High α-diversity of skin microbiome and mycobiome in Japanese patients with vitiligo.

BACKGROUND: Vitiligo is an acquired pigmentary disorder characterized by depigmented patches on the skin that majorly impact patients' quality of life. Although its etiology involves genetic and environmental factors, the role of microorganisms as environmental factors in vitiligo pathology remains under-researched. OBJECTIVES: Our study explored the presence of characteristic bacterial and fungal flora in vitiligo-affected skin and investigated their potential roles in vitiligo pathogenesis. METHODS: We sequenced bacterial 16S rRNA and the fungal ITS1 region from skin swabs collected at frequently affected sites, namely the forehead and back, of patients with vitiligo. We analyzed bacterial and fungal flora in lesional and non-lesional areas of patients with vitiligo compared with corresponding sites in age- and sex-matched healthy subjects. RESULTS: Our findings revealed elevated α-diversity in both bacterial and fungal flora within vitiligo lesions compared with healthy controls. Notably, bacterial flora exhibited a distinctive composition in patients with vitiligo, and the proportional representation of Enterococcus was inversely correlated with the degree of vitiligo progression. Gammaproteobacteria, Staphylococcus spp., and Corynebacterium spp. were more abundant in vitiligo patients, with notable Staphylococcus spp. prevalence during the stable phase on the forehead. Conversely, the proportion of Malassezia sympodialis was lower and that of Malassezia globosa was higher in the progressive phase on the back of vitiligo patients. CONCLUSION: Our study identified some characteristic bacterial and fungal groups associated with vitiligo activity and prognosis, highlighting the potential roles of microorganisms in pathogenesis and offering insights into personalized disease-management approaches.

A Connecting Link between Hyaluronan Synthase 3-Mediated Hyaluronan Production and Epidermal Function.

Hyaluronan (HA), an essential component of the extracellular matrix of the skin, is synthesized by HA synthases (HAS1-3). To date, epidermal HA has been considered a major player in regulating cell proliferation and differentiation. However, a previous study reported that depletion of epidermal HA by Streptomyces hyaluronidase (St-HAase) has no influence on epidermal structure and function. In the present study, to further explore roles of epidermal HA, we examined effects of siRNA-mediated knockdown of HAS3, as well as conventional HA-depletion methods using St-HAase and 4-methylumbelliferone (4MU), on epidermal turnover and architecture in reconstructed skin or epidermal equivalents. Consistent with previous findings, HA depletion by St-HAase did not have a substantial influence on the epidermal architecture and turnover in skin equivalents. 4MU treatment resulted in reduced keratinocyte proliferation and epidermal thinning but did not seem to substantially decrease the abundance of extracellular HA. In contrast, siRNA-mediated knockdown of HAS3 in epidermal equivalents resulted in a significant reduction in epidermal HA content and thickness, accompanied by decreased keratinocyte proliferation and differentiation. These results suggest that HAS3-mediated HA production, rather than extracellularly deposited HA, may play a role in keratinocyte proliferation and differentiation, at least in the developing epidermis in reconstructed epidermal equivalents.

Antiwrinkle efficacy of 1-ethyl-ß-N-acetylglucosaminide, an inducer of epidermal hyaluronan production.

BACKGROUND: Hyaluronan (HA) has a unique hydration capacity that contributes to firmness and bounciness of the skin. Epidermal HA declines with skin aging, which may lead to clinical signs of aging including skin wrinkles and loss of hydration and elasticity. Recently, we developed a new cosmetic agent 1-ethyl-ß-N-acetylglucosaminide (ß-NAG2), which enhances HA production in cultured human keratinocytes. The aim of this study was to explore antiaging potential of ß-NAG2 in reconstructed human epidermal models and human clinical trial. MATERIALS AND METHODS: The amount of HA in ß-NAG2-treated epidermal models by topical application was analyzed by enzyme-linked immunosorbent assay (ELISA)-like assay. A randomized, double-blind and placebo-controlled study was conducted in Japanese females (n = 33) by topically treating each side of the face with a lotion formulated with ß-NAG2 or placebo for 8 weeks. RESULTS: Topically applied ß-NAG2 dose dependently increased HA production in epidermal models. Treatment with ß-NAG2-formulated lotion significantly improved skin hydration and elasticity and reduced skin wrinkling in crow's foot areas when compared to the placebo formulation. CONCLUSION: Topically applied ß-NAG2 promoted epidermal HA production in vitro and showed antiwrinkle activity in vivo accompanying the improvement in skin hydration and elasticity. Our study provides a novel strategy for antiwrinkle care through ß-NAG2-induced epidermal HA production.

A Lower Irradiation Dose of 308 nm Monochromatic Excimer Light Might Be Sufficient for Vitiligo Treatment: A Novel Insight Gained from In Vitro and In Vivo Analyses.

A 308 nm monochromatic excimer light (MEL) is widely used to treat patients with vitiligo. However, dose optimization still needs to be clarified. This study aimed to obtain objective evidence regarding various doses of MEL irradiation, induced cell level changes in vitro, and skin level alterations in vivo. Cultured human keratinocytes were irradiated with MEL using various doses. After irradiation at low doses, stem cell factor, endothelin-1, and glycoprotein nonmetastatic melanoma protein B, factors that activate and protect melanocytes, were found to be significantly elevated in keratinocytes. After irradiation using medium and high doses, inflammatory cytokines were induced. The amount of ATP released and the level of inflammasome activation, which are known to be related to interleukin-1ß activation, were also increased. The back skin of guinea pigs and mice were irradiated with MEL at varying doses. After irradiation, an increase of epidermal melanin and epidermal melanocytes was confirmed, using the minimal erythemal dose or less. In rhododendrol-induced leukoderma guinea pigs, a much lower dose of MEL irradiation was effective, when compared with the effective dose for control guinea pigs. Our results suggest that a lower irradiation dose of MEL might be sufficient and more suitable for repigmentation in vitiligo treatment.

1-Ethyl-ß-N-acetylglucosaminide increases hyaluronan production in human keratinocytes by being converted to N-acetylglucosamine via ß-N-acetylglucosaminidase-dependent manner.

Regulation of hyaluronan (HA) is important for the maintenance of epidermal homeostasis. Here, we examined the mechanism by which 1-ethyl-ß-N-acetylglucosaminide (ß-NAG2), a newly developed N-acetylglucosamine (NAG) derivative, increases HA production in cultured human epidermal keratinocytes. When keratinocytes were treated with ß-NAG2, mRNA expression of HA synthase 3, which is responsible for HA production in human keratinocytes, was not influenced, but the intracellular level of UDP-NAG, a substrate used for HA synthesis, was increased. By using a synthetic substrate for ß-N-acetylglucosaminidase (ß-NAGase), keratinocytes were found to possess ß-NAGase activity, and treatment of o-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino N-phenyl carbamate (PUGNAc), an inhibitor of ß-NAGase, abolished the release of NAG from ß-NAG2 in keratinocytes. Furthermore, PUGNAc attenuated the ß-NAG2-induced intracellular UDP-NAG and HA production in keratinocytes. These results suggest that ß-NAG2 is converted to NAG by endogenous ß-NAGase in keratinocytes, and the resulting NAG is further metabolized to UDP-NAG and utilized for HA production.

Accelerated human epidermal turnover driven by increased hyaluronan production.

BACKGROUND: Hyaluronan (HA) is an essential component of extracellular matrix in the skin, but its functions in the epidermis remain elusive. OBJECTIVE: We examined the interaction of increased HA production mediated by 1-ethyl-ß-N-acetylglucosaminide (ß-NAG2), a newly developed highly selective inducer of HA production which is intracellularly converted to UDP-N-acetylglucosamine, a substrate of HA, with epidermal proliferation and differentiation. METHODS: The amount, molecular size and epidermal tissue distribution of HA and expression of CD44, a cell surface receptor for HA, were analyzed in ß-NAG2-treated organ cultured human skin, reconstructed human skin equivalents or cultured human skin keratinocytes. The relationship between HA and epidermal proliferation or differentiation was examined. RESULTS: ß-NAG2 significantly increased HA production in the epidermis of skin explants or skin equivalents without affecting molecular size of HA (>2000 kDa) or CD44 mRNA expression. Histochemical experiments revealed that ß-NAG2 enhances HA signals in the basal to granular layers of the epidermis of skin equivalents, accompanying increased epidermal stratification. Immunohistochemical experiments demonstrated that signals of Ki67, transglutaminase 1 and filaggrin are increased in ß-NAG2-treated skin equivalents, and these observations were confirmed by the data showing that mRNA expression of PCNA, transglutaminase 1 (TGM1) and filaggrin (FLG) is significantly up-regulated by ß-NAG2 in skin equivalents. Importantly, blockade of HA production by inhibiting conversion of ß-NAG2 to UDP-NAG abolished ß-NAG2-mediated up-regulation of PCNA, TGM1 and FLG mRNA expression in cultured keratinocytes. CONCLUSION: These results suggest that increased epidermal HA production plays a key role in epidermal morphogenesis and homeostasis by accelerating keratinocyte proliferation and differentiation.

Depth-resolved investigation of multiple optical properties and wrinkle morphology in eye-corner areas with multi-contrast Jones matrix optical coherence tomography.

BACKGROUND: Multi-contrast Jones matrix optical coherence tomography (JM-OCT) can provide quantitative depth-resolved local optical properties by improving the measurement algorithm. MATERIALS AND METHODS: We examined the relationship between depth-resolved local optical properties of eye-corner skin measured by JM-OCT and corresponding wrinkle morphology of aged women (n = 21; age range, 71.7 ± 1.7 years). Wrinkle morphology was analyzed by measuring the surface topography of three-dimensional replicas. The same regions were measured three-dimensionally by JM-OCT, and the local optical properties at each depth were computed. RESULTS: Birefringence (BR) and mean wrinkle depth correlated significantly at a depth of 88.2-138.6 µm from the skin surface, and attenuation coefficient (AC) and mean wrinkle depth correlated significantly at a depth of 12.6-18.9 µm and 189-459.9 µm from the skin surface, although a degree of polarization uniformity (DOPU) did not. Stepwise multiple regression analysis demonstrated that a significant regression equation (R2  = 0.649, P < .001) for predicting mean wrinkle depth was determined by BR at 107.1 µm depth (BR 107.1 µm ), DOPU at 170.1 µm (DOPU 170.1µm ), and AC at 252 µm (AC 252 µm ) as independent variables and that these standardized beta regression coefficients were -0.860, -0.593, and -0.440, respectively, suggesting that BR, DOPU, and AC sufficiently explained mean wrinkle depth. CONCLUSION: These results suggest that BR 107.1 µm , DOPU 170.1 µm, and AC 252 µm may indicate collagen-related structure in the papillary, upper-reticular dermis, and microstructure or tissue density in reticular dermis, respectively, and may be involved in wrinkle formation.

HYBID (alias KIAA1199/CEMIP) and hyaluronan synthase coordinately regulate hyaluronan metabolism in histamine-stimulated skin fibroblasts.

The immune-regulatory compound histamine is involved in the metabolism of the essential skin component hyaluronan (HA). We previously reported that histamine up-regulates the expression of HYBID (hyaluronan-binding protein involved in hyaluronan depolymerization, also called CEMIP or KIAA1199), which plays a key role in HA degradation. However, no information is available about histamine's effects on HA synthase (HAS) expression, the molecular sizes of HA species produced, and histamine receptors and their signaling pathways in skin fibroblasts. Moreover, histamine's effects on photoaged skin remain elusive. Here, we show that histamine increases HA degradation by up-regulating HYBID and down-regulating HAS2 in human skin fibroblasts in a dose- and time-dependent manner and thereby decreases the total amounts and sizes of newly produced HA. Histamine H1 blocker abrogated the histamine effects on HYBID up-regulation, HAS2 suppression, and HA degradation. Histamine H1 agonist exhibited effects on HA levels, composition, and breakdown similar to those of histamine. Of note, blockade of protein kinase Cδ or PI3K-Akt signaling abolished histamine-mediated HYBID stimulation and HAS2 suppression, respectively. Immunohistochemical experiments revealed a significant ∼2-fold increase in tryptase-positive mast cells in photoaged skin, where HYBID and HAS2 expression levels were increased and decreased, respectively, compared with photoprotected skin. These results indicate that histamine controls HA metabolism by up-regulating HYBID and down-regulating HAS2 via distinct signaling pathways downstream of histamine receptor H1. They further suggest that histamine may contribute to photoaged skin damage by skewing HA metabolism toward degradation.

Inhibition of HYBID (KIAA1199)-mediated hyaluronan degradation and anti-wrinkle effect of Geranium thunbergii extract.

BACKGROUND: Hyaluronan (HA) is an essential constituent of extracellular matrix in the skin. HA reduction in the dermis and overexpression of HYBID (KIAA1199), a key molecule for HA degradation in skin fibroblasts, are implicated in facial skin wrinkling. AIMS: We aimed to obtain anti-wrinkle agent(s) by screening for inhibition of HYBID-mediated HA degradation. METHODS: Various plant extracts were screened for inhibition of HA degradation in HYBID-stable transfectants in HEK293 (HYBID/HEK293). Inhibition of HA-degrading activity and HYBID mRNA and protein expression by Geranium thunbergii extract was studied in skin fibroblasts and HYBID/HEK293 cells. Size distribution of newly produced HA was evaluated by preparing metabolically radiolabeled HA in skin fibroblasts. A double-blind, randomized, and placebo-controlled study was performed in healthy Japanese women (n = 21) by topically treating each side of the face with a lotion formulated with G. thunbergii extract or placebo for 8 weeks. RESULTS: Among the plant extracts tested, only G. thunbergii extract abolished HA depolymerization in skin fibroblasts and HYBID/HEK293 cells by down-regulating HYBID mRNA and protein expression and by inhibiting HYBID-mediated HA-degrading activity. Although untreated skin fibroblasts produced polydispersed HA, G. thunbergii extract-treated cells produced high-molecular-weight HA. Treatment with G. thunbergii extract-formulated lotion significantly improved skin elasticity and reduced skin wrinkling scores at the outer eye corner compared with the placebo formulation. CONCLUSIONS: Geranium thunbergii extract inhibited HYBID-mediated HA degradation in vitro and showed anti-wrinkle activity in vivo accompanying the improvement in skin elasticity. Our study provides a possible strategy for anti-wrinkle care through inhibition of HYBID-mediated HA degradation.

Lutein, a nonprovitamin A, activates the retinoic acid receptor to induce HAS3-dependent hyaluronan synthesis in keratinocytes.

Carotenoids have been reported to have potent antioxidant activities and to protect tissues and cells from certain diseases and environmental insults. The molecular mechanism of the action of provitamin A carotenoids such as ß-carotene and ß-cryptoxanthin is mediated in part by retinoic acid, an active form of provitamin A, but the molecular basis of the biological activities of non-provitamin A carotenoids such as lutein, zeaxanthin, and astaxanthin is not fully understood. In this study, we investigated to determine whether the actions of non-provitamin A carotenoids are mediated via retinoid signaling by monitoring retinoic acid receptor (RAR)-dependent hyaluronan production in cultured human keratinocytes. Not only ß-carotene and ß-cryptoxanthin, but also lutein, zeaxanthin, and astaxanthin, upregulated HAS3 gene expression and were followed by hyaluronan synthesis. We found that LE540, an antagonist of retinoic acid receptors, abolished lutein dependent hyaluronan synthesis and that lutein significantly increased retinoic acid responsive element (RARE)-driven transcript acitivity. In addition, we found that citral, an inhibitor of retinal dehydrogenases, decreased lutein-stimulated hyaluronan synthesis, indicating that lutein metabolites rather than lutein itself act as an RAR ligand in RAR-mediated transcription activity in keratinocytes. A series of non-provitamin A can be substituted for retinoids and should be considered as a potential means of improving skin health.

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.

Changes in epidermal hyaluronan metabolism following UVB irradiation.

BACKGROUND: Hyaluronan (HA) plays a role in keratinocyte proliferation and differentiation, and have shown different biological activities depending on its molecular mass. While many studies have shown changes in the amount of HA after UVB irradiation, molecular mass change remains to be elucidated. OBJECTIVE: To investigate the change in the molecular mass of HA after UVB irradiation in mouse epidermis. METHODS: The mice were irradiated with a single dose of UVB (0.15J/cm(2)). The amount of HA was examined using HABP sandwich assay. The molecular mass distribution was estimated by Sephacryl S-1000 chromatography. Has and Hyal mRNA expressions were detected by real-time PCR. RESULTS: On day 2 after UVB irradiation, both the amount of HA and the up-regulation of Has3 mRNA expression reached their maximum. The average HA molecular mass was about 1000 kDa, a level similar to that of the non-irradiated epidermis. On day 3, the average HA molecular mass drastically decreased to 100 kDa, while Hyal1, Hyal2, and Hyal3 mRNA expressions slightly increased. The amount of HA, however, remained high. On days 4 and 5, the amount of HA gradually decreased, but the molecular mass of HA remained low. A drastic reduction of the HA molecular mass after UVB irradiation was confirmed. CONCLUSION: UVB irradiation elicits remarkable changes in the molecular mass of HA, as well as amount. These qualitative and quantitative changes of HA might play an important role in UVB-induced cell proliferation and differentiation. Further study will be required to resolve the mechanism of HA degradation in the epidermis.

The presence of N(ε)-(Carboxymethyl) lysine in the human epidermis.

It is well known that advanced glycation end products (AGEs) are formed in long-lived dermal proteins such as collagen, and that their formation is related to skin aging. To examine the distribution of AGEs in skin tissue, we performed immunofluorescence studies on the human skin using an anti-AGEs antibody. Interestingly, AGEs signals were observed not only in the dermis but also in the epidermis. The objectives of this study were to confirm the presence of N(ε)-(Carboxymethyl) lysine (CML), an AGE structure, in the epidermis and to characterize the CML-modified proteins. The presence of CML in the stratum corneum (SC) was examined using liquid chromatography-electrospray ionization time-of-flight mass spectrometry. Concordance between the retention times of a compound in the SC hydrolysate and authentic CML, as well as with the specific mass transition of CML, was detected. This result showed that CML is present in the epidermis. In order to characterize the CML-modified proteins in the epidermis, protein samples extracted from the SC were analyzed using two-dimensional electrophoresis followed by an amino acid sequence analysis. The clarified peptide sequences covered approximately 27% of the amino acid sequences of cytokeratin 10 (K10). In the immunoblotting experiment following the two-dimensional electrophoresis, where protein samples extracted from whole epidermis were used, the position of the major CML-positive spots corresponded to those of K10. Taken together these results showed that CML is present in the human epidermis, and suggest that K10 is one of the target molecules for CML modification in the epidermis.

Adiponectin resides in mouse skin and upregulates hyaluronan synthesis in dermal fibroblasts.

Adipose tissue is a hormonally active tissue that produces adipokines that influence the activity of other tissues. Adiponectin is an adipocyte-specific adipokine involved in systemic metabolism. We detected the expression of adiponectin receptors (AdipoR1 and AdipoR2) mRNA in cultured dermal fibroblasts. The full-length adiponectin (fAd), but not the globular adiponectin (gAd), increased hyaluronan (HA) production and upregulated HA synthase (HAS) 2 mRNA expression. AdipoR1 and AdipoR2 mRNAs were also expressed in keratinocytes, though neither fAd nor gAd had any effect on HA synthesis. In mouse skin, we found that adiponectin was present and decreased markedly with aging. The age-dependent pattern of adiponectin decrease in skin, correlated well with that of HA in skin. Our experiments were also the first to identify adiponectin production in cultured mouse sebocytes, a finding that suggests that skin adiponectin may derive not only from plasma and/or subcutaneous adipose tissue, but also from the sebaceous gland. These results indicated that adiponectin plays an important role in the HA metabolism of skin.

Conditional inactivation of Has2 reveals a crucial role for hyaluronan in skeletal growth, patterning, chondrocyte maturation and joint formation in the developing limb.

The glycosaminoglycan hyaluronan (HA) is a structural component of extracellular matrices and also interacts with cell surface receptors to directly influence cell behavior. To explore functions of HA in limb skeletal development, we conditionally inactivated the gene for HA synthase 2, Has2, in limb bud mesoderm using mice that harbor a floxed allele of Has2 and mice carrying a limb mesoderm-specific Prx1-Cre transgene. The skeletal elements of Has2-deficient limbs are severely shortened, indicating that HA is essential for normal longitudinal growth of all limb skeletal elements. Proximal phalanges are duplicated in Has2 mutant limbs indicating an involvement of HA in patterning specific portions of the digits. The growth plates of Has2-deficient skeletal elements are severely abnormal and disorganized, with a decrease in the deposition of aggrecan in the matrix and a disruption in normal columnar cellular relationships. Furthermore, there is a striking reduction in the number of hypertrophic chondrocytes and in the expression domains of markers of hypertrophic differentiation in the mutant growth plates, indicating that HA is necessary for the normal progression of chondrocyte maturation. In addition, secondary ossification centers do not form in the central regions of Has2 mutant growth plates owing to a failure of hypertrophic differentiation. In addition to skeletal defects, the formation of synovial joint cavities is defective in Has2-deficient limbs. Taken together, our results demonstrate that HA has a crucial role in skeletal growth, patterning, chondrocyte maturation and synovial joint formation in the developing limb.

Increased hyaluronan production and decreased E-cadherin expression by cytokine-stimulated keratinocytes lead to spongiosis formation.

The pathogenesis of spongiosis, which is a well-known hallmark of acute eczema, is not fully understood. We sought to clarify the mechanism for the influx of tissue fluid into the epidermis and the loss of cohesion between keratinocytes in acute eczema that result in spongiosis. We first demonstrated increased intercellular accumulation of hyaluronan (HA) in the spongiotic epidermis by immunochemical staining using hyaluronic-acid-binding protein (HABP) and augmented hyaluronan synthase 3 (HAS3) mRNA expression by spongiotic keratinocytes using in situ hybridization. We also showed that the epidermis where the intercellular space was strongly stained with HABP showed weaker expression of membrane E-cadherin. Next, we demonstrated--by a sandwich assay using HABP, real-time PCR, and flow cytometry--that, among various cytokines, only IL-4, IL-13, and IFN-gamma increased HA production, enhanced HAS3 mRNA expression, and decreased membrane E-cadherin expression by normal human epidermal keratinocytes in both low- and high-Ca media. Finally, we demonstrated IL-4, IL-13, their combination, and IFN-gamma could induce intercellular space widening of the epidermis with increased HA accumulation and decreased E-cadherin expression in the organotypic culture. These results suggest that the augmented production of HA and the decreased E-cadherin expression by keratinocytes stimulated with IL-4/IL-13 or IFN-gamma cause spongiosis in acute eczema.

Characteristics of the epidermis and stratum corneum of hairless mice with experimentally induced diabetes mellitus.

Diabetes mellitus induces many pathophysiologic changes in the skin. Even so, dermatologists still lack an animal model of diabetes that enables the direct evaluation of the various functional properties of the skin. Our group induced two types of an experimental type 1 diabetes model in hairless mice by administering either streptozotocin or alloxan, in order to examine the properties of the stratum corneum and epidermis of these animals. The plasma glucose concentrations of the mice at 3 wk after their i.v. injection were significantly higher than those of control mice (streptozotocin, 3.2-fold; alloxan, 3.7-fold). The stratum corneum water content was significantly reduced in both types of diabetic mice, whereas the transepidermal water loss remained unchanged. The amino acid content with normal epidermal profilaggrin processing was either normal or elevated in the stratum corneum of the streptozotocin-treated mice. In contrast, the stratum corneum triglyceride content in the streptozotocin-treated mice was significantly lower than the control level, even though the levels of ceramides, cholesterols, and fatty acids in the stratum corneum were all higher than the control levels. The streptozotocin-treated group also exhibited decreases in basal cell proliferation and epidermal DNA content linked with an increase in the number of corneocyte layers in the stratum corneum, suggesting that the rates of epidermal and stratum corneum turnover were slower in the streptozotocin-treated animals than in the normal controls. In contrast, there were no remarkable changes in any of the epidermal differentiation marker proteins examined. This finding in diabetic mice, namely, reduction in both the epidermal proliferation and stratum corneum water content without any accompanying impairment in the stratum corneum barrier function, is similar to that found in aged human skin. Our new animal model of diabetes will be useful for the study of diabetic dermopathy as well as the mechanisms of stratum corneum moisturization.

Hyaluronan synthase 3 regulates hyaluronan synthesis in cultured human keratinocytes.

Three human hyaluronan synthase genes (HAS1, HAS2, and HAS3) have been cloned, but the functional differences between these HAS genes remains obscure. The purpose of this study was to examine which of the HAS genes are selectively regulated in epidermis. We examined the relation of changes between hyaluronan production and HAS gene expression when cytokines were added to cultured human keratinocytes. Interferon-gamma increased hyaluronan production whereas transforming growth factor beta decreased it. Both cytokines affected preferentially high-molecular-mass (> 106 Da) hyaluronan production. Consistent with the change in hyaluronan synthesis, we found that interferon-gamma markedly upregulated HAS3 mRNA whereas transforming growth factor beta downregulated HAS3 transcript levels. The expression of HAS1 mRNA was not significantly affected by either cytokine, and HAS2 mRNA expression was undetectable under either basal or cytokine-stimulated conditions by northern blot using total RNA. Furthermore, in situ mRNA hybridization showed that mouse epidermal keratinocytes abundantly expressed HAS3 mRNA from the basal to the granular cell layers, suggesting that HAS3 functions in epidermis. These findings suggest that HAS3 gene expression plays a crucial role in the regulation of hyaluronan synthesis in the epidermis.