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 ...

We examined in situ expression of putative hyaluronan synthase genes, Has1 and Has2, and effects of transforming growth factor-beta on their expression. In situ mRNA hybridization showed that mouse skin expressed both Has1 and Has2 mRNA in dermis and epidermis. In dermis, the number of cells expressing the Has1 mRNA was less than that of the Has2 mRNA, and in epidermis, some strong signals from both mRNA were seen in stratum granulosum. Northern blot analysis showed that cultured human skin fibroblasts expressed Has1 mRNA of 2.4 kb and Has2 mRNA of 3.2 and 4.8 kb, whereas human keratinocytes expressed Has1 mRNA of 4.8 but not 2.4 kb and a trace of Has2 mRNA. When the cultures were stimulated with transforming growth factor-beta, both Has1 and Has2 mRNA were upregulated in fibroblasts, and only Has1 mRNA of 2.4 but not 4.8 kb was induced in keratinocytes. The maximal amount of the upregulated Has1 mRNA in keratinocytes at 2 h after stimulation decreased time-dependently to the nonstimulated level at 18 h, although the stimulation for 18 h of fibroblasts was effective on the expression of both Has mRNA. Differences in expression pattern of Has and Has2 mRNA in mouse skin and a higher response of fibroblasts to transforming growth factor-beta suggest that Has1 and Has2 genes are regulated independently and synthesized hyaluronan may have a different function in epidermis and dermis.

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.

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.

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.