembly and cleavage of the focal adhesion kinase (FAK). This is the first demonstration, to our knowledge, of different isoforms of calpain being activated by distinct signals in the same cell to accomplish the same phenotypic end point, cell migration. We also show for the first time that IP-9 increased the intracellular calcium flux and resulted in triggering of the μ-calpain isoform, in turn resulting in productive cell motility.