In ADAMTS-4 and a 7.1-fo d increase in ADAMTS-5 mRNA as compared to contro chondrocytes (data not shown). To determine whether particu ar subpopu ations of Proteasome Inhibitors chondrocytes respond different y to HA o igosaccharides, articu ar chondrocytes were iso ated from the upper ayers ( 30%) and the ower, midd e/deep zone ayers of bovine carti age. Ce s iso ated from these 2 zones as we as fu -thickness carti age s ices were incubated for 24 hours in the presence or absence of 250 g/m of HA o igosaccharides and ana yzed for changes in ADAMTS-4 and ADAMTS-5 mRNA expression.
As shown in Figure 2, chondrocytes derived from the upper midd e/superficia ayers were risedronate more responsive to HA o igosaccharides than were chondrocytes iso ated from the midd e and deep zones. Chondrocytes derived from.HA o igosaccharide enhancement of the eve s of ADAMTS-4 and ADAMTS-5 protein re eased into the medium. Conditioned media from bovine articu ar chondrocyte cu tures were ana yzed for aggrecanases by Western b otting. ADAMTS-4 is synthesized in a pro form (fu ength; p100), which is processed in a mu tistep manner, inc uding furin c eavage to a p68 form and subsequent conversion to species (p53 and p40 forms) with significant aggrecanase activity (G u373–A a374 c eaving activity) (21). As shown in Figure 3A, chondrocytes purchase Nattokinase produced and secreted immunoreactive ADAMTS-4 that was present as p68, p53, and p40 species in media from 24-hour contro cu tures.
After incubation with HA o igosaccharides, there was an increase in ADAMTS-4 re eased as compared to untreated chondrocytes, with increases in the p53 and p40 species being the most prominent, especia y at 24 hours. The p53 and p40 species a so increased in Sodium Danshensu 67920-52-9 proportion to the HA o igosaccharide concentration (Figure 3C), reaching a maximum eve at 250 g/m . ADAMTS-5 was visua ized as a sing e 70-kd protein band (Figure 3B). HA o igosaccharides a so induced an increase in the eve of ADAMTS-5 in the conditioned cu ture medium, a eve that was maxima fo owing treatment of chondrocytes with 250 g/m of HA o igosaccharides (Figures 3B and D). Interesting y, no significant stimu ation of aggrecanase protein was observed in the ce ysates (data not shown). HA o igosaccharide enhancement of the accumu ation of aggrecanase degradation products, G1-ITEGE domains. As a measure of aggrecanase activity, ADAMTS-4– or ADAMTS-5–generated fragments of aggrecan can be detected by use of antibodies that recognize the neoepitope G1-ITEGE domains (22).
Accumuation of G1-ITEGE domains re eased into the conditioned medium of bovine carti age exp ant cu tures was ana yzed by Western b otting. As shown in Figure 4, the gross domestic product G1-ITEGE domain present in the medium from contro exp ants after 5 days of cu ture was represented as a heterogeneous band between 68 kd and 72 kd. This 68–72-kd band is simi ar in size to the G1-ITEGE domain iso ated from the conditioned medium of treated bovine exp ant cu tures (data not shown) (see a so ref. 27). When carti age exp ants were exposed to HA o igosaccharides for 1, 3, or 5 days, a progressive increase in the eve of G1-ITEGE domains re eased into the medium was observed (Figure 4). These resu ts suggest that ADAMTS-mediated aggrecan degradation occurred in these tissues and increased.