Mutations in SPINK5, encoding the serine protease inhibitor LEKTI, cause Netherton syndrome, a severe autosomal recessive genodermatosis. Spink5(-/-) mice faithfully replicate key features of Netherton syndrome, including altered desquamation, impaired keratinization, hair malformation and a skin barrier defect. LEKTI deficiency causes abnormal desmosome cleavage in the upper granular layer through degradation of desmoglein 1 due to stratum corneum tryptic enzyme and stratum corneum chymotryptic enzyme-like hyperactivity. This leads to defective stratum corneum adhesion and resultant loss of skin barrier function. Profilaggrin processing is increased and implicates LEKTI in the cornification process. This work identifies LEKTI as a key regulator of epidermal protease activity and degradation of desmoglein 1 as the primary pathogenic event in Netherton syndrome.

The tetraspanin CD151 is a cell-surface molecule known for its strong lateral interaction with the laminin-binding integrin α3β1. Patients with a nonsense mutation in CD151 display end-stage kidney failure associated with regional skin blistering and sensorineural deafness, and mice lacking the integrin α3 subunit die neonatally because of severe abnormalities in the lung and kidney epithelia. We report the generation of Cd151-null mice that recapitulate the renal pathology of human patients, i.e., with age they develop massive proteinuria caused by focal glomerulosclerosis, disorganization of the glomerular basement membrane, and tubular cystic dilation. However, neither skin integrity nor hearing ability are impaired in the Cd151-null mice. Furthermore, we generated podocyte-specific conditional knockout mice for the integrin α3 subunit that show renal defects similar to those in the Cd151 knockout mice. Our results support the hypothesis that CD151 plays a key role in strengthening α3β1-mediated adhesion in podocytes.

flox/flox; K14-Cre mice lacking the α3 subunit specifically in the basal layer of the epidermis. These mice are viable but display several skin defects, including local inflammation, hair loss, basement membrane duplication and microblistering at the dermalepidermal junction, whereas hemidesmosome assembly and keratinocyte differentiation are not impaired. Wound healing is slightly faster in the absence of integrin α3β1, whereas proliferation, the distribution of other integrins and the deposition of basement membrane proteins in the wound bed are unaltered. In vitro, cell spreading is rescued by increased surface expression of α6β1 integrin in the absence of integrin α3. The α3-deficient keratinocytes migrate with an increased velocity and persistence, whereas proliferation, growth factor signaling, hemidesmosome assembly, and laminin-332 deposition appeared to be normal. We suggest that integrin α3β1 delays keratinocyte migration during wound reepithelialization, by binding to the laminin-332 that is newly deposited on the wound bed.Supplementary material available online at

The family of integrin transmembrane receptors is essential for the normal function of multicellular organisms by facilitating cellextracellular matrix adhesion. The vitronectin-binding integrin αVβ5 localizes to focal adhesions (FAs) as well as poorly characterized flat clathrin lattices (FCLs). Here, we show that, in human keratinocytes, αVβ5 is predominantly found in FCLs, and formation of the αVβ5containing FCLs requires the presence of vitronectin as ligand, Ca 2+ , and the clathrin adaptor proteins ARH (also known as LDLRAP1), Numb and EPS15/EPS15L1. Integrin chimeras, containing the extracellular and transmembrane domains of β5 and the cytoplasmic domains of β1 or β3, almost exclusively localize in FAs. Interestingly, lowering actomyosin-mediated contractility promotes integrin redistribution to FLCs in an integrin tail-dependent manner, while increasing cellular tension favors αVβ5 clustering in FAs. Our findings strongly indicate that clustering of integrin αVβ5 in FCLs is dictated by the β5 subunit cytoplasmic domain, cellular tension and recruitment of specific adaptor proteins to the β5 subunit cytoplasmic domains.

Integrins connect the extracellular matrix with the cell interior, and transduce signals through interactions of their cytoplasmic tails with cytoskeletal and signaling proteins. Using the yeast two-hybrid system, we isolated a novel splice variant (filamin-Bvar-1) of the filamentous actin cross-linking protein, filamin-B, that interacts with the cytoplasmic domain of the integrin β1A and β1D subunits. RT-PCR analysis showed weak, but wide, expression of filamin-Bvar-1 and a similar splice variant of filamin-A (filamin-Avar-1) in human tissues. Furthermore, alternative splice variants of filamin-B and filamin-C, from which the flexible hinge-1 region is deleted (ΔH1), were induced during in vitro differentiation of C2C12 mouse myoblasts. We show that both filamin-Avar-1 and filamin-Bvar-1 bind more strongly than their wild-type isoforms to different integrin β subunits. The mere presence of the high-affinity binding site for β1A is not sufficient for targeting the filamin-Bvar-1 construct to focal contacts. Interestingly, the simultaneous deletion of the H1 region is required for the localization of filamin-B at the tips of actin stress fibers. When expressed in C2C12 cells, filamin-Bvar-1(ΔH1) accelerates their differentiation into myotubes. Furthermore, filamin-B variants lacking the H1 region induce the formation of thinner myotubes than those in cells containing variants with this region. These findings suggest that specific combinations of filamin mRNA splicing events modulate the organization of the actin cytoskeleton and the binding affinity for integrins.

Progression through the various stages of skin tumorigenesis is correlated with an altered expression of the integrin α3β1, suggesting that it plays an important role in the tumorigenic process. Using epidermis-specific Itga3 KO mice subjected to the 7,12-dimethylbenzanthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate two-stage skin carcinogenesis protocol, we demonstrate that efficient tumor development is critically dependent on the presence of α3β1. In the absence of α3β1, tumor initiation is dramatically decreased because of increased epidermal turnover, leading to a loss of DMBA-initiated label-retaining keratinocytes. Lineage tracing revealed emigration of α3-deficient keratinocytes residing in the bulge of the hair follicle toward the interfollicular epidermis. Furthermore, tumor growth and cell proliferation were strongly reduced in mice with an epidermis-specific deletion of Itga3. However, the rate of progression of α3β1-null squamous cell carcinomas to undifferentiated, invasive carcinomas was increased. Therefore, α3β1 critically affects skin carcinogenesis with opposing effects early and late in tumorigenesis.skin cancer | cell adhesion | cell migration | laminin receptor | hair cycling S kin cancer is the most common form of cancer among white populations, with basal cell carcinomas and squamous cell carcinomas (SSCs) being the most common subtypes. Although early detection and surgical resection can prevent most complications associated with this disease, SCCs frequently metastasize and then cannot be effectively treated. Understanding the molecular basis of skin tumorigenesis is a prerequisite for future prevention and therapy. The well-characterized 7,12-dimethylbenzanthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) protocol models the multistep nature of human skin carcinogenesis in mice. Oncogenic mutations (e.g., Hras), induced by a single treatment with the carcinogen DMBA confer growth advantage to the initiated cells, which form benign papillomas under repetitive tumorpromoting treatments with the phorbol ester TPA. Subsequent progression to SCCs involves mutation of Trp53 and trisomization of chromosomes 6 and 7 (1-5).Integrins are αβ heterodimeric adhesion receptors that play an important role in maintaining epithelial integrity. In the skin, the major integrins α2β1, α3β1, and α6β4 connect the cytoskeleton of basal keratinocytes to the underlying basement membrane (6). Besides their key function in skin physiology, these integrins also have been implicated in the development and progression of SCCs (7). Mouse models in which different integrins are either overexpressed in the suprabasal epidermis or mutated in the whole animal showed altered susceptibilities to chemically induced skin tumorigenesis (8-10). Increased expression of α2β1, α3β1, and α6β4 has been observed in hyperproliferating human cancers of the head and neck (11). Integrins thus seem to play a role in initiation and promotion of tumors. Surprisingly, the role of α3β1 in basal keratinocytes in skin tumorigene...

The two NPxY motifs of β1 play distinct and separable roles in controlling the function and trafficking of α5β1. Whereas talin binding to the membrane-proximal NPxY is crucial for connecting α5β1 to the actin cytoskeleton and thus permit the tension required for fibronectin fibrillogenesis and cell migration, kindlin binding to the membrane-distal NPxY is dispensable for these events but regulates α5β1 surface expression and degradation.

Migration of keratinocytes requires a regulated and dynamic turnover of hemidesmosomes (HDs). We and others have previously identified three serine residues on the integrin ␤4 cytoplasmic domain that play a critical role in the regulation of HD disassembly. In this study we show that only two of these residues (Ser-1356 and Ser-1364) are phosphorylated in keratinocytes after stimulation with either PMA or EGF. Furthermore, in direct contrast to previous studies performed in vitro, we found that the PMA-and EGF-stimulated phosphorylation of ␤4 is not mediated by PKC, but by ERK1/2 and its downstream effector kinase p90RSK1/2. EGF-stimulated phosphorylation of ␤4 increased keratinocyte migration, and reduced the number of stable HDs. Furthermore, mutation of the two serines in ␤4 to phospho-mimicking aspartic acid decreased its interaction with the cytoskeletal linker protein plectin, as well as the strength of ␣6␤4-mediated adhesion to laminin-332. During mitotic cell rounding, when the overall cell-substrate area is decreased and the number of HDs is reduced, ␤4 was only phosphorylated on Ser-1356 by a distinct, yet unidentified, kinase. Collectively, these data demonstrate an important role of ␤4 phosphorylation on residues Ser-1356 and Ser-1364 in the formation and/or stability of HDs. Hemidesmosomes (HDs)3 are specialized junctional complexes that mediate firm adhesion of epithelial cells to the underlying basement membrane. Two types of HDs have been characterized: type I and II (1). Type I (classical) HDs are present in squamous and complex epithelia, such as the skin and the bladder. They contain integrin ␣6␤4, plectin, the bullous pemphigoid antigens 180 (BP180) and 230 (BP230), and the tetraspanin CD151 (2). Type II HDs lack BP180 or BP230 and are present in simple epithelia, such as the intestine (3). As the integrin ␣6␤4 binds to Ln-332 in the extracellular matrix (ECM) and associates intracellularly with plectin, which in turn interacts with the keratin filament system, a protein complex is formed that protects the cell against mechanical stress. The importance of this linkage for epidermal-dermal cohesion is substantiated by the finding that in both humans and genetically modified mice, mutations in the genes for these proteins that either prevent their expression or function, result in a skin blistering disorder known as epidermolysis bullosa (2, 4).The primary interaction between plectin and ␤4 occurs through the first pair of fibronectin type III (FnIII) domains and a small part of the connecting segment (CS) of ␤4 and the actin binding domain of plectin (plectin-ABD) (5-7). Indeed, mice carrying a specific deletion of the C-terminal portion of the ␤4 cytoplasmic domain, which still contains the plectin-ABD binding site, can still form normal HDs (8). However, binding of ␤4 to the plectin-ABD is stabilized by adjacent binding sites in the CS and the C-tail of the ␤4 subunit that interact with the plakin domain of plectin (9, 10). In type I HDs, the interaction of ␤4 with plectin is further reinfor...