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. HA is ubiquitously present as a major constituent of the extracellular matrix (ECM) in vertebrate tissues, providing structural and functional integrity to cells and organs. Although many organs maintain high concentrations of HA, skin contains approximately half the total body HA (1). HA is rapidly depolymerized within tissues, from extralarge native molecules of 1,000-10,000 kDa, to intermediate-size fragments of 10-100 kDa present in the extracellular milieu (2). Approximately one-third of total body HA is replaced daily, and the skin is a major determinant organ for HA turnover, with a metabolic half-life of 1-1.5 d (2). HA degradation is enhanced under certain pathological conditions and its lower molecular weight products are commonly detected in diseases, such as arthritis and cancers (3-5). The reduced average molecular weight of HA (as low as 200 kDa) in synovial fluids from patients with osteoarthritis (OA) or rheumatoid arthritis (RA) leads to decreased synovial viscosity and is associated with synovial inflammation (6). In addition, much lower molecular weight HA fragments (∼20 kDa) are known to stimulate neovascularization and facilitate tumor cell motility and invasion (5,7,8).There are six human hyaluronidase-related genes clustered on two chromosomal loci, 3p21.3 (HYAL1, HYAL2, and HYAL3) and 7q31.3 (HYAL4, HYALP1, and SPAM1) (9). However, because HYALP1 is a pseudogene (9), and HYAL4 and SPAM1 have restricted expression patterns, HYALP1, HYAL4, and SPAM1 are unlikely to have major roles in constitutive HA degradation in vivo. HYAL3 has a restricted expression pattern (9) and its ability to degrade HA is questionable (10). Therefore, HYAL1 and HYAL2 are most likely ...

Mongolian gerbils were used to evaluate brain edema during restoration of flow following bilateral carotid occlusion for 1 h. We have modified the method for fluorometric measurement of Evans blue to monitor vascular protein leakage (vasogenic edema). The extraction of extravasated Evans blue was performed by homogenizing the whole brain in 50% trichloroacetic acid. The supernatant was diluted fourfold with ethanol and the Evans blue fluorescence was measured. The tissue blank was negligible. Evans blue content of the plasma was similarly determined and the ratio of tissue to plasma Evans blue content was calculated. Furthermore, Evans blue fluorescence was used for microscopic investigation. It is suggested that Evans blue fluorescence can be applied for quantification of protein leakage with much more sensitivity and accuracy than the colorimetric absorbance method, as well as for tissue localization of protein leakage.

Subscribe to PCMR and stay up-to-date with the only journal committed to publishing basic research in melanoma and pigment cell biology As a member of the IFPCS or the SMR you automatically get online access to PCMR. Sign up as a member today at www.ifpcs.org or at www.societymelanomaresarch.org SummaryRhododendrol, 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.

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-␤1, 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-␤1 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-␤1 receptors correlated positively with HAS2 expression and inversely with HYBID expression. On the other hand, TGF-␤1 upregulated 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-␤1, basic FGF, EGF, and PDGF-BB enhance HA production in skin fibroblasts, TGF-␤1 most efficiently contributes to production of high molecular weight HA by HAS up-regulation and HYBID down-regulation and suggests that inefficient downregulation of HYBID by TGF-␤1 in arthritic synovial fibroblasts may be linked to accumulation of depolymerized HA in synovial fluids in arthritis patients. Hyaluronan (HA)3 is a nonsulfated linear glycosaminoglycan composed of repeating disaccharide units of ␤-(1,3)-linked-Dglucuronic acid and ␤-(1,4)-linked-N-acetyl-D-glucosamine. HA is ubiquitously present as a major component in vertebrate connective tissues such as skin and synovial membrane and contributes to space filling, lubrication, and cell proliferation and migration (1). The turnover of HA in most tissues is extraordinarily rapid; the half-life of HA in the skin, which contains about half of all HA in the body, is 1-1.5 days (2). Thus, the tight control of HA synthesis and degradation is necessary for this turnover and seems to finely balance the amounts of high molecular mass HA (1,000 -10,000 kDa) within tissues (1, 2). On the other hand, an imbalance of synthesis and degradation causes the accumulation of HA with different molecular weights, which is commonly observed in diseases such as arthritis and cancers (3-5). Synovial fluids from patients with osteoarthritis (OA) or rheumatoid arthritis (RA) contain lower molecular mass HA (as low as 200 kDa) than that from normal subjects, leading to decreased synovial viscosity and increased inflammatory responses (6). HA is synthesized by HA synthases (Has1, Has2, and Has3) at the cell membrane, and the expression of Has enzyme...

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.