duration of exposure of the tissues to HA o igosaccharides. Enhanced interactions Rhein between MT4-MMP and ADAMTS-4 by HA o igosaccharide treatment of chondrocytes. To examine whether the association of GPIanchored MT4-MMP with ADAMTS-4 is a so affected by HA o igosaccharide–induced activation of ADAMTS-4 aggrecanase activity, we examined the interaction between ADAMTS-4 and MT4-MMP by coimmunoprecipitation. Protein  ysates were prepared and incubated with anti–ADAMTS-4–conjugated magnetic beads, and the resu ting bound and unbound fractions were ana yzed by Western b otting with anti–MT4-MMP antibody. As shown in Figure 5A, a sing e 63-kd band for MT4-MMP was detected in the ADAMTS-4 immunoprecipitate of contro  and HA o igosaccharide–treated chondrocytes.

The  eve  of MT4-MMP recovered from ADAMTS-4 precipitates increased fo  owing 12 AMN-107 hours of HA o igosaccharide treatment of chondrocytes (Figures 5A and B). MT4-MMP was a so recovered in the unbound fraction but presented as a doub et band. These doub et bands are indicative of the 65-kd ( atent form) and the 63-kd (activated form) proteins of MT4-MMP  . No changes in the  eve  of the doub et bands present in the unbound fraction were observed in immunoprecipitates of  ysates from HA o igosac charide–treated chondrocytes. The  even  of actin in the unbound fraction a so was not affected. Interesting y, the addition of HA o igosaccharides had no effect on the expression of MT4–MMP mRNA (Figure 5C) or MT4-MMP protein (Figure 5D). As a positive contro , treatment with I 1 for 24 hours significant y increased the expression of MT4-MMP mRNA (Figure 5C).

These data suggest that HA o igosaccharides not on y increase the synthesis of buy Rutin ADAMTS-4, but a so promote the interaction of ADAMTS-4 and the activated form (63 kd) of MT4- MMP.We have previous y shown that chemica  inhibitors of the NF- B and p38 MAPK pathways b ock HA o igosaccharide–induced increases in MMP-13 mRNA, protein, and caseino ytic activity in bovine articu ar chondrocytes (12). Converse y, inhibition of Akt activation (but not NF- B, p38 MAP, or p44/42 kinases) was shown to b ock HA o igosaccharide activation of HAS-2 and HA production (13). To identify the genera  signa ing pathway responsib e for HA o igosaccharide–induced stimu ation of aggrecanase, bovine articu ar chondrocytes were pretreated with optimized concentrations of chemica  pathway inhibitors. In contro  chondrocyte cu tures treated with HA o igosaccharides but without added inhibitor, there was an 11-fo d and a 2.7-fo d increase in ADAMTS-4 and smallpox ADAMTS-5 mRNA, respective y, fo  owing 24 hours of exposure to HA o igosaccharides (Figures 6A and B). He ena in pretreatment effective y b ocked the stimu ation of both ADAMTS-4 and ADAMTS-5 mRNA.

In contrast, foowing pretreatment with SB203580, an increase in HA o igosaccharide–induced ADAMTS-4 and ADAMTS-5 mRNA expression was still  observed. Additionay, neither the p44/42 MAPK inhibitor PD98059 nor the PI3K/Akt pathway inhibitor  Y294002   supplier Asarylaldehyde bocked the stimu ation of ADAMTS-4 or ADAMTS-5 mRNA. As shown in Figures 6C and D, pretreatment with he ena in a so b ocked the HA o igosaccharide–stimu ated as we   as basa   eve s of ADAMTS-4 and ADAMTS-5 protein recovered in the conditioned medium ( ane 3).