Delayed epidermal permeability barrier formation and hair follicle aberrations in Inv-Cldn6 mice

Homozygous mice overexpressing Claudin-6 (Cldn6) exhibit a perturbation in the epidermal differentiation program leading to a defective epidermal permeability barrier (EPB) and dehydration induced death ensuing within 48 h of birth [Turksen, K., Troy, T.C., 2002. Permeability barrier dysfunction in transgenic mice overexpressing claudin 6. Development 129, 1775-1784]. Their heterozygous counterparts are also born with an incomplete EPB; however, barrier formation continues after birth and normal hydration levels are achieved by postnatal day 12 allowing survival into adulthood. Heterozygous Inv-Cldn6 mice exhibit a distinct coat phenotype and histological analysis shows mild epidermal hyperkeratosis. Expression of K5 and K14 is aberrant, extending beyond the basal layer into the suprabasal layer where they are not co-localized suggesting that their expression is uncoupled. There is also atypical K17 and patchy K15 expression in the basal layer with no K6 expression in the interfollicular epidermis; together with marked changes in late differentiation markers (e.g. profilaggrin/filaggrin, loricrin, transglutaminase 3) indicating that the normal epidermal differentiation program is modified. The expression compartment of various Cldns is also perturbed although overall protein levels remained comparable. Most notably induction of Cldn5 and Cldn8 was observed in the Inv-Cldn6 epidermis. Heterozygous Inv-Cldn6 animals also exhibit subtle alterations in the differentiation program of the hair follicle including a shorter anagen phase, and altered hair type distribution and length compared to the wild type; the approximately 20% increase in zig-zag hair fibers at the expense of guard hairs and the approximately 30% shorter guard hairs contribute to coat abnormalities in the heterozygous mice. In addition, the transgenic hair follicles exhibit a decreased expression of K15 as well as some hair-specific keratins and express Cldn5 and Cldn18, which are not detectable in the wild type. These data indicate that Cldn6 plays a role in the differentiation processes of the epidermis and hair follicle and supports the notion of a link between Cldn regulation and EPB assembly/maintenance as well as the hair cycle.     

Broad yet high substrate specificity: the challenge of AAA+ proteins

AAA+ proteins remodel target substrates in an ATP-dependent manner, an activity that is of central importance for a plethora of cellular processes. While sharing a similar hexameric structure AAA+ proteins must exhibit differences in substrate recognition to fulfil their diverse biological functions. Here we describe strategies of AAA+ proteins to ensure substrate specificity. AAA domains can directly mediate substrate recognition, however, in general extra domains, added to the core AAA domain, control substrate interaction. Such extra domains may either directly recognize substrates or serve as a platform for adaptor proteins, which transfer bound substrates to their AAA+ partner proteins. The positioning of adaptor proteins in substrate recognition can enable them to control the activity of their partner proteins by coupling AAA+ protein activation to substrate availability.     

Overexpression of the calcium sensing receptor accelerates epidermal differentiation and permeability barrier formation in vivo

The calcium sensing receptor (CaSR) has emerged as an important mediator of a wide range of Ca(2+)-dependent physiological responses (Ca(2+) signaling) in various tissues. To explore the role of CaSR in the epidermis, we utilised the keratin 14 promoter to express CaSR cDNA constitutively in the basal cells of the stratified squamous epithelium of transgenic mice. Analysis of the transgenic mice revealed that a sensitized response to CaSR signaling accelerates the epidermal differentiation program with the precocious formation of the epidermal permeability barrier (EPB) during development and an accelerated hair growth at birth. Our observations indicate that overexpression of CaSR in the undifferentiated basal cells leads to changes in the differentiation program of the transgenic epidermis, including the stimulation of keratins 1 and 6 as well as the overexpression of several markers of terminal differentiation such as filaggrin, loricrin and involucrin. Our data suggest that the observed modifications in the differentiation pathway are a consequence of a CaSR-induced enhancement of Ca(2+) signaling involving cross-talk with other signaling pathways (e.g. EGF and Wnt/Ca(2+)). These studies provide new insights into the role of CaSR in epidermal differentiation including EPB development and hair follicle morphogenesis.     

Claudin immunolocalization in neonatal mouse epithelial tissues

Emerging evidence supports the notion that claudins (Cldns) are dynamically regulated under normal conditions to respond to the selective permeability requirements of various tissues, and that their expression is developmentally controlled. We describe the localization of those Cldns that we have previously demonstrated to be functionally important in epidermal differentiation and the formation of the epidermal permeability barrier, e.g., Cldn1, Cldn6, Cldn11, and Cldn18, and the presence of Cldn3 and Cldn5 in various neonatal mouse epithelia including the epidermis, nail, oral mucosa, tongue, and stomach. Cldn1 is localized in the differentiated and/or undifferentiated compartments of the epidermis and nail and in the dorsal surface of the tongue and glandular compartment of the stomach but is absent from the oral mucosa and the keratinized compartment of the stomach. Cldn3 is present in the basal cells of the nail matrix and both compartments of the murine stomach but not in the epidermis, oral mucosa, or tongue. Cldn5 is found in the glandular compartment of the stomach but not in the epidermis, nail unit, oral mucosa, forestomach, and tongue. Cldn6, Cldn11, and Cldn18 occur in the differentiating suprabasal compartment of the epidermis, nail, and oral mucosa and in the dorsal and ventral surfaces of the tongue and the keratinized squamous epithelium of the stomach. The simple columnar epithelium of the glandular stomach stains for Cldn18 and reveals a non-membranous pattern for Cldn6 and Cldn11 expression. Our results demonstrate differential Cldn protein profiles in various epithelial tissues and their differentiation stages. Although the molecular mechanisms regulating Cldn expression are unknown, elucidation of their differential localization patterns in tissues with diverse permeability requirements should provide a better understanding of the role of tight junctions in tissue function. This work was supported by a research grant from the Canadian Institutes of Health Research (MOP-69087).     

Junctions gone bad: claudins and loss of the barrier in cancer

The tight junctions (TJs) of epithelia are responsible for regulating the "fence and gate" function of polarized epithelial cells. It is now well-established that dysregulation of these functions contributes to initiation and progression of cancer. Recently, it has become clear that the Claudins, members of a large family of 27 closely related transmembrane proteins, play a crucial role in formation, integrity and function of TJs, the epithelial permeability barrier and epithelial polarization. A growing body of data indicates that Claudin expression is altered in numerous epithelial cancers in a stage- and tumor-specific manner. While a single universal mechanism is still lacking, accumulating evidence supports a role for epigenetic regulation of Claudin expression in tumorgenesis, with concomitant alterations in barrier function. We review here new insights and challenges in understanding Claudin function in normal physiology and cancer.     

Insights into the Role of the Calcium Sensing Receptor in Epidermal Differentiation in Vivo

While the important role of calcium (Ca⁺⁺) signaling is fundamental in epidermal cell physiology, a detailed knowledge of precisely how epidermal cells respond to Ca⁺⁺ levels is not clear. Using peptide-specific antibodies that we generated, we set out to evaluate the temporal and spatial distribution pattern of the Ca⁺⁺-sensing receptor (CaSR) during epidermogenesis and to assess its involvement in the mature epidermis (e.g., in acute injury and tumorigenesis). Our data indicate a developmentally regulated expression of CaSR: up-regulation occurs in specific epidermal cells and cell layers in normal development or in response to injury when epidermal cells are induced to undergo commitment and early differentiation events, and down-regulation occurs in terminal differentiation stages. These results provide a new perspective on the role of the CaSR in these processes and describe a novel tool for evaluating Ca⁺⁺-mediated epidermal differentiation.     

Revisiting the bulge

Two recent papers--one from the group of Elaine Fuchs in Science and the other from the group of George Costarelis in Nature Biotechnology--on the isolation and multipotentiality of adult hair follicle stem cells catapult us ahead in our understanding of epidermal and hair follicle lineages.     

The targeted overexpression of a Claudin mutant in the epidermis of transgenic mice elicits striking epidermal and hair follicle abnormalities

Skin is one of the largest organs of the body, and is formed during development through a highly orchestrated process involving mesenchymal-epithelial interactions, cell commitment, and terminal differentiation. It protects against microorganism invasion and UV irradiation, inhibits water loss, regulates body temperature, and is an important part of the immune system. Using transgenic mouse technology, we have demonstrated that Claudin (Cldn)-containing tight junctions (TJs) are intricately involved in cell signaling during epidermal differentiation and that an epidermal suprabasal overexpression of Cldn6 results in a perturbed epidermal terminal differentiation program with distinct phenotypic abnormalities. To delineate the role of the Cldn cytoplasmic tail domain in epidermal differentiation, we engineered transgenic mice targeting the overexpression of a Cldn6 cytoplasmic tail-truncation mutant in the epidermis. Transgenic mice were characterized by a lethal barrier dysfunction in addition to the existence of hyperproliferative squamous invaginations/cysts replacing hair follicles. Immunohistochemical analysis revealed an epidermal cytoplasmic accumulation of Cldn6, Cldn11, Cldn12, and Cldn18, downregulation of Cldn1 and aberrant expression of various classical markers of epidermal differentiation; namely the basal keratins as well as K1, involucrin, loricrin, and filaggrin. Collectively these studies suggest an important role for Cldns in epidermal/hair follicle differentiation programs likely involving cross talk to signaling pathways (e.g., Notch) directing cell fate selection and differentiation.     

The molecular basis and genotype–phenotype correlations of congenital adrenal hyperplasia (CAH) in Anatolian population

Molecular Biology Reports - Congenital adrenal hyperplasia (CAH) is an autosomal recessive genetic disorder due to presence of mutations in the genes involved in the metabolism of steroid hormones...