Tight junctions in epidermis: from barrier to keratinization.

Tight junctions (TJs) are one type of intercellular junction. TJs prevent diffusion of solutes through the intercellular spaces in simple and stratified epithelia. Mice lacking claudin-1 (an adhesion molecule at TJs) show dehydration from the skin by impaired barrier function of epidermal TJs. Meanwhile, a human hereditary disease with ichthyosis (NISCH syndrome) has been found to be a claudin-1-deficient disease. The two models of lacking-claudin-1 indicate that TJs are related not only to the epidermal barrier but also to keratinization. Research of TJs in epidermis is reviewed in view of the barrier to keratinization.

Tight junctions in Schwann cells of peripheral myelinated axons: a lesson from claudin-19-deficient mice.

Tight junction (TJ)-like structures have been reported in Schwann cells, but their molecular composition and physiological function remain elusive. We found that claudin-19, a novel member of the claudin family (TJ adhesion molecules in epithelia), constituted these structures. Claudin-19-deficient mice were generated, and they exhibited behavioral abnormalities that could be attributed to peripheral nervous system deficits. Electrophysiological analyses showed that the claudin-19 deficiency affected the nerve conduction of peripheral myelinated fibers. Interestingly, the overall morphology of Schwann cells lacking claudin-19 expression appeared to be normal not only in the internodal region but also at the node of Ranvier, except that TJs completely disappeared, at least from the outer/inner mesaxons. These findings have indicated that, similar to epithelial cells, Schwann cells also bear claudin-based TJs, and they have also suggested that these TJs are not involved in the polarized morphogenesis but are involved in the electrophysiological "sealing" function of Schwann cells.

Expression of CCR5 in graft-versus-host disease (GVHD) of the skin: immunohistochemical staining of 38 cases.

To ascertain the involvement of CCR5 in prolongation of graft-versus-host disease (GVHD), we performed immunohistochemical staining of CCR5 in 38 GVHD samples (23 acute and 15 chronic). A total of seven out of 15 cases of chronic GVHD were positive for CCR5; however, only two out of 23 in acute GVHD were positive for CCR5. In three cases, expression of CCR5 in infiltrating lymphocytes was negative in the acute phase, but positive in the chronic phase of GVHD. These findings suggest that the immunopathological mechanism that differentiates between acute and chronic GVHD is a CCR5-mediated immunoreaction.

HAN11 binds mDia1 and controls GLI1 transcriptional activity.

BACKGROUND: The Hedgehog pathway is important in normal and diseased skin. One of the key transcription factors in the pathway is GLI1. GLI1-dependent transcription is positively regulated by DYRK1A, which is reported to bind HAN11. HAN11 is the human homologue of AN11, which controls flavonoid synthesis in plants. OBJECTIVE: We wanted to identify other binding partners of HAN11 and investigate whether HAN11 regulates GLI1-dependent transcription. METHODS: We used TAP-tag purification and GST pull down to identify protein-protein interactions and performed luciferase assays of transcriptional activity. We used immunofluorescence microscopy to examine the subcellular distribution of HAN11, mDia1 and GLI1. We performed in situ hybridisation to compare expression of HAN11 with GLI1 and patched in mouse embryos. RESULTS: We identified the cytoskeletal regulator mDia1 as a binding partner of HAN11. We showed that HAN11 binds the FH2 actin binding domain of mDia1 and confirmed that HAN11 also interacts with DYRK1A. Overexpression of mDia1 or active RhoA caused translocation of HAN11 from nucleus to cytoplasm. HAN11 and mDia1 repressed DYRK1A-dependent GLI1 transcriptional activity. HAN11 overexpression decreased SZ95 sebocyte proliferation and increased cytoplasmic GLI1. AN11 was highly expressed in E10.5 mouse embryo limb buds, in an overlapping pattern with Ptc and GLI1. CONCLUSION: These results suggest that AN11 may be a physiological regulator of GLI1 transcriptional activity.

Molecular architecture of tight junctions of periderm differs from that of the maculae occludentes of epidermis.

Occludin and claudins are tetraspan-transmembrane proteins in tight junctions. Maculae occludentes, which are less-developed tight junctions, occur in the granular cell layer of the epidermis. The periderm, which overlies the developing epidermis and functions as a protective layer for the embryo, carries developed tight junctions as observed in simple epithelia. In both periderm and epidermis, occludin is expressed at the cell-cell border. To determine the difference between tight junctions of periderm and epidermis, claudin-6 expression was examined in periderm and epidermis. Immunofluorescence staining showed claudin-6 expression at the cell-cell border of the periderm, but not in the epidermis. Reverse transcription-polymerase chain reaction confirmed that claudin-6 was not expressed in mouse adult skin, whereas immunoelectron microscopy revealed that claudin-6 was localized at tight junctions of the periderm. Furthermore, L fibroblasts with stable expression of exogenous claudin-6 formed developed tight junctions at cell-cell borders. These findings indicate that molecular architecture of tight junctions of the periderm is different from that of the maculae occludentes of the epidermis, and that claudin-6 is important in the formation of tight junctions of the periderm.

Tight junctions in the skin.

Tight junctions (=zonulae occludentes, TJs) function as an effective barrier in simple epithelia. Recent developments in the molecular biology of TJs revealed that TJs also exist in the stratum granulosum and contribute to barrier function in epidermis. Furthermore, several TJ-related junctions were identified in epidermis. In this review article, the history of the investigation into TJs in epidermis and the perspectives of investigation into TJs in dermatology are described.

Expression patterns of claudins, tight junction adhesion molecules, in the inner ear.

Tight junctions (TJs) are indispensable for the establishment of compositionally distinct fluid compartments in the inner ear, but our knowledge of the claudins, TJ adhesion molecules, in the inner ear is still fragmentary. We examined the expression and distribution of claudin-1 to claudin-18 (except for claudin-7, -13 and -17) in the inner ear by immunofluorescence microscopy. In the cochlea, the organ of Corti expressed claudin-1, -2, -3, -9, -10, -12, -14 and -18. In the stria vascularis, claudin-1, -2, -3, -8, -9, -10, -12, -14 and -18 were expressed in the marginal cells, whereas the basal cells were positive only for claudin-11. In Reissner's membrane and the spiral limbus, the expression of claudin-1, -2, -3, -8, -9, -10, -12, -14 and -18 was detected. Furthermore, in the vestibule, claudin-1, -3, -9, -12, -14 and -18 were expressed in the sensory epithelia, whereas in the dark cell area claudin-1, -3, -8, -9, -12, -14 and -18 were detectable. These findings, i.e., very complex expression patterns of claudin species in the inner ear, would reflect the importance and the complexity of the barrier function of TJs in the inner ear.

Seventeen cases of alopecia areata: combination of SADBE topical immunotherapy with other therapies.

Topical immunotherapy is effective for severe alopecia areata. However, there are patients with alopecia areata refractory to topical immunotherapy alone. We tried SADBE (squaric acid dibutylester) topical immunotherapy combined with topical dry ice cryotherapy, carpronium chloride (a parasympathetic nerve stimulant) and/or oral cepharanthin (a biscoclaur alkaloid) in alopecia areata refractory to topical SADBE. Seventeen patients with alopecia areata (3 multiple, 3 ophiasis, 5 totalis and 6 universalis) were treated with SADBE in our department in 1999 to 2001. In 3 cases (2 multiple and 1 universalis) out of the 17 cases, cosmetically acceptable regrowth of hair was observed in several months with topical SADBE alone. In the other 14 cases, the SADBE therapy alone for several months (mean: 6.9 months) resulted in no or poor regrowth of hair. However, with subsequent combination therapy of topical SADBE for several months (mean: 7.6 months), satisfactory regrowth of hair was observed in 6 of the 14 cases. Our cases indicate that combination therapy of topical SADBE with other therapies can be a choice for alopecia areata which is refractory to topical SADBE therapy alone.

A case of chronic perianal pyoderma treated with the recycled skin graft method.

We present here a case of chronic perianal pyoderma (CPP), who was treated with a novel skin graft. The patient was a 28-year-old male who had suffered from painful abscesses and nodules on his buttocks for over 10 years. Although the abscesses and nodules were initially restricted to one buttock, they gradually spread over both buttocks. He visited our hospital in July of 2000. He was clinically diagnosed as CPP and treated with oral doses of antibiotics. When admitted to our hospital in September of 2000, an anal fistula was also found. The operations for CPP and anal fistula were simultaneously performed. After the anal fistula treatment, the lesions of CPP were totally resected. In this procedure, we removed the epidermis and upper dermis from the excised CPP lesions and grafted them on the defects of the excised lesion. There has been no recurrence of the CPP. This operation procedure, which we call the "recycled skin graft method" is less invasive, because a donor site for the skin graft is unnecessary. We considered it to be as one of the effective treatments for CPP.

Characterization of tight junctions and their disruption by UVB in human epidermis and cultured keratinocytes.

It has not been confirmed whether tight junctions (TJs) function as a paracellular permeability barrier in adult human skin. To clarify this issue, we performed a TJ permeability assay using human skin obtained from abdominal plastic surgery. Occludin, a marker protein of TJs, was expressed in the granular layer, in which a subcutaneously injected paracellular tracer, Sulfo-NHS-LC-Biotin (556.59 Da), was halted. Incubation with ochratoxin A decreased the expression of claudin-4, an integral membrane protein of TJs, and the diffusion of paracellular tracer was no longer prevented at the TJs. These results demonstrate that human epidermis possesses TJs that function as an intercellular permeability barrier at least against small molecules (∼550 Da). UVB irradiation of human skin xenografts and human skin equivalents (HSEs) resulted in functional deterioration of TJs. Immunocytochemical staining of cultured keratinocytes showed that occludin was localized into dot-like shapes and formed a discontinuous network when exposed to UVB irradiation. Furthermore, UVB irradiation downregulated the active forms of Rac1 and atypical protein kinase C, suggesting that their inactivation caused functional deterioration of TJs. In conclusion, TJs function as a paracellular barrier against small molecules (∼550 Da) in human epidermis and are functionally deteriorated by UVB irradiation.

An activating beta1 integrin mutation increases the conversion of benign to malignant skin tumors.

Identifying the physiologic relevance of cancer-associated genetic polymorphisms is a major challenge. Several changes in the coding sequence of beta integrin subunits have now been described in human tumors. One of these, T188Ibeta1, was identified as a heterozygous mutation in a poorly differentiated squamous cell carcinoma (SCC) and shown to activate extracellular matrix adhesion and inhibit keratinocyte differentiation in vitro. To study its contribution to tumor development, we overexpressed the mutant or wild-type (WT) human beta1 subunit in the basal layer of mouse epidermis using the keratin 14 promoter. The transgenic integrins were expressed at the cell surface and were functional, with the T188Ibeta1 subunit promoting cell spreading to a greater extent than WTbeta1. Epidermal proliferation and differentiation were unaffected and no expansion of the stem cell compartment was detected. During chemical carcinogenesis, both transgenes increased papilloma formation, but only the T188Ibeta1 transgene stimulated the conversion of papillomas to SCCs. Papillomas bearing the mutation showed increased Erk activity and reduced differentiation. SCCs expressing T188Ibeta1 were less well-differentiated than those expressing WTbeta1. These observations establish that the expression of a genetic variant in the I-like domain of beta1 integrins does not affect normal epidermal homeostasis, but increases tumor susceptibility and influences tumor type.

Tight junction proteins in keratinocytes: localization and contribution to barrier function.

Recent research suggests that tight junctions (TJs) are located in the stratum granulosum, where they contribute to the barrier function of the epidermis. In this study, we investigated the formation of functional TJs in cultured normal human epidermal keratinocytes. We observed the development of permeability barrier function through the process of Ca(2+)-induced differentiation. Immunofluorescence analyses at 96 h after Ca(2+)-induced differentiation revealed concentrated portions of occludin, a TJ-specific marker, arranged as continuous lines circumscribing individual flattened suprabasal cells in areas with high concentrations of claudin-1 and -4. Transient Ca(2+) depletion reversibly disrupted the continuous network of TJ proteins and the permeability barrier. We also found that the addition of ochratoxin A weakened the permeability barrier and the expression of claudin-4. Our findings suggest that TJ proteins contribute to the permeability barrier in epidermal keratinocytes.