Keigo Kawabata

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-β-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.

Rhododendrol, a depigmentation‐inducing phenolic compound, exerts melanocyte cytotoxicity via a tyrosinase‐dependent mechanism

Rhododendrol, an inhibitor of melanin synthesis developed for lightening/whitening cosmetics, was recently reported to induce a depigmentary disorder principally at the sites of repeated chemical contact. Rhododendrol competitively inhibited mushroom tyrosinase and served as a good substrate, while it also showed cytotoxicity against cultured human melanocytes at high concentrations sufficient for inhibiting tyrosinase. The cytotoxicity was abolished by phenylthiourea, a chelator of the copper ions at the active site, and by specific knockdown of tyrosinase with siRNA. Hence, the cytotoxicity appeared to be triggered by the enzymatic conversion of rhododendrol to active product(s). No reactive oxygen species were detected in the treated melanocytes, but up‐regulation of the CCAAT‐enhancer‐binding protein homologous protein gene responsible for apoptosis and/or autophagy and caspase‐3 activation were found to be tyrosinase dependent. These results suggest that a tyrosinase‐dependent accumulation of ER stress and/or activation of the apoptotic pathway may contribute to the melanocyte cytotoxicity.

In vivo Three-Dimensional Birefringence Analysis Shows Collagen Differences between Young and Old Photo-Aged Human Skin

Polarization-sensitive optical coherence tomography (PS-OCT) permits non-invasive visualization of dermal birefringence, mainly due to collagenous structures. The purpose of this study is to use PS-OCT to assess intrinsic-age-related and photo-age-related differences in three-dimensional dermal birefringence. We measured dermal birefringence of the cheek skin and photo-protected interior upper arm skin from old and young volunteers. The algorithm that we used automatically produces the transversal dermal birefringence map from the polarization-sensitive OCT volume. This allowed quantitative comparison and visualization of the transverse distribution of the dermal birefringence. We found that dermal birefringence of the cheek skin was significantly smaller in the old group than in the young group (young group, 0.295+/-0.037 degrees microm(-1); old group, 0.207+/-0.03 degrees microm(-1); P=0.003), whereas the interior upper arm showed no age-dependent difference. The transversal map of the cheek showed a heterogeneous decrease in dermal birefringence due to photoaging. The maps suggested that the peripheral regions of some infundibula were surrounded by a strong collagen network. Three-dimensional analyses of dermal birefringence using PS-OCT help to quantify the diagnosis of photoaging.

The presence of Nε-(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.

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-β1, 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-β1 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-β1 receptors correlated positively with HAS2 expression and inversely with HYBID expression. On the other hand, TGF-β1 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-β1, basic FGF, EGF, and PDGF-BB enhance HA production in skin fibroblasts, TGF-β1 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-β1 in arthritic synovial fibroblasts may be linked to accumulation of depolymerized HA in synovial fluids in arthritis patients.

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.

A new objective histological scale for studying human photoaged skin

A quantitative understanding of the histological alteration of the skin is important for assessing the severity of photoaging.

Glutathione maintenance is crucial for survival of melanocytes after exposure to rhododendrol

Rhododendrol is a phenolic compound that shows a tyrosinase‐dependent toxicity for melanocytes and occasionally induces a vitiligo‐like skin depigmentation. The post‐tyrosinase mechanisms determining melanocyte death or survival, however, are far from clear. Here, we find that rhododendrol treatment leads to a reduction in the levels of cellular glutathione but also induces a cellular antioxidant response that eventually increases glutathione levels. We further find that rhododendrol toxicity is enhanced when glutathione levels are experimentally reduced and alleviated when glutathione levels are increased. Hence, it appears that the size of the preexisting glutathione pool along with the capacity to supply glutathione via the antioxidant response determines whether melanocytes survive or die after rhododendrol exposure. It is conceivable, therefore, that rhododendrol‐induced leukoderma depends on the capacity to maintain appropriate glutathione levels and that enhancement of glutathione levels may preserve a patients melanocytes and potentially help in repigmentation.

Cartilage Oligomeric Matrix Protein Increases in Photodamaged Skin

Cartilage oligomeric matrix protein (COMP) is a structural component of cartilage. Recent studies have described COMP as a pathogenic factor that promotes collagen deposition in fibrotic skin disorders such as scleroderma and keloid skin. Although collagen, a major dermis component, is thought to decrease in photoaged skin, recent reports have demonstrated the presence of tightly packed collagen fibrils with a structural resemblance to fibrosis in the papillary dermis of photoaged skin. Here we examined how photoaging damage relates to COMP expression and localization in photoaged skin. In situ hybridization revealed an increase in COMP-mRNA-positive cells with the progress of photoaging in preauricular skin (sun-exposed skin). The signal intensity of immunostaining for COMP increased with photoaging in not only the papillary dermis but also the reticular dermis affected by advancing solar elastosis. Immunoelectron microscopy detected the colocalization of COMP with both elastotic materials and collagen fibrils in photoaged skin. Ultraviolet light A irradiation of human dermal fibroblasts induced COMP expression at both the mRNA and protein levels. Ultraviolet light A-induced COMP expression was inhibited by an anti-transforming growth factor-β antibody or SB431542, an activin receptor-like kinase 5 inhibitor. These results suggest that the transforming growth factor-β-mediated upregulation of COMP expression may contribute to the modulation of dermal extracellular matrix in the photoaging process.

Relationship of hyaluronan and HYBID (KIAA1199) expression with roughness parameters of photoaged skin in Caucasian women

Hyaluronan (HA) is an important constituent of extracellular matrix (ECM) in the skin, and HA degradation mediated by HYBID (KIAA1199) is suggested to be implicated in facial skin wrinkling in Japanese women. Ethnic difference in skin wrinkle formation is known between Caucasian and Japanese women, but no information is available for the relations of HA and HYBID expression levels with skin wrinkling in Caucasian women.