Cell motility and cell shape adaptations are crucial during wound healing, inflammation, and malignant progression. These processes require the remodeling of the keratin cytoskeleton to facilitate cellcell and cell-matrix adhesion. However, the role of keratins for biomechanical properties and invasion of epithelial cells is only partially understood. In this study, we address this issue in murine keratinocytes lacking all keratins on genome engineering. In contrast to predictions, keratin-free cells show about 60% higher cell deformability even for small deformations. This response is compared with the less pronounced softening effects for actin depolymerization induced via latrunculin A. To relate these findings with functional consequences, we use invasion and 3D growth assays. These experiments reveal higher invasiveness of keratin-free cells. Reexpression of a small amount of the keratin pair K5/K14 in keratin-free cells reverses the above phenotype for the invasion but does not with respect to cell deformability. Our data show a unique role of keratins as major players of cell stiffness, influencing invasion with implications for epidermal homeostasis and pathogenesis. This study supports the view that down-regulation of keratins observed during epithelial-mesenchymal transition directly contributes to the migratory and invasive behavior of tumor cells.

The keratin-hemidesmosome interaction is crucial for cell-matrix adhesion and migration in several epithelia including the epidermis. Mutations in constituent proteins cause severe blistering skin disorders by disrupting the adhesion complex. Despite extensive studies, the role of keratins in hemidesmosome assembly and maintenance is only partially understood. Here, we address this issue in keratinocytes in which all keratins are depleted by genome engineering. Unexpectedly, such keratinocytes maintain many characteristics of normal counterparts. The absence of the entire keratin cytoskeleton, however, leads to loss of plectin from the hemidesmosomal plaque and scattering of the hemidesmosome transmembrane core along the basement membrane zone. To investigate the functional consequences, we performed migration and adhesion assays. These revealed that in the absence of keratins, keratinocytes adhere much faster to ECM substrates and migrate ~2 times faster compared to wildtype cells. Re-expression of the single keratin pair K5 and K14 fully reversed the above phenotype. Our data uncover a novel role of keratins in the maintenance of hemidesmosomes upstream of plectin with implications for epidermal homeostasis and pathogenesis. They support the view that the downregulation of keratins observed during epithelial-mesenchymal transition supports the migratory and invasive behavior of tumor cells.

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Low environmental humidity aggravates symptoms of the inflammatory skin disease atopic dermatitis (AD). Using mice that develop AD-like signs, we show that an increase in environmental humidity rescues their cutaneous inflammation and associated epidermal abnormalities. Quantitative proteomics analysis of epidermal lysates of mice kept at low or high humidity identified humidity-regulated proteins, including chloride channel accessory 3A2 (CLCA3A2), a protein with previously unknown function in the skin. The epidermis of patients with AD, organotypic skin cultures under dry conditions, and cultured keratinocytes exposed to hyperosmotic stress showed up-regulation of the nonorthologous human homolog CLCA2. Hyperosmolarity-induced expression occurred via p38/c-Jun N-terminal kinase-activating transcription factor 2 signaling. CLCA2 knockdown promoted keratinocyte apoptosis induced by hyperosmotic stress through impairment of cell-cell adhesion. These findings provide a mechanistic explanation for the beneficial effect of high environmental humidity for AD patients and identify CLCA3A2/CLCA2 up-regulation as a mechanism to protect keratinocytes from damage induced by low humidity.

Epidermal integrity and wound healing depend on remodeling of cell-matrix contacts including hemidesmosomes. Mutations in β4-integrin and plectin lead to severe epidermolysis bullosa (EB). Whether mutations in keratins K5 or K14, which cause EB simplex, also compromise cell-matrix adhesion through altering hemidesmosomal components is not well investigated. In particular, the dependence of β4-integrin endocytosis and turnover on keratins remains incompletely understood. Here, we show that the absence of keratins causes loss of plectin-β4-integrin interaction and elevated β4-integrin phosphorylation at Ser1354 and Ser1362. This triggered a caveolin-dependent endocytosis of β4-integrin but not of other integrins through Rab5 and Rab11 compartments in keratinocytes. Expressing a phospho-deficient β4-integrin mutant reduces β4-integrin endocytosis and rescues plectin localization in keratin-free cells. β4-integrin phosphorylation in the absence of keratins resulted from elevated Erk1/2 activity downstream of increased EGFR and PKCα signaling. Further, increased Erk1/2 phosphorylation and altered plectin localization occur in keratin-deficient mouse epidermis in vivo. Strikingly, expression of the K14-R125P EBS mutant also resulted in plectin mislocalization and elevated β4-integrin turnover, suggesting disease relevance. Our data underscore a major role of keratins in controlling β4-integrin endocytosis involving a plectin-Erk1/2-dependent mechanism relevant for epidermal differentiation and pathogenesis.