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.     

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.     

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.     

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.     

Adhesiveness and distribution of vinculin and spectrin in retinal pigmented epithelial cells during growth and differentiation in vitro

Colonies of chick retinal pigmented epithelial (RPE) cells offer an excellent model system for studying the organization of cytoskeleton in sheets of differentiating epithelial cells. The cells occupying the center of the colony resemble RPE cells in vivo and are cuboidal, pigmented, and relatively nonadherent while those toward the periphery gradually become flatter, nonpigmented, motile, and strongly adherent to the substratum. Immunofluorescence microscopy with antiserum against chicken erythrocyte alpha-spectrin reveals that this protein is present in the cortex of RPE cells in all parts of the colony. It is neither concentrated in, nor excluded from the regions occupied by the major microfilament bundles, and its distribution is not related to the adhesion patterns visualized by surface reflection interference microscopy. In contrast, the distribution of vinculin is closely correlated with the adhesiveness of RPE cells in different parts of the colony. Immunofluorescence microscopy reveals that in the RPE cells vinculin may be diffusely distributed in the cytoplasm; present in a cortical band outlining the cell borders; and present in focal contacts and adhesions. The distribution of vinculin is affected by the length of time the colonies grow in culture, by the degree of cell packing and by the adhesiveness of cells to the substratum. In RPE cells grown in vitro for short periods (less than or equal to 3 days) vinculin is found in focal contacts and adhesions in both the undifferentiated, well spread peripheral cells as well as in the differentiated, polygonally packed central cells of the colony. In RPE cells cultured for longer periods (greater than or equal to 14 days) vinculin is present in focal contacts and adhesions only in strongly adherent, undifferentiated cells at the edge of the colony. In packed central cells of both short- and long-term cultures vinculin is found in the cortical band which circumscribes the apical ends of cells at the level of the adherens type intercellular junctions. Its appearance in the cortical bands does not depend on the length of time the colonies are grown in vitro but on the presence of cell-cell contacts resulting from an increased degree of cell packing within the central part of the colony. These results are discussed in relation to the development and the role of extracellular matrix in determining the adhesiveness of RPE cells in vitro.     

Forskolin has biphasic effects on osteoprogenitor cell differentiation in vitro

Cells isolated from fetal rat calvaria (RC) and maintained in vitro in medium containing ascorbic acid and B-glycerophosphate form three-dimensional, mineralized nodules having the histological, immunohistological, and ultrastructural characteristics of woven bone. We have studied the effects of forskolin (FSK), a diterpene that activates adenylate cyclase, in this system. While 10(-7)-10(-5) M FSK significantly stimulated cAMP levels in RC cells, lower concentrations did not. cAMP levels with 10(-5) M FSK reached a maximum by 30 min at 37 degrees C and returned to basal level in 2-3 hr. Changes in cAMP levels correlated with changes in cellular shape: cells treated with 10(-5) M FSK assumed a stellate morphology, lost microfilament bundles, and reduced their substrate adhesiveness, while cells treated with 10(-9) M were not affected. Exponential growth and saturation densities of FSK-treated cultures were similar to untreated cultures, indicating that FSK was neither toxic nor stimulatory to the population. The effect on bone nodule formation of FSK present continuously depended on concentration: 10(-5) M FSK significantly inhibited the number of nodules formed, while 10(-9) M FSK significantly stimulated bone nodule formation. Single short treatments with either 10(-5) M or 10(-9) M FSK had no effect on nodule formation, but repeated short duration treatments (1 hr every 2 days for 21 days) gave results similar to continuous exposure. These results indicate that intermittent elevations in intracellular cAMP have an inhibitory effect on bone formation. In addition, our work indicates that low concentrations of FSK stimulate differentiation of osteoprogenitor cells possibly through a non-cAMP-dependent process.     

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...     

Microtubules, microfilaments and adhesion patterns in differentiating chick retinal pigment epithelial (RPE) cells in vitro

The distribution of microtubules (MT), microfilaments (MF), and patterns of cell-to-substratum adhesion were studied by tubulin antibody labeling, NBD-phallacidin staining and by reflection interference contrast (RIC) microscopy respectively in colonies of differentiating RPE cells obtained from explants after 10 days in culture. In each colony three zones could be identified: a central zone of packed well-differentiated cuboidal cells (zone 1), an intermediate zone of more flattened, pleomorphic cells (zone 2) and a peripheral zone of very spread cells at the edge of the colony (zone 3). As visualized with antibodies to tubulin, the MT distribution in cells of each zone was distinctly different and correlated well with differences in cell shape. Changes in the distribution of MF were more striking. In the cuboidal well-differentiated cells of zone 1, prominent cortical bands but no stress fibers were observed after staining with NBD-phallacidin and RIC microscopy showed that the cells lacked strong adhesion to the substratum. Stress fibers, in addition to cortical bands of MF, were seen in the more spread, less differentiated cells of zone 2 and focal contacts were observed when these cells were examined by RIC microscopy. The flattened least differentiated cells in zone 3 lacked cortical bands but had prominent stress fibers. These cells displayed a variety of adhesion forms ranging from a mosaic of far and close contacts to numerous focal contacts and broad focal adhesions. Our results show that as the RPE cells display less differentiated morphologies, i.e. are more flattened and less densely packed towards the edge of the colony, there is a gradual decrease in the cortical bands of MF and an increase in the number and prominence of stress fibers. This increase in numbers of stress fibers is correlated with an increase in the cell adhesiveness to the substratum, as estimated by RIC microscopy. These results strongly support the general observation that normal epithelial cells in colonies tend to adhere to the substratum more strongly by marginal cells than by the more differentiated centrally located cuboidal cells which have well developed intercellular contacts.