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 ...

Vascular endothelial growth factor (VEGF) is a strong angiogenic mitogen and plays important roles in angiogenesis under various pathophysiological conditions. The in vivo angiogenic activity of secreted VEGF may be regulated by extracellular inhibitors, because it is also produced in avascular tissues such as the cartilage. To seek the binding inhibitors against VEGF, we screened the chondrocyte cDNA library by a yeast two‐hybrid system by using VEGF165 as bait and identified connective tissue growth factor (CTGF) as a candidate. The complex formation of VEGF165 with CTGF was first established by immunoprecipitation from the cells overexpressing both binding partners. A competitive affinity‐binding assay also demonstrated that CTGF binds specifically to VEGF165 with two classes of binding sites (Kd = 26 ± 11 nM and 125 ± 38 nM). Binding assay using deletion mutants of CTGF indicated that the thrombospondin type‐1 repeat (TSP‐1) domain of CTGF binds to the exon 7‐coded region of VEGF165 and that the COOH‐terminal domain preserves the affinity to both VEGF165 and VEGF121. The interaction of VEGF165 with CTGF inhibited the binding of VEGF165 to the endothelial cells and the immobilized KDR/IgG Fc; that is, a recombinant protein for VEGF165 receptor. By in vitro tube formation assay of endothelial cells, full‐length CTGF and the deletion mutant possessing the TSP‐1 domain inhibited VEGF165‐induced angiogenesis significantly in the complex form. This antiangiogenic activity of CTGF was demonstrated further by in vivo angiogenesis assay by using Matrigel injection model in mice. These data demonstrate for the first time that VEGF165 binds to CTGF through a protein‐to‐protein interaction and suggest that the angiogenic activity of VEGF165 is regulated negatively by CTGF in the extracellular environment.

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We investigated interhemispheric interactions between the human hand motor areas using transcranial cortical magnetic and electrical stimulation. A magnetic test stimulus was applied over the motor cortex contralateral to the recorded muscle (test motor cortex), and an electrical or magnetic conditioning stimulus was applied over the ipsilateral hemisphere (conditioning motor cortex). We investigated the effects of the conditioning stimulus on responses to the test stimulus. Two effects were elicited at different interstimulus intervals (ISIs): early facilitation (ISI = 4–5 ms) and late inhibition (ISI ≥ 11 ms). The early facilitation was evoked by a magnetic or anodal electrical conditioning stimulus over the motor point in the conditioning hemisphere, which suggests that the conditioning stimulus for early facilitation directly activates corticospinal neurones. The ISIs for early facilitation taken together with the time required for activation of corticospinal neurones by I3‐waves in the test hemisphere are compatible with the interhemispheric conduction time through the corpus callosum. Early facilitation was observed in responses to I3‐waves, but not in responses to D‐waves nor to I1‐waves. Based on these results, we conclude that early facilitation is mediated through the corpus callosum. If the magnetic conditioning stimulus induced posteriorly directed currents, or if an anodal electrical conditioning stimulus was applied over a point 2 cm anterior to the motor point, then we observed late inhibition with no early facilitation. Late inhibition was evoked in responses to both I1‐ and I3‐waves, but was not evoked in responses to D‐waves. The stronger the conditioning stimulus was, the greater was the amount of inhibition. These results are compatible with surround inhibition at the motor cortex.

Paired-pulse magnetic stimulation techniques have been used to study the intracortical circuitry of the motor cortex in humans. There are several paired stimulation methods. Two of them have been used for studying inhibitory and facilitatory connections in the motor cortex at short interstimulus intervals (ISIs). When the first stimulus (S1) is subthreshold and the second (S2) suprathreshold, electromyographic (EMG) responses to both stimuli are smaller than the responses to S2 alone at short ISIs (1-5 ms; intracortical inhibition) and larger at longer ISIs (Kujirai et al. 1993). In contrast, when S1 is suprathreshold and S2 subthreshold, EMG responses to both stimuli can be larger than the control responses at ISIs of 1.3, 2.6 and 4.0 ms (Tokimura et al. 1996; Nakamura et al. 1997b; Ziemann et al. 1998;Rothwell, 1999). These two effects were not observed when S2 was a low intensity anodal electrical stimulus, which tends to evoke D-waves (direct waves: descending volleys produced by direct activation of pyramidal tract neurones), but were very clear when S2 was a magnetic stimulus that elicited I-waves (indirect waves: descending volleys produced by indirect activation of pyramidal tract neurones via presynaptic neurones). Based on these results, both effects were considered to be produced at the motor cortex. The latter effect has been termed 'intracortical I-wave facilitation ' (Ziemann et al. 1998).Several studies have shown that later I-waves are more affected by intracortical inhibition than early I-waves (Nakamura et al. 1997a; Hanajima et al. 1998; Di Lazzaro et al. 1998). I3-waves appear to be particularly susceptible to intracortical inhibition, whereas I1-waves are little affected (Hanajima et al. 1998). However, it remains to be determined whether there are differences in intracortical I-wave facilitation among different I-waves. In this paper, in order to clarify details of this effect, we studied intracortical I-wave facilitation of I1-and I3-waves using both single motor unit and surface EMG recordings. METHODS SubjectsTen healthy volunteers (8 men and 2 women; 28-46 years old; height, 143-180 cm; weight, 45-95 kg) were studied. Written informed consent was obtained from all the subjects. Surface EMG recordings were done in all subjects. Single motor unit studies were performed in nine subjects, one subject (a In order to elucidate the mechanisms underlying intracortical I-wave facilitation elicited by pairedpulse magnetic stimulation, we compared intracortical facilitation of I1-waves with that of I3-waves using single motor unit and surface electromyographic (EMG) recordings from the first dorsal interosseous muscle (FDI). We used a suprathreshold first stimulus (S1) and a subthreshold second stimulus (S2). In most experiments, both stimuli induced currents in the same direction. In others, S1 induced posteriorly directed currents and S2 induced anteriorly directed currents. When both stimuli induced anteriorly directed currents (I1-wave effects), an interstimulus interval (ISI) of 1.5 ms res...