Formation of apical compartments underlies the morphogenesis of most epithelial organs during development. The extracellular matrix (ECM), particularly the basement membrane (BM), plays an important role in orienting the apico-basal polarity and thereby the positioning of apical lumens. Integrins have been recognized as essential mediators of matrix-derived polarity signals. The importance of β1-integrins in epithelial polarization is well established but the significance of the accompanying α-subunits have not been analyzed in detail.

The hemidesmosomal transmembrane component collagen XVII (ColXVII) plays an important role in the anchorage of the epidermis to the underlying basement membrane. However, this adhesion protein seems to be also involved in the regulation of keratinocyte migration, since its expression in these cells is strongly elevated during reepithelialization of acute wounds and in the invasive front of squamous cell carcinoma, while its absence in ColXVII-deficient keratinocytes leads to altered cell motility. Using a genetic model of murine Col17a1⁻/⁻ keratinocytes we elucidated ColXVII mediated signaling pathways in cell adhesion and migration. Col17a1⁻/⁻ keratinocytes exhibited increased spreading on laminin 332 and accelerated, but less directed cell motility. These effects were accompanied by increased expression of the integrin subunits β4 and β1. The migratory phenotype, as evidenced by formation of multiple unstable lamellipodia, was associated with enhanced phosphoinositide 3-kinase (PI3K) activity. Dissection of the signaling pathway uncovered enhanced phosphorylation of the β4 integrin subunit and the focal adhesion kinase (FAK) as activators of PI3K. This resulted in elevated Rac1 activity as a downstream consequence. These results provide mechanistic evidence that ColXVII coordinates keratinocyte adhesion and directed motility by interfering integrin dependent PI3K activation and by stabilizing lamellipodia at the leading edge of reepithelializing wounds and in invasive squamous cell carcinoma.

Bullous pemphigoid is an autoimmune blistering skin disease associated with autoantibodies against the dermal-epidermal junction. Passive transfer of antibodies against BP180/collagen (C) XVII, a major hemidesmosomal pemphigoid antigen, into neonatal mice results in dermal-epidermal separation upon applying gentle pressure to their skin, but not in spontaneous skin blistering. In addition, this neonatal mouse model precludes treatment and observation of diseased animals beyond 2-3 days. Therefore, in the present study we have developed a new disease model in mice reproducing the spontaneous blistering and the chronic course characteristic of the human condition. Adult mice were pre-immunized with rabbit IgG followed by injection of BP180/CXVII rabbit IgG. Mice pre-immunized against rabbit IgG and injected 6 times every second day with the BP180/CXVII-specific antibodies (n = 35) developed spontaneous sustained blistering of the skin, while mice pre-immunized and then treated with normal rabbit IgG (n = 5) did not. Blistering was associated with IgG and complement C3 deposits at the epidermal basement membrane and recruitment of inflammatory cells, and was partly dependent on Ly-6G-positive cells. We further used this new experimental model to investigate the therapeutic potential of luteolin, a plant flavonoid with potent anti-inflammatory and anti-oxidative properties and good safety profile, in experimental BP. Luteolin inhibited the Fcγ-dependent respiratory burst in immune complex-stimulated granulocytes and the autoantibody-induced dermal-epidermal separation in skin cryosections, but was not effective in suppressing the skin blistering in vivo. These studies establish a robust animal model that will be a useful tool for dissecting the mechanisms of blister formation and will facilitate the development of more effective therapeutic strategies for managing pemphigoid diseases.

The properties of epithelial cells within tissues are regulated by their immediate microenvironment, which consists of neighboring cells and the extracellular matrix (ECM). Integrin heterodimers orchestrate dynamic assembly and disassembly of cell-ECM connections and thereby convey biochemical and mechanical information from the ECM into cells. However, the specific contributions and functional hierarchy between different integrin heterodimers in the regulation of focal adhesion dynamics in epithelial cells are incompletely understood. Here, we have studied the functions of RGD-binding αV-integrins in a Madin Darby Canine Kidney (MDCK) cell model and found that αV-integrins regulate the maturation of focal adhesions (FAs) and cell spreading. αV-integrin-deficient MDCK cells bound collagen I (Col I) substrate via α2β1-integrins but failed to efficiently recruit FA components such as talin, focal adhesion kinase (FAK), vinculin and integrin-linked kinase (ILK). The apparent inability to mature α2β1-integrin-mediated FAs and link them to cellular actin cytoskeleton led to disrupted mechanotransduction in αV-integrin deficient cells seeded onto Col I substrate.