Tricellulin and its role in the epididymal epithelium of the rat.

Tricellulin is a tight-junction protein present at tricellular tight junctions. It has been suggested that basal cells are implicated in the blood-epididymis barrier. Basal cells express claudins, a component of tight junctions; however, there is no information regarding the potential architecture or regulation of basal cell-principal cell interactions. The present objectives were to determine the expression and localization of tricellulin in rat epididymis in relation to occludin, basal cell-principal cell interactions, and other junctional proteins. Tricellulin levels were similar in all segments of the adult epididymis, and the protein was localized to the apical region of the epithelium. Postnatal development showed that tricellulin levels increased with age and localization changed from cytoplasmic to membrane-bound as a function of age. Colocalization with occludin indicated that both proteins are in the region of the tight junction. In the initial segment, the proteins did not colocalize compared to the epididymis where they were both colocalized. Tricellulin did not colocalize with cytokeratin 5, a marker of basal cells, in any region of the epididymis, including the corpus and cauda epididymidis, where apical projections of basal cells were apparent. Tricellulin knockdown studies using small interfering RNA in rat caput epididymal principal cells resulted in decreased transepithelial resistance and was correlated with decreased levels of Cldn3, Cldn1, and occludin. Tight-junction protein1, also known as ZO-1, and cadherin1 levels were unchanged. This is the first report of tricellulin in the epididymis and on the interaction between tricellulin and other tight-junction proteins.

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.

Defining keratin protein function in skin epithelia: epidermolysis bullosa simplex and its aftermath.

Epidermolysis bullosa simplex (EBS) is a rare genetic condition typified by superficial bullous lesions following incident frictional trauma to the skin. Most cases of EBS are due to dominantly acting mutations in keratin 14 (K14) or K5, the type I and II intermediate filament (IF) proteins that copolymerize to form a pancytoplasmic network of 10 nm filaments in basal keratinocytes of epidermis and related epithelia. Defects in K5-K14 filament network architecture cause basal keratinocytes to become fragile, and account for their rupture upon exposure to mechanical trauma. The discovery of the etiology and pathophysiology of EBS was intimately linked to the quest for an understanding of the properties and function of keratin filaments in skin epithelia. Since then, continued cross-fertilization between basic science efforts and clinical endeavors has highlighted several additional functional roles for keratin proteins in the skin, suggested new avenues for effective therapies for keratin-based diseases, and expanded our understanding of the remarkable properties of the skin as an organ system.

Epidermolysis bullosa simplex: a paradigm for disorders of tissue fragility.

Epidermolysis bullosa (EB) simplex is a rare genetic condition typified by superficial bullous lesions that result from frictional trauma to the skin. Most cases are due to dominantly acting mutations in either keratin 14 (K14) or K5, the type I and II intermediate filament (IF) proteins tasked with forming a pancytoplasmic network of 10-nm filaments in basal keratinocytes of the epidermis and in other stratified epithelia. Defects in K5/K14 filament network architecture cause basal keratinocytes to become fragile and account for their trauma-induced rupture. Here we review how laboratory investigations centered on keratin biology have deepened our understanding of the etiology and pathophysiology of EB simplex and revealed novel avenues for its therapy.

Base behavior behind budding breasts: integrins and mammary stem cell activity.

In a recent Nature Cell Biology paper, Taddei et al. (2008) reveal that deletion of beta1 integrin from K5-expressing mammary epithelial basal cells specifically attenuates ductal stem cell activity, without dramatically altering the basal cell layer, or morphogenesis overall.

Induction of inflammatory cytokines by a keratin mutation and their repression by a small molecule in a mouse model for EBS.

Epidermolysis bullosa simplex (EBS) is a skin disorder caused by mutations in keratin (K) 5 or K14 genes. It is widely regarded as a mechanobullous disease, resulting from a weakened cytoskeleton, causing extensive cytolysis. It was postulated by others that certain K14 mutations induce tumor necrosis factor-alpha (TNF-alpha) and increase apoptosis. Here, we report that in K5-/- mice and in a cell culture model of EBS, the mRNA and protein levels of TNF-alpha remain unaltered. Transcriptome analysis of K5-/- mice revealed, however, that the proinflammatory cytokines IL-6 and IL-1beta were significantly upregulated at the mRNA level in K5-/- mouse skin. These results were confirmed by TaqMan real-time PCR and ELISA assays. We hypothesize that keratin mutations contribute to EBS in a mouse model by inducing local inflammation that mediates a stress response. Following clinical reports, we applied the small molecule doxycycline to K5-/- mice. We demonstrate that doxycycline extended the survival of neonatal K5-/- mice from less than 1 to up to 8 hours. Microarray and TaqMan real-time PCR showed a downregulation of matrix metalloproteinase 13 and IL-1beta, indicating an effect of doxycycline on transcription. Our data offer a novel small molecule-based therapy approach for EBS.

Homozygous K5Cre transgenic mice have wavy hair and accelerated malignant progression in a murine model of skin carcinogenesis.

Mice with conditional gene deletions have been extremely valuable in allowing investigators to study the genes of interest in a tissue-specific manner. The Cre-loxP recombination system provides a powerful tool to produce targeted rearrangements of particular genes. The keratin 5-Cre recombinase (K5Cre) transgenic mouse line has been used to generate skin specific gene deletions. We found that the K5Cre mice display a unique phenotype when bred to homozygosity. The K5Cre(+/+) mice have a wavy hair coat and curly whiskers. Histologically, the hair follicles appear disoriented. Over time, the K5Cre(+/+) mice develop patches of alopecia. These mice are also runted when compared to wild-type controls. Fostering the K5Cre(+/+) pups to wild-type mothers results in normal weight gain, suggesting a maternal defect in milk production. When the K5Cre(+/+) mammary glands were examined, we not only found a significant decrease in the number of mammary branches in the virgin females, but also a greater number of quiescent alveoli units in the lactating glands. When the K5Cre(+/+) mice were bred to v-Ha-ras (Tg . AC) transgenic mice, the resulting Tg . AC(+/-) K5Cre(+/+) offspring were utilized in a chemically induced skin carcinogenesis model. The mice were treated with 2.5 microg of 12-O-tetradecanoylphorbol-13-acetate (TPA) weekly for 10 wk. No difference was observed in the time to onset of papilloma formation, the number of papillomas and the average papilloma volume between the Tg . AC(+/-) K5Cre(+/+) mice and their corresponding controls. Surprisingly, however, the K5Cre(+/+) papillomas displayed an accelerated tendency to malignant progression; in addition, the frequency of malignant transformation of the papillomas is significantly enhanced. Although the K5Cre(+/+) mice resemble waved-1 and -2 mutants, the molecular basis for the K5Cre(+/+) phenotype is probably different. In conclusion, we discovered a unique phenotype associated with the K5Cre(+/+) transgenic line.

Matrix metalloproteinase-19 expression in keratinocytes is repressed by transcription factors Tst-1 and Skn-1a: implications for keratinocyte differentiation.

Matrix metalloproteinase-19 (MMP-19), unlike other members of the MMP family, is expressed in basal keratinocytes of intact epidermis whereas keratinocytes in suprabasal and higher epidermal layers express this enzyme only during cutaneous disorders. As the activity of MMP-19 effects proliferation, migration, and adhesion of keratinocytes we examined whether transcription factors involved in keratinocyte differentiation repress the expression of MMP-19. Using luciferase reporter assays, POU transcription factors Tst-1 (Oct-6) and Skn-1a (Oct-11) markedly downregulated the activity of MMP-19 promoter in COS-7 cells and HaCaT keratinocytes. Tst-1 alone was able to inhibit 85% of the promoter activity. Skn-1a exhibited a weak inhibitory effect although it synergistically increased effects of Tst-1. HaCaT cells stably transfected with Tst-1 showed a strong decrease of activity of MMP-19 promoter that correlated with suppression of MMP-19, cytokeratin 14 and 5, decreased cell proliferation, and altered expression of involucrin and loricrin. The expression of MMP-9 was also significantly reduced in Tst-1 expressing keratinocytes. MMP-2 was substantially affected during its activation whereas the expression of MMP-28 was unchanged. Our results suggest that Tst-1 and Skn-1a regulate expression of MMPs in keratinocytes and effect both the expression and activation of these proteolytic enzymes.