Transport of CLCA2 to the nucleus by extracellular vesicles controls keratinocyte survival and migration.

Chloride channel accessory 2 (CLCA2) is a transmembrane protein, which promotes adhesion of keratinocytes and their survival in response to hyperosmotic stress. Here we show that CLCA2 is transported to the nucleus of keratinocytes via extracellular vesicles. The nuclear localization is functionally relevant, since wild-type CLCA2, but not a mutant lacking the nuclear localization signal, suppressed migration of keratinocytes and protected them from hyperosmotic stress-induced cell death. In the nucleus, CLCA2 bound to and activated ß-catenin, resulting in enhanced expression of Wnt target genes. Mass-spectrometry-based interaction screening and functional rescue studies identified RNA binding protein 3 as a key effector of nuclear CLCA2. This is of likely relevance in vivo because both proteins co-localize in the human epidermis. Together, these results identify an unexpected nuclear function of CLCA2 in keratinocytes under homeostatic and stress conditions and suggest a role of extracellular vesicles and their nuclear transport in the control of key cellular activities.

Interaction of the NRF2 and p63 transcription factors promotes keratinocyte proliferation in the epidermis.

Epigenetic regulation of cell and tissue function requires the coordinated action of transcription factors. However, their combinatorial activities during regeneration remain largely unexplored. Here, we discover an unexpected interaction between the cytoprotective transcription factor NRF2 and p63- a key player in epithelial morphogenesis. Chromatin immunoprecipitation combined with sequencing and reporter assays identifies enhancers and promoters that are simultaneously activated by NRF2 and p63 in human keratinocytes. Modeling of p63 and NRF2 binding to nucleosomal DNA suggests their chromatin-assisted interaction. Pharmacological and genetic activation of NRF2 increases NRF2-p63 binding to enhancers and promotes keratinocyte proliferation, which involves the common NRF2-p63 target cyclin-dependent kinase 12. These results unravel a collaborative function of NRF2 and p63 in the control of epidermal renewal and suggest their combined activation as a strategy to promote repair of human skin and other stratified epithelia.

Humidity-regulated CLCA2 protects the epidermis from hyperosmotic stress.

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 CLCA2 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.

Keratins Stabilize Hemidesmosomes through Regulation of ß4-Integrin Turnover.

Epidermal integrity and wound healing depend on remodeling of cell-matrix contacts including hemidesmosomes. Mutations in ß4-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 ß4-integrin endocytosis and turnover on keratins remains incompletely understood. Here, we show that the absence of keratins causes loss of plectin-ß4-integrin interaction and elevated ß4-integrin phosphorylation at Ser1354 and Ser1362. This triggered a caveolin-dependent endocytosis of ß4-integrin but not of other integrins through Rab5 and Rab11 compartments in keratinocytes. Expressing a phospho-deficient ß4-integrin mutant reduces ß4-integrin endocytosis and rescues plectin localization in keratin-free cells. ß4-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 ß4-integrin turnover, suggesting disease relevance. Our data underscore a major role of keratins in controlling ß4-integrin endocytosis involving a plectin-Erk1/2-dependent mechanism relevant for epidermal differentiation and pathogenesis.

Regulation of keratin network organization.

Keratins form the major intermediate filament cytoskeleton of epithelia and are assembled from heterodimers of 28 type I and 26 type II keratins in cell- and differentiation-dependent patterns. By virtue of their primary sequence composition, interactions with cell adhesion complexes and components of major signaling cascades, keratins act as targets and effectors of mechanical force and chemical signals to determine cell mechanics, epithelial cohesion and modulate signaling in keratin isotype-specific manners. Therefore, cell-specific keratin expression and organization impact on cell growth, migration and invasion. Here, we review the recent literature, focusing on the question how keratin networks are regulated and how the interplay of keratins with adhesion complexes affects these processes and provides a framework to understand keratins contribution to blistering and inflammatory disorders and to tumor metastasis.

Keratins significantly contribute to cell stiffness and impact invasive behavior.

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 cell-cell 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.

Keratins mediate localization of hemidesmosomes and repress cell motility.

The keratin (K)-hemidesmosome (HD) 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 HD 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 their normal counterparts. However, the absence of the entire keratin cytoskeleton leads to loss of plectin from the hemidesmosomal plaque and scattering of the HD 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 extracellular matrix substrates and migrate approximately two times faster compared with wild-type cells. Reexpression of the single keratin pair K5 and K14 fully reversed the above phenotype. Our data uncover a role of keratins, which to our knowledge is previously unreported, in the maintenance of HDs 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.