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.

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.

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.

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.

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.

Mouse homologue of skin-specific retroviral-like aspartic protease involved in wrinkle formation.

Retroviral proteases are encoded in the retroviral genome and are responsible for maturation and assembly of infectious virus particles. A number of retroviral protease sequences with retroviral elements are integrated in every eukaryotic genome as endogenous retroviruses. Recently, retroviral-like aspartic proteases that were not embedded within endogenous retroviral elements were identified throughout the eukaryotic and prokaryotic genomes. However, the physiological role of this novel protease family, especially in mammals, is not known. During the high throughput in situ hybridization screening of mouse epidermis, as a granular layer-expressing clone, we identified a mouse homologue of SASPase (Skin ASpartic Protease), a recently identified retroviral-like aspartic protease. We detected and purified the endogenous 32-kDa (mSASP32) and 15-kDa (mSASP15) forms of mSASP from mouse stratum corneum extracts and determined their amino acid sequences. Next, we bacterially produced recombinant mSASP15 via autoprocessing of GST-mSASP32. Purified recombinant mSASP15 cleaved a quenched fluorogenic peptide substrate, designed from the autoprocessing site for mSASP32 maximally at pH 5.77, which is close to the pH of the epidermal surface. Finally, we generated mSASP-deficient mice that at 5 weeks of age showed fine wrinkles that ran parallel on the lateral trunk without apparent epidermal differentiation defects. These results indicate that the retroviral-like aspartic protease, SASPase, is involved in prevention of fine wrinkle formation via activation in a weakly acidic stratum corneum environment. This study provides the first evidence that retroviral-like aspartic protease is functionally important in mammalian tissue organization.

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.

Identification of novel keratinocyte-secreted peptides dermokine-alpha/-beta and a new stratified epithelium-secreted protein gene complex on human chromosome 19q13.1.

We performed high-throughput in situ hybridization screening of sections of mouse epidermis using an equalized skin cDNA library as probes and identified a novel gene giving rise to two splicing variants, both of which are expressed in the spinous layer. This gene was mapped between two genes encoding keratinocyte-related peptides, suprabasin and keratinocyte differentiation-associated protein (Kdap), on human chromosome 19q13.1. These gene products appeared to carry functional signal sequences. We then designated these two splicing variants as dermokine-alpha and -beta. Northern blotting and quantitative RT-PCR revealed that dermokine-alpha/-beta, suprabasin, and Kdap were highly expressed in stratified epithelia. In mouse embryonic development, dermokine-alpha/-beta began to be expressed during the period of stratification. Also, in differentiating primary cultured human keratinocytes, transcription of dermokine-alpha/-beta, suprabasin, and Kdap was induced. These findings indicated that dermokine-alpha/-beta, suprabasin, and Kdap are secreted from the spinous layer of the stratified epithelia and that these genes form a novel gene complex on the chromosome.