Epidermal keratinocytes regulate hyaluronan metabolism via extracellularly secreted hyaluronidase 1 and hyaluronan synthase 3.

Hyaluronan (HA) is a high-molecular-weight (HMW) glycosaminoglycan, which is a fundamental component of the extracellular matrix that is involved in a variety of biological processes. We previously showed that the HYBID/KIAA1199/CEMIP axis plays a key role in the depolymerization of HMW-HA in normal human dermal fibroblasts (NHDFs). However, its roles in normal human epidermal keratinocytes (NHEKs) remained unclear. HYBID mRNA expression in NHEKs was lower than that in NHDFs, and NHEKs showed no depolymerization of extracellular HMW-HA in culture, indicating that HYBID does not contribute to extracellular HA degradation. In this study, we found that the cell-free conditioned medium of NHEKs degraded HMW-HA under weakly acidic conditions (pH 4.8). This degrading activity was abolished by hyaluronidase 1 (HYAL1) knockdown but not by HYAL2 knockdown. Newly synthesized HYAL1 was mainly secreted extracellularly, and the secretion of HYAL1 was increased during differentiation, suggesting that epidermal interspace HA is physiologically degraded by HYAL1 according to pH decrease during stratum corneum formation. In HA synthesis, hyaluronan synthase 3 (HAS3) knockdown reduced HA production by NHEKs, and interferon-γ-dependent HA synthesis was correlated with increased HAS3 expression. Furthermore, HA production was increased by TMEM2 knockdown through enhanced HAS3 expression. These results indicate that NHEKs regulate HA metabolism via HYAL1 and HAS3, and TMEM2 is a regulator of HAS3-dependent HA production.

Disorganisation of basement membrane zone architecture impairs melanocyte residence in vitiligo.

The basement membrane zone is the interface between the epidermis and dermis, and it is disrupted in several skin conditions. Here, we report the results of a comprehensive investigation into the structural and molecular factors of the basement membrane zone in vitiligo, a dermatological disorder characterised by depigmented patches on the skin. Using electron microscopy and immunofluorescence staining, we confirmed abnormal basement membrane zone morphology and disrupted basement membrane zone architecture in human vitiliginous skin. Furthermore, we identified elevated expression of matrix metalloproteinase 2 (MMP2) in human dermal fibroblasts as a key factor responsible for basement membrane zone matrix degradation. In our in vitro and ex vivo models, overexpression of MMP2 in fibroblasts led to basement membrane zone disruption and melanocyte disappearance. Importantly, we reveal that the loss of melanocytes in vitiligo is primarily linked to their weakened adhesion to the basement membrane, mediated by binding between integrin ß1 and laminin and discoidin domain receptor 1 and collagen IV. Finally, inhibition of matrix metalloproteinase 2 expression reversed depigmentation in a mouse model of vitiligo. In conclusion, our research shows the importance of basement membrane zone integrity in melanocyte residence and offers new avenues for therapeutic interventions to address this challenging skin condition. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Human TMEM2 is not a catalytic hyaluronidase, but a regulator of hyaluronan metabolism via HYBID (KIAA1199/CEMIP) and HAS2 expression.

Cutaneous hyaluronan (HA) is depolymerized to intermediate sizes in the extracellular matrix, and further fragmented in the regional lymph nodes. Previously, we showed that the HA-binding protein involved in HA depolymerization (HYBID), also known as KIAA1199/CEMIP, is responsible for the first step of HA depolymerization. Recently, mouse transmembrane 2 (mTMEM2) with high structural similarity to HYBID was proposed to be a membrane-bound hyaluronidase. However, we showed that the knockdown of human TMEM2 (hTMEM2) conversely promoted HA depolymerization in normal human dermal fibroblasts (NHDFs). Therefore, we examined the HA-degrading activity and function of hTMEM2 using HEK293T cells. We found that human HYBID and mTMEM2, but not hTMEM2, degraded extracellular HA, indicating that hTMEM2 does not function as a catalytic hyaluronidase. Analysis of the HA-degrading activity of chimeric TMEM2 in HEK293T cells suggested the importance of the mouse GG domain. Therefore, we focused on the amino acid residues that are conserved in active mouse and human HYBID and mTMEM2 but are substituted in hTMEM2. The HA-degrading activity of mTMEM2 was abolished when its His248 and Ala303 were simultaneously replaced by the corresponding residues of inactive hTMEM2 (Asn248 and Phe303). In NHDFs, enhancement of hTMEM2 expression by proinflammatory cytokines decreased HYBID expression and increased hyaluronan synthase 2-dependent HA production. The effects of proinflammatory cytokines were abrogated by hTMEM2 knockdown. A decreased HYBID expression by interleukin-1ß and transforming growth factor-ß was canceled by hTMEM2 knockdown. In conclusion, these results indicate that hTMEM2 is not a catalytic hyaluronidase, but a regulator of HA metabolism.

Naked mole-rat TMEM2 lacks physiological hyaluronan-degrading activity.

Mouse transmembrane protein 2 (mTMEM2) has been identified as a hyaluronidase, which has extracellularly G8 and GG domains and PbH1 repeats; however, our previously study showed that human TMEM2 (hTMEM2) is not a catalytic hyaluronidase due to the absence of the critical amino acid residues (His248/Ala303) in the GG domain. Naked mole-rats (NMRs) accumulate abundant high-molecular weight hyaluronan (HA) in their tissues, suggesting decreased HA degradation. Therefore, we aimed to evaluate the HA-degrading activity of NMR TMEM2 (nmrTMEM2) and compare it with those of mTMEM2 and hTMEM2. The amino acid residues of nmrTMEM2 (Asn247/Val302) are similar to Asn248/Phe303 of hTMEM2, and nmrTMEM2-expressing HEK293T cells showed negligible activity. We confirmed the significance of these amino acid residues using an inactive chimeric TMEM2 with the human GG domain, which acquired catalytic activity when Asn248/Phe303 was substituted with His248/Ala303. Semi-quantitative comparison of the activities of the membrane-fractions derived from m/h/nmrTMEM2-expressing HEK293T cells revealed that at least 20- and 14-fold higher amounts of nmr/ hTMEM2 were required to degrade HA to the same extent as by mTMEM2. Thus, unlike mTMEM2, nmrTMEM2 is not a physiological hyaluronidase. The inability of nmrTMEM2 to degrade HA might partially account for the high-molecular-weight HA accumulation in NMR tissues.

An indigo-producing plant, Polygonum tinctorium, possesses a flavin-containing monooxygenase capable of oxidizing indole.

Polygonum tinctorium (P. tinctorium) is an indigo plant that is cultivated for a specific metabolite that it produces i.e., indoxyl ß-D-glucoside (indican). In this study, flavin-containing monooxygenase (PtFMO) from P. tinctorium was cloned. When recombinant PtFMO was expressed in E. coli in the presence of tryptophan, indigo production was observed. Furthermore, we measured the activity of PtFMO using the membrane fraction from E. coli and found that it could produce indigo using indole as a substrate. The co-expression of PtFMO with indoxyl ß-D-glucoside synthase (PtIGS), which catalyzes the glucosylation of indoxyl, brought about the formation of indican in E. coli. The results showed that indican was synthesized by sequential reactions of PtFMO and PtIGS. In three-week-old P. tinctorium specimens, the first leaves demonstrated higher levels of PtFMO expression than the subsequent leaves. This result coincided with that of our prior study on PtIGS expression level. Our study provides evidence that PtFMO might contribute to indican biosynthesis.

Pro-inflammatory cytokines suppress HYBID (hyaluronan (HA) -binding protein involved in HA depolymerization/KIAA1199/CEMIP) -mediated HA metabolism in human skin fibroblasts.

In the skin, the metabolism of hyaluronan (HA) is highly regulated. Aging leads to chronic low-grade inflammation, which is characterized by elevated levels of pro-inflammatory cytokines; however, the relationship between inflammation and HA metabolism is not clear. Herein, we investigated the effects of a mixture of pro-inflammatory cytokines containing TNF-α, IL-1ß, and IL-6 on HA metabolism in human skin fibroblasts. Treatment with the cytokine mixture for 24 h suppressed HA depolymerization via downregulation of HYBID (HA-binding protein involved in HA depolymerization/KIAA1199/CEMIP) and promoted HA synthesis via upregulation of HAS2 in human skin fibroblasts. Moreover, HAS2-dependent HA synthesis was driven mainly by IL-1ß with partial contribution from TNF-α. Transmembrane protein 2 (TMEM2/CEMIP2), which was previously reported as a candidate hyaluronidase, was upregulated by the cytokine mixture, suggesting that TMEM2 might not function as a hyaluronidase in human skin fibroblasts. Furthermore, the effects of the cytokine mixture on HA metabolism were observed in fibroblasts after 8 days of treatment with cytokines during three passages. Thus, we have shown that HYBID-mediated HA metabolism is negatively regulated by the pro-inflammatory cytokine mixture, providing novel insights into the relationship between inflammation and HA metabolism in the skin.

Interferon-γ downregulates tight junction function, which is rescued by interleukin-17A.

Although atopic dermatitis (AD) has been reported to be a typical type 2 immune response disease, it is also an inflammatory skin disease that involves cytokines, such as Th1, Th17 and Th22. However, little is known about the mechanism by which the candidate cytokines, alone or in combination, are involved in AD pathology. Differences in cytokine balance, which contribute to the complexity of AD pathology, may influence the stratum corneum barrier function through tight junction (TJ) functional stability and contribute to disease severity. To confirm the regulatory mechanism of TJ protein expression in AD, we investigated the Th1 and Th17 pathways, which are the initiation factors of chronic AD pathology. We examined the effects of these cytokines on TJ protein expression in normal human epidermal keratinocytes in vitro, and also examined their function in a human skin equivalent model. We observed a time- and dose-dependent inhibitory effect of IFN-γ on claudin-1 expression via the IFN-γ receptor/JAK/STAT signalling pathway. IFN-γ impaired TJ function in a human skin equivalent model. Moreover, we investigated co-stimulation with IL-17A, which is highly expressed in AD skin lesions and found that IL-17A restores IFN-γ-induced TJ dysfunction. This restoration of TJ function was mediated by atypical protein kinase C zeta activation without recovery of TJ protein expression. These results are informative for personalized AD treatment via systemic therapies using anti-cytokine antibodies and/or JAK inhibitors.

Acorus calamus extract and its component α-asarone attenuate murine hippocampal neuronal cell death induced by l-glutamate and tunicamycin.

The Asian traditional medicinal plant Acorus calamus and its component α-asarone exhibited various biological activities, such as antiinflammation and antioxidant effects. In the present study, we investigated the in vitro effects of A. calamus extract and α-asarone on oxidative stress- and endoplasmic reticulum (ER) stress-induced cell death in hippocampal HT22 cells. A. calamus extract and α-asarone both significantly suppressed cell death induced by the oxidative stress inducer l-glutamate and ER stress inducer tunicamycin. A. calamus extract and α-asarone also significantly reduced reactive oxygen species (ROS) production induced by l-glutamate. Moreover, A. calamus extract and α-asarone suppressed the phosphorylation of protein kinase RNA-like ER kinase (PERK) induced by tunicamycin. These results suggest that A. calamus extract and α-asarone protect hippocampal cells from oxidative stress and ER stress by decreasing ROS production and suppressing PERK signaling, respectively. α-Asarone has potential as a potent therapeutic candidate for neurodegenerative diseases, including Alzheimer's disease.

Fish allergy tolerance 16 months after diagnosis.

Fish allergy is generally thought to be persistent, and approximately 80% of patients with fish allergies do not develop tolerance even 10 years after diagnosis. There have been no reports of rapid tolerance development in patients with severe fish allergies. We report the development of tolerance 16 months after the diagnosis of fish allergies. A 13-month-old boy was diagnosed with rosefish allergy (Sebastes matsubarae) and Japanese jack mackerel allergy (Trachurus japonicus). To find out which species of fish he could consume safely, he underwent several oral food challenge (OFC) tests. It was determined that he could consume tuna, salmon, cod, sardine, chub mackerel (Scomber japonicus), and Japanese amberjack (Seriola quinqueradiata) without eliciting signs of allergy. He continued to eat the fish that did not produce allergic reactions three to four times a week. The titer of serum allergen-specific immunoglobulin E (IgE) to fish had decreased in a subsequent ImmunoCAP®-specific IgE blood test performed 16 months after the diagnosis of the rosefish allergy. Following this test result, he underwent OFCs with rosefish and Japanese jack mackerel, both of which turned out to be negative, and it was determined that he had developed tolerance to fish. In this case, the repeated OFCs were useful in identifying fish species that were safe for consumption. In addition, the decrease in allergen-specific IgE was useful in predicting the development of tolerance. We hypothesize that proactive consumption of available fish species may lead to this rapid induction of tolerance to fish allergens.

GPNMB Extracellular Fragment Protects Melanocytes from Oxidative Stress by Inhibiting AKT Phosphorylation Independent of CD44.

Glycoprotein non-metastatic melanoma protein B (GPNMB) is a type I transmembrane glycoprotein that plays an important role in cancer metastasis and osteoblast differentiation. In the skin epidermis, GPNMB is mainly expressed in melanocytes and plays a critical role in melanosome formation. In our previous study, GPNMB was also found to be expressed in skin epidermal keratinocytes. In addition, decreased GPNMB expression was observed in the epidermis of lesional skin of patients with vitiligo. However, the exact role of keratinocyte-derived GPNMB and its effect on vitiligo is still unknown. In this study, we demonstrated that GPNMB expression was also decreased in rhododendrol-induced leukoderma, as seen in vitiligo. The extracellular soluble form of GPNMB (sGPNMB) was found to protect melanocytes from cytotoxicity and the impairment of melanogenesis induced by oxidative stress. Furthermore, the effect of rGPNMB was not altered by the knockdown of CD44, which is a well-known receptor of GPNMB, but accompanied by the suppressed phosphorylation of AKT but not ERK, p38, or JNK. In addition, we found that oxidative stress decreased both transcriptional GPNMB expression and sGPNMB protein expression in human keratinocytes. Our results suggest that GPNMB might provide novel insights into the mechanisms related to the pathogenesis of vitiligo and leukoderma.

The role and regulation of TMEM2 (transmembrane protein 2) in HYBID (hyaluronan (HA)-binding protein involved in HA depolymerization/ KIAA1199/CEMIP)-mediated HA depolymerization in human skin fibroblasts.

We have previously reported that HYBID (hyaluronan (HA)-binding protein involved in HA depolymerization/KIAA1199/CEMIP) is a specific HA-binding protein that is essential for HA depolymerization in skin and synovial fibroblasts. HA is incorporated into cells in the presence of HYBID and clathrin, degraded in endosomes, and excreted into the extracellular space. However, it is not yet clear whether HYBID itself catalytically cleaves HA. A recent report on transmembrane protein 2 (TMEM2)-a novel cell surface hyaluronidase-prompted us to investigate whether TMEM2 is essential for HYBID-mediated HA depolymerization. In the present study, we found that transforming growth factor beta 1 (TGF-ß1), which suppressed HA depolymerization with a concomitant decrease in HYBID expression, upregulated TMEM2 expression conversely in human skin fibroblasts. TMEM2 expression was not affected by histamine, which significantly increased HA depolymerization accompanied by an increase in HYBID expression. We confirmed a similar response in two other cell lines: KEL FIB keloid fibroblasts and HT1080 fibrosarcoma cells. TGF-ß1 was the only inducer of TMEM2 expression among growth factors including epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and platelet-derived growth factor-BB (PDGF-BB), which suppressed HYBID expression. Moreover, HYBID knockdown completely suppressed HA depolymerization, whereas TMEM2 knockdown unexpectedly enhanced it. These findings clearly indicate that HYBID is indispensable, but TMEM2 is not involved in the HYBID-mediated HA depolymerization system as a catalytic hyaluronidase in human skin fibroblasts.

Targeted deletion of HYBID (hyaluronan binding protein involved in hyaluronan depolymerization/ KIAA1199/CEMIP) decreases dendritic spine density in the dentate gyrus through hyaluronan accumulation.

HYBID (hyaluronan binding protein involved in hyaluronan [HA] depolymerization, KIAA1199/CEMIP) is a key player in HA depolymerization of the skin fibroblasts, arthritic synovial fibroblasts, and brain. Our previous study demonstrated that Hybid knock-out (KO) mice showed spatial memorial impairment, which is accompanied by the accumulation of high molecular weight HA in the hippocampus. However, the mechanism underlying cognitive impairment by Hybid deficiency remains unclear. In the present study, we examined the HA distribution patterns in the brains of wild-type (WT) and Hybid KO mice by HA staining using HA binding protein, and found that in Hybid KO mice, HA is accumulated and doublecortin-positive immature neurons are significantly decreased in the dentate gyrus of the hippocampus, where Hybid mRNA is highly expressed in WT mice. The Golgi-Cox staining demonstrated that the dendritic spine density is significantly decreased in the dentate gyrus granule cells in Hybid KO mice. These results suggest that Hybid-mediated HA degradation is critical for the synaptic formation process by contributing to cognitive functions, such as learning and memory, in the mouse brain.

Hyaluronan-Binding Protein Involved in Hyaluronan Depolymerization Controls Endochondral Ossification through Hyaluronan Metabolism.

Hyaluronan (HA) plays an important role in the development and maintenance of tissues, and its degradation is implicated in many pathologic conditions. We recently reported that HA-binding protein involved in HA depolymerization (CEMIP; alias HYBID/KIAA1199) is a key molecule in HA depolymerization, but its developmental and pathologic functions remain elusive. We generated Hybid-deficient mice using the Cre/locus of crossover in P1 (loxP) system and analyzed their phenotypes. Hybid-deficient mice were viable and fertile, but their adult long bones were shorter than those of wild-type animals. Hybid-deficient mice showed lengthening of hypertrophic zone in the growth plate until 4 weeks after birth. There were fewer capillaries and osteoclasts at the chondroosseous junction in the Hybid-deficient mice compared with the wild-type mice. In situ hybridization demonstrated that Hybid was expressed by hypertrophic chondrocytes at the chondroosseous junction. Cultured primary chondrocytes expressed higher levels of Hybid than did osteoblasts or osteoclasts, and the Hybid expression in the chondrocytes was up-regulated after maturation to hypertrophic chondrocytes. High-molecular-weight HA was accumulated in the lengthened hypertrophic zone in Hybid-deficient mice. In addition, high-molecular-weight HA significantly reduced cell growth and tube formation in vascular endothelial growth factor-stimulated or -nonstimulated endothelial cells. HA metabolism by HYBID is involved in endochondral ossification during postnatal development by modulation of angiogenesis and osteoclast recruitment at the chondroosseous junction.

Regulation of Hyaluronan (HA) Metabolism Mediated by HYBID (Hyaluronan-binding Protein Involved in HA Depolymerization, KIAA1199) and HA Synthases in Growth Factor-stimulated Fibroblasts.

Regulation of hyaluronan (HA) synthesis and degradation is essential to maintenance of extracellular matrix homeostasis. We recently reported that HYBID (HYaluronan-Binding protein Involved in hyaluronan Depolymerization), also called KIAA1199, plays a key role in HA depolymerization in skin and arthritic synovial fibroblasts. However, regulation of HA metabolism mediated by HYBID and HA synthases (HASs) under stimulation with growth factors remains obscure. Here we report that TGF-ß1, basic FGF, EGF, and PDGF-BB commonly enhance total amount of HA in skin fibroblasts through up-regulation of HAS expression, but molecular size of newly produced HA is dependent on HYBID expression levels. Stimulation of HAS1/2 expression and suppression of HYBID expression by TGF-ß1 were abrogated by blockade of the MAPK and/or Smad signaling and the PI3K-Akt signaling, respectively. In normal human skin, expression of the TGF-ß1 receptors correlated positively with HAS2 expression and inversely with HYBID expression. On the other hand, TGF-ß1 up-regulated HAS1/2 expression but exerted only a slight suppressive effect on HYBID expression in synovial fibroblasts from the patients with osteoarthritis or rheumatoid arthritis, resulting in the production of lower molecular weight HA compared with normal skin and synovial fibroblasts. These data demonstrate that although TGF-ß1, basic FGF, EGF, and PDGF-BB enhance HA production in skin fibroblasts, TGF-ß1 most efficiently contributes to production of high molecular weight HA by HAS up-regulation and HYBID down-regulation and suggests that inefficient down-regulation of HYBID by TGF-ß1 in arthritic synovial fibroblasts may be linked to accumulation of depolymerized HA in synovial fluids in arthritis patients.

KIAA1199, a deafness gene of unknown function, is a new hyaluronan binding protein involved in hyaluronan depolymerization.

Hyaluronan (HA) has an extraordinarily high turnover in physiological tissues, and HA degradation is accelerated in inflammatory and neoplastic diseases. CD44 (a cell surface receptor) and two hyaluronidases (HYAL1 and HYAL2) are thought to be responsible for HA binding and degradation; however, the role of these molecules in HA catabolism remains controversial. Here we show that KIAA1199, a deafness gene of unknown function, plays a central role in HA binding and depolymerization that is independent of CD44 and HYAL enzymes. The specific binding of KIAA1199 to HA was demonstrated in glycosaminoglycan-binding assays. We found that knockdown of KIAA1199 abolished HA degradation by human skin fibroblasts and that transfection of KIAA1199 cDNA into cells conferred the ability to catabolize HA in an endo-ß-N-acetylglucosaminidase-dependent manner via the clathrin-coated pit pathway. Enhanced degradation of HA in synovial fibroblasts from patients with osteoarthritis or rheumatoid arthritis was correlated with increased levels of KIAA1199 expression and was abrogated by knockdown of KIAA1199. The level of KIAA1199 expression in uninflamed synovium was less than in osteoarthritic or rheumatoid synovium. These data suggest that KIAA1199 is a unique hyaladherin with a key role in HA catabolism in the dermis of the skin and arthritic synovium.

Low-flux electron diffraction study for the intercellular lipid organization on a human corneocyte.

Human skin stratum corneum (SC) structures were investigated by electron diffraction (ED) with a very low-flux electron beam with the help of high-sensitivity detectors, the imaging plate and the CCD camera. This low-flux electron diffraction (LFED) method made it possible to minimize the unfavorable effect of electron beam damage and to give a reliable diffraction pattern from a small selected area (0.2µm(2)) on a corneocyte. Dependence of the 2-dimensional ED pattern on the size of the selected area showed that orientational correlation between lipid packing domains can persist over the area much larger than their domain size. The LFED method also allowed us to trace the detailed structural change induced by the electron beam damage. The ED diffraction peak for the lattice constant of about 4.1nm decayed in three steps. The detailed analysis of these three steps suggested that a different type of orthorhombic structure exists interacted with the well-described hexagonal and orthorhombic structures, in the process of decay resulting from electron beam damage.

JAK inhibitors for the treatment of vitiligo.

Vitiligo is an autoimmune disease involving melanocyte-targeting T cells initiated by environmental and genetic factors. Steroids and tacrolimus have been used as topical treatments. Recently, novel topical agents targeting Janus kinase (JAK), a family of tyrosine kinases that regulates cytokine signaling, have emerged. Ruxolitinib is the first approved in vitiligo therapy. Furthermore, ritlecitinib is currently under clinical trials for oral treatment of active vitiligo. In this review, we discuss the possibility of topical JAK inhibitors as promising options for the treatment of vitiligo with regard to their mechanism of action, efficacy and safety.

Involvement of aquaporin-7 in the cutaneous primary immune response through modulation of antigen uptake and migration in dendritic cells.

Dendritic cells (DCs) have the ability to present antigen and play a critical role in the induction of the acquired immune response. Skin DCs uptake antigen and subsequently migrate to regional draining lymph nodes (LNs), where they activate naive T cells. Here we show that the water/glycerol channel protein aquaporin 7 (AQP7) is expressed on epidermal and dermal DCs and involved in the initiation of primary immune responses. AQP7-deficient DCs showed a decreased cellular uptake of low-molecular-mass compounds (fluorescein isothiocyanate and Lucifer yellow) and high-molecular-mass substances (ovalbumin and dextran), suggesting that AQP7 is involved in antigen uptake. AQP7-deficient DCs also exhibited reduced chemokine-dependent cell migration in comparison to wild-type DCs. Consistent with these in vitro results, AQP7-deficient mice demonstrated a reduced accumulation of antigen-retaining DCs in the LNs after antigen application to the skin, which could be attributed to decreased antigen uptake and migration. Coincidentally, AQP7-deficient mice had impaired antigen-induced sensitization in a contact hypersensitivity model. These observations suggested that AQP7 in skin DCs is primarily involved in antigen uptake and in the subsequent migration of DCs and is responsible for antigen presentation and the promotion of downstream immune responses.

Activation of TLR2 enhances tight junction barrier in epidermal keratinocytes.

The epidermis has developed physical and immunological barriers that prevent infiltration of deleterious chemicals and pathogens. As a first step to understanding the relationship between these barriers, we investigated whether TLR2 activation functionally alters tight junctions (TJs) in cultured human keratinocytes. Stimulation with peptidoglycan, a ligand for TLR2, elevated the TJ-associated barrier in the space of 3 h. The increase in TJ-associated barrier function due to peptidoglycan stimulation was suppressed by the knockdown of TLR adaptor MyD88 or the pretreatment with TLR2-neutralizing Ab, indicating that TLR2 activation enhanced TJ-associated barrier. One and 3 h after peptidoglycan stimulation, expression levels of the TJ proteins occludin, claudin-1, claudin-4, and ZO-1 were unchanged. However, immunoprecipitation studies demonstrated that the association of phospho-atypical protein kinase Cζ/ι, crucial for TJ biogenesis, with occludin was increased. Significantly, inhibition of atypical protein kinase Cζ/ι activity completely blocked the immediate elevation of the TJ-associated barrier. Finally, peptidoglycan was applied to the stratum corneum surface of a human skin equivalent, and the TJ barrier was evaluated. In the space of 3 h after the stimulation, the amount of intercellular tracer in the stratum corneum incubated from the dermal side was reduced, indicating that the TJ barrier is strengthened via TLR2 activation. Taken together, our findings indicated that infiltration of pathogens into the epidermis immediately enhanced TJ function via TLR2 signaling. Furthermore, the dynamically controlled TJs in skin are considered fundamental in preventing further invasion of pathogens and maintaining cutaneous barrier homeostasis.