Akiharu Kubo

Claudin-based tight junctions are crucial for the mammalian epidermal barrier: a lesson from claudin-1–deficient mice

The tight junction (TJ) and its adhesion molecules, claudins, are responsible for the barrier function of simple epithelia, but TJs have not been thought to play an important role in the barrier function of mammalian stratified epithelia, including the epidermis. Here we generated claudin-1–deficient mice and found that the animals died within 1 d of birth with wrinkled skin. Dehydration assay and transepidermal water loss measurements revealed that in these mice the epidermal barrier was severely affected, although the layered organization of keratinocytes appeared to be normal. These unexpected findings prompted us to reexamine TJs in the epidermis of wild-type mice. Close inspection by immunofluorescence microscopy with an antioccludin monoclonal antibody, a TJ-specific marker, identified continuous TJs in the stratum granulosum, where claudin-1 and -4 were concentrated. The occurrence of TJs was also confirmed by ultrathin section EM. In claudin-1–deficient mice, claudin-1 appeared to have simply been removed from these TJs, leaving occludin-positive (and also claudin-4–positive) TJs. Interestingly, in the wild-type epidermis these occludin-positive TJs efficiently prevented the diffusion of subcutaneously injected tracer (∼600 D) toward the skin surface, whereas in the claudin-1–deficient epidermis the tracer appeared to pass through these TJs. These findings provide the first evidence that continuous claudin-based TJs occur in the epidermis and that these TJs are crucial for the barrier function of the mammalian skin.

A homozygous frameshift mutation in the mouse Flg gene facilitates enhanced percutaneous allergen priming

Loss-of-function mutations in the FLG (filaggrin) gene cause the semidominant keratinizing disorder ichthyosis vulgaris and convey major genetic risk for atopic dermatitis (eczema), eczema-associated asthma and other allergic phenotypes. Several low-frequency FLG null alleles occur in Europeans and Asians, with a cumulative frequency of ∼9% in Europe. Here we report a 1-bp deletion mutation, 5303delA, analogous to common human FLG mutations, within the murine Flg gene in the spontaneous mouse mutant flaky tail (ft). We demonstrate that topical application of allergen to mice homozygous for this mutation results in cutaneous inflammatory infiltrates and enhanced cutaneous allergen priming with development of allergen-specific antibody responses. These data validate flaky tail as a useful model of filaggrin deficiency and provide experimental evidence for the hypothesis that antigen transfer through a defective epidermal barrier is a key mechanism underlying elevated IgE sensitization and initiation of cutaneous inflammation in humans with filaggrin-related atopic disease.

Odf2-deficient mother centrioles lack distal/subdistal appendages and the ability to generate primary cilia

Outer dense fibre 2 (Odf2; also known as cenexin) was initially identified as a main component of the sperm tail cytoskeleton, but was later shown to be a general scaffold protein that is specifically localized at the distal/subdistal appendages of mother centrioles. Here we show that Odf2 expression is suppressed in mouse F9 cells when both alleles of Odf2 genes are deleted. Unexpectedly, the cell cycle of Odf2−/− cells does not seem to be affected. Immunofluorescence and ultrathin-section electron microscopy reveals that in Odf2−/− cells, distal/subdistal appendages disappear from mother centrioles, making it difficult to distinguish mother from daughter centrioles. In Odf2−/− cells, however, the formation of primary cilia is completely suppressed, although ∼25% of wild-type F9 cells are ciliated under the steady-state cell cycle. The loss of primary cilia in Odf2−/− F9 cells can be rescued by exogenous Odf2 expression. These findings indicate that Odf2 is indispensable for the formation of distal/subdistal appendages and the generation of primary cilia, but not for other cell-cycle-related centriolar functions.

A peculiar internalization of claudins, tight junction-specific adhesion molecules, during the intercellular movement of epithelial cells.

Tight junctions (TJs) seal the intercellular space of epithelial cells, while individual epithelial cells move against adjacent cells in cellular sheets. To observe TJs in live epithelial cellular sheets, green fluorescent protein (GFP) was fused to the N-terminus of claudin-3 (a major cell adhesion molecule of TJs), which was stably expressed at a level that was approximately 50% of that of endogenous claudin-3 in mouse Eph4 epithelial cells. Under confluent culture conditions, individual cells moved within cellular sheets, which was associated with the remodeling of TJs. However, during this remodeling, GFP-positive TJs did not lose their structural continuity. When TJs between two adjacent cells decreased in length during this remodeling, GFP-claudin-3 was frequently pinched off as a granular structure from GFP-positive TJs together with endogenous claudins. Co-culture experiments, as well as electron microscopy, revealed that the two apposed membranes of TJs were not detached, but co-endocytosed into one of the adjacent cells. Interestingly, other TJ components such as occludin, JAM and ZO-1 appeared to be dissociated from claudins before this endocytosis. The endocytosis of claudins was facilitated when the intercellular motility was upregulated by wounding the cellular sheets. These findings suggest that this peculiar internalization of claudins plays a crucial role in the remodeling of TJs, and that the fine regulation of this endocytosis is important for TJs to seal the intercellular space of epithelial cells that are moving against adjacent cells within cellular sheets.

Skin Barrier Disruption: A Requirement for Allergen Sensitization?

For at least half a century, noninvasive techniques have been available to quantify skin barrier function, and these have shown that a number of human skin conditions and disorders are associated with defects in skin permeability. In the last decade, several genes responsible for skin barrier defects observed in both monogenetic and complex, polygenic disorders have been elucidated and functionally characterized. This has led to an explosion of work in the last six years that has identified pathways connecting epidermal barrier disruption and antigen uptake as well as the quality and/or magnitude of the antigen-specific adaptive immune response. This review will introduce the notion that diseases arise from the dynamic crosstalk that occurs between the skin barrier and immune system using atopic dermatitis or eczema as the disease prototype. Nevertheless, the concepts put forth are highly relevant to a number of antigen-driven disorders for which skin barrier is at least transiently compromised such as psoriasis, allergic contact dermatitis and blistering disorders.

Stress-induced production of chemokines by hair follicles regulates the trafficking of dendritic cells in skin

Langerhans cells (LCs) are epidermal dendritic cells with incompletely understood origins that associate with hair follicles for unknown reasons. Here we show that in response to external stress, mouse hair follicles recruited Gr-1hi monocyte-derived precursors of LCs whose epidermal entry was dependent on the chemokine receptors CCR2 and CCR6, whereas the chemokine receptor CCR8 inhibited the recruitment of LCs. Distinct hair-follicle regions had differences in their expression of ligands for CCR2 and CCR6. The isthmus expressed the chemokine CCL2; the infundibulum expressed the chemokine CCL20; and keratinocytes in the bulge produced the chemokine CCL8, which is the ligand for CCR8. Thus, distinct hair-follicle keratinocyte subpopulations promoted or inhibited repopulation with LCs via differences in chemokine production, a feature also noted in humans. Pre-LCs failed to enter hairless skin in mice or humans, which establishes hair follicles as portals for LCs.

Recruitment of PCM1 to the Centrosome by the Cooperative Action of DISC1 and BBS4 A Candidate for Psychiatric Illnesses

CONTEXT A role for the centrosome has been suggested in the pathology of major mental illnesses, especially schizophrenia (SZ). OBJECTIVES To show that pericentriolar material 1 protein (PCM1) forms a complex at the centrosome with disrupted-in-schizophrenia 1 (DISC1) and Bardet-Biedl syndrome 4 protein (BBS4), which provides a crucial pathway for cortical development associated with the pathology of SZ. To identify mutations in the PCM1 gene in an SZ population. DESIGN Interaction of DISC1, PCM1, and BBS proteins was assessed by immunofluorescent staining and coimmunoprecipitation. Effects of PCM1, DISC1, and BBS on centrosomal functions and corticogenesis in vivo were tested by RNA interference. The PCM1 gene was examined by sequencing 39 exons and flanking splice sites. SETTING Probands and controls were from the collection of one of us (A.E.P.). PATIENTS Thirty-two probands with SZ from families that had excess allele sharing among affected individuals at 8p22 and 219 white controls. MAIN OUTCOME MEASURES Protein interaction and recruitment at the centrosome in cells; neuronal migration in the cerebral cortex; and variant discovery in PCM1 in patients with SZ. RESULTS PCM1 forms a complex with DISC1 and BBS4 through discrete binding domains in each protein. DISC1 and BBS4 are required for targeting PCM1 and other cargo proteins, such as ninein, to the centrosome in a synergistic manner. In the developing cerebral cortex, suppression of PCM1 leads to neuronal migration defects, which are phenocopied by the suppression of either DISC1 or BBS4 and are exacerbated by the concomitant suppression of both. Furthermore, a nonsense mutation that segregates with SZ spectrum psychosis was found in 1 family. CONCLUSIONS Our data further support for the role of centrosomal proteins in cortical development and suggest that perturbation of centrosomal function contributes to the development of mental diseases, including SZ.

Flaky tail mouse denotes human atopic dermatitis in the steady state and by topical application with Dermatophagoides pteronyssinus extract.

The barrier abnormality, a loss-of-function mutation in the gene encoding filaggrin (FLG), which is linked to the incidence of atopic dermatitis (AD), is a recently discovered but important factor in the pathogenesis of AD. Flaky tail (Flg(ft)) mice, essentially deficient in filaggrin, have been used to investigate the role of filaggrin on AD. However, the relevancy of Flg(ft) mice to human AD needs to be determined further. In this study, we observed the clinical manifestations of Flg(ft) mice in the steady state and their cutaneous immune responses against external stimuli, favoring human AD. Under specific pathogen-free conditions, the majority of Flg(ft) mice developed clinical and histological eczematous skin lesions similar to human AD with outside-to-inside skin barrier dysfunction evaluated by newly devised methods. In addition, cutaneous hapten-induced contact hypersensitivity as a model of acquired immune response and a mite extract-induced dermatitis model physiologically relevant to a human AD were enhanced in Flg(ft) mice. These results suggest that the Flg(ft) mouse genotype has potential as an animal model of AD corresponding with filaggrin mutation in human AD.

Langerhans cell antigen capture through tight junctions confers preemptive immunity in experimental staphylococcal scalded skin syndrome.

Epidermal LCs but not dermal DCs take up skin surface protein through intact tight junctions and mediate IgG1 antibody responses to bacterial antigen, conferring protective immunization.

SASPase regulates stratum corneum hydration through profilaggrin-to-filaggrin processing

The stratum corneum (SC), the outermost layer of the epidermis, acts as a barrier against the external environment. It is hydrated by endogenous humectants to avoid desiccation. However, the molecular mechanisms of SC hydration remain unclear. We report that skin‐specific retroviral‐like aspartic protease (SASPase) deficiency in hairless mice resulted in dry skin and a thicker and less hydrated SC with an accumulation of aberrantly processed profilaggrin, a marked decrease of filaggrin, but no alteration in free amino acid composition, compared with control hairless mice. We demonstrated that recombinant SASPase directly cleaved a linker peptide of recombinant profilaggrin. Furthermore, missense mutations were detected in 5 of 196 atopic dermatitis (AD) patients and 2 of 28 normal individuals. Among these, the V243A mutation induced complete absence of protease activity in vitro, while the V187I mutation induced a marked decrease in its activity. These findings indicate that SASPase activity is indispensable for processing profilaggrin and maintaining the texture and hydration of the SC. This provides a novel approach for elucidating the complex pathophysiology of atopic dry skin.