Type XVII Collagen Regulates Lamellipod Stability, Cell Motility, and Signaling to Rac1 by Targeting Bullous Pemphigoid Antigen 1e to α6β4 Integrin

Abstract

Rac1 activity, polarity, lamellipodial dynamics, and directed motility are defective in keratinocytes exhibiting deficiency in ␤4 integrin or knockdown of the plakin protein Bullous Pemphigoid Antigen 1e (BPAG1e). The activity of Rac, formation of stable lamellipodia, and directed migration are restored in ␤4 integrin-deficient cells by inducing expression of a truncated form of ␤4 integrin, which lacks binding sites for BPAG1e and plectin. In these same cells, BPAG1e, the truncated ␤4 integrin, and type XVII collagen (Col XVII), a transmembrane BPAG1e-binding protein, but not plectin, colocalize along the substratum-attached surface. This finding suggested to us that Col XVII mediates the association of BPAG1e and ␣6␤4 integrin containing the truncated ␤4 subunit and supports directed migration. To test these possibilities, we knocked down Col XVII expression in keratinocytes expressing both full-length and truncated ␤4 integrin proteins. Col XVII-knockdown keratinocytes exhibit a loss in BPAG1e-␣6␤4 integrin interaction, a reduction in lamellipodial stability, an impairment in directional motility, and a decrease in Rac1 activity. These defects are rescued by a mutant Col XVII protein truncated at its carboxyl terminus. In summary, our results suggest that in motile cells Col XVII recruits BPAG1e to ␣6␤4 integrin and is necessary for activation of signaling pathways, motile behavior, and lamellipodial stability.Re-epithelialization, the process by which epidermal cells undergo directed migration and move over an exposed wound bed, is a critical aspect in the closure of epithelial wounds. Factors that impact the ability of a cell to migrate can be roughly grouped into those involved in the basic mechanism of motility and those involved in the steering mechanism (1). To migrate efficiently, in a directed manner, cells must establish and maintain an asymmetric morphology with defined leading and trailing edges (2). This leading edge is characterized by a sheet-like extension termed a lamellipodium (1). Moreover, the rates of extension, the stability of the formed lamellipodium, and the size of the extension are the determining factors in defining the migration characteristics of a cell (3, 4). Indeed, the rate of lamellipodium extension correlates with migration rate, whereas lamellipodium stability (persistence) correlates with processivity or turning frequency (1).Lamellipodium formation requires localized rearrangement of the actin cytoskeleton, which involves signaling mediated by Rac1 (a small GTPase of the Rho family) to the actin severing protein cofilin (5). Through severing of actin filaments, cofilin increases the number of free actin barbed ends from which actin filaments extend while simultaneously increasing the depolymerization rate of older actin filaments, thereby increasing the local pool of free actin monomers (6). Spatial control of Rac1 and cofilin activity can therefore drive localized extension of the actin-rich lamellipodium. In addition to rearrangement of the actin cytoskeleton, lamellipo...

α6β4 Integrin, a Master Regulator of Expression of Integrins in Human Keratinocytes

“…S3). Interestingly, in prior studies we did not observe a comparable difference in velocity between wild-type keratinocytes and skin cells deficient in BPAG1e or BP180 (34,35). Again the current result was not due to nonspecific effects of the ␣6 integrin shRNA because velocity and normal migratory behavior was restored in the clones following expression of ␣6 integrin mRNA that is refractory to the ␣6 integrin shRNA (Fig.…”

Plakophilin 2 Affects Cell Migration by Modulating Focal Adhesion Dynamics and Integrin Protein Expression

“…These changes disrupted the formation and maturation of desmosome junctions. We hypothesized that the global change in RhoA activity may also affect cell–substrate attachments, including integrin-based focal adhesions, as such adhesions are known to regulate cell behavior via functional interactions with small GTPases and the actin cytoskeleton (Hamill et al, 2009; Hamill et al, 2010; Hamill et al, 2011; Tsuruta et al, 2011; Hong et al, 2012). We set out to address the possibility that PKP2 functions to integrate signals within the keratinocyte adhesive network of both cadherin- and integrin-based adhesions.…”

Focal Contact and Hemidesmosomal Proteins in Keratinocyte Migration and Wound Repair

“…Although surface expression of b 4 and a 3 integrins is unchanged in the knockdown cells, loss of expression of collagen XVII results in a decreased expression of BPAG1e. 115 This indicates a role for collagen XVII in assembly of a a 6 b 4 integrin/ BPAG1e signaling complex in motile skin cells.…”

“…95,96 Collagen XVII is also able to link b 4 integrin and BPAG1e when the b 4 integrin binding site for BPAG1e has been deleted. 115 The role of collagen XVII in migration is controversial. Keratinocytes derived from patients lacking the protein show enhanced migration over controls.…”