Tetsuya Sayo

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.

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.

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.

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.

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.

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 β-carotene and β-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 β-carotene and β-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.

N-Methyl-L-Serine Stimulates Hyaluronan Production in Human Skin Fibroblasts

We examined the effects of N-methyl-L-serine (NMS), an amino acid derivative, on hyaluronan (HA) synthesis in human skin fibroblasts. NMS (1–10 mM), but not L-serine, stimulated the incorporation of [3H]glucosamine into HA dose-dependently, with a maximum stimulation of 1.5-fold compared to the control. The effect of NMS was specific for HA production, because there was no change in sulfated glycosaminoglycan formation. Neither the N-methyl derivatives of L-glycine or L-alanine, nor N-methyl-D-serine, could stimulate HA synthesis, indicating that the β-hydroxyl group and the L-configuration were essential for the activity. Gel filtration of the products showed that NMS stimulated the production of high-molecular-mass HA (>106 D) without affecting the production of low-molecular-mass HA. NMS required 24 h to stimulate HA production, and when fibroblasts were pretreated for 10–24 h with NMS (1–10 mM), membrane-associated HA synthase activity was increased dose-dependently. Thus, a second messenger is likely to be involved in the stimulation of HA production by NMS.

Reduction of hyaluronan and increased expression of HYBID (alias CEMIP and KIAA1199) correlate with clinical symptoms in photoaged skin

Hyaluronan (HA) metabolism in skin fibroblasts is mediated by HYBID (hyaluronan binding protein involved in hyaluronan depolymerization, alias CEMIP and KIAA1199) and the HA synthases HAS1 and HAS2. However, photoageing‐dependent changes in HA and their molecular mechanisms, and the relationship between HA metabolism and clinical symptoms in photoaged skin remain elusive.

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.