Abstract
The release of aggrecan catabolites from cartilage is an early event in the pathogenesis of degenerative joint diseases. The enzymes involved in this process are unknown, controversial, and the subject of intense investigation. In this paper we have utilized a recombinant substrate containing the interglobular domain (IGD) of aggrecan to study specifically aggrecanase versus matrix metalloproteinase (MMP) catabolism in this domain of aggrecan. Our studies have shown that (i) there are species differences in the expression of latent versus active MMP activity on the aggrecan IGD; (ii) interleukin-1alpha exposure induces both aggrecanase and MMP activities, whereas retinoic acid induces only aggrecanase activity and inhibits the MMP activity on the aggrecan IGD; (iii) activators of latent MMP activity (p-aminophenylmercuric acetate and trypsin) significantly reduce aggrecanase activity; (iv) the time course of the appearance of aggrecanase versus the MMP catabolism of aggrecan IGD differs; (v) aggrecanase is a protease with metalloprotease characteristics; however (vi) the physiological (tissue) inhibitors of MMPs show weak inhibition (TIMP-1) or no inhibition (TIMP-2) of aggrecanase activity. Collectively, these studies show that aggrecanase and MMP catabolism of the aggrecan IGD are independent and uncoupled.
Highlights
The loss of aggrecan from the articular cartilage matrix is one of the earliest pathophysiological hallmarks of diseases such as osteoarthritis and rheumatoid arthritis
Our studies have shown that (i) there are species differences in the expression of latent versus active matrix metalloproteinase (MMP) activity on the aggrecan interglobular domain (IGD); (ii) interleukin-1␣ exposure induces both aggrecanase and MMP activities, whereas retinoic acid induces only aggrecanase activity and inhibits the MMP activity on the aggrecan IGD; (iii) activators of latent MMP activity (p-aminophenylmercuric acetate and trypsin) significantly reduce aggrecanase activity; (iv) the time course of the appearance of aggrecanase versus the MMP catabolism of aggrecan IGD differs; (v) aggrecanase is a protease with metalloprotease characteristics; (vi) the physiological inhibitors of MMPs show weak inhibition (TIMP-1) or no inhibition (TIMP-2) of aggrecanase activity
Conditioned medium from porcine chondrocyte cultures treated with IL-1␣ was incubated with a range of specific metalloproteinase inhibitors over the dose ranges indicated: 1,10-phenanthroline dissolved in Me2SO, EDTA dissolved in Dulbecco’s modified Eagle’s medium (DMEM) (10 – 0.1 mM), actinonin dissolved in distilled water (500 –1 M), TIMP-1 dissolved in distilled water (255 nM), and TIMP-2 dissolved in distilled water (285 nM)
Summary
Materials—Alkaline phosphatase-conjugated second antibody and substrate used in Western blot analysis were obtained from Promega as the Protoblot Western blot AP system Activation of MMPs in Conditioned Medium from Bovine Chondrocyte Cultures—Conditioned medium from control and RA-treated cultures was incubated with p-aminophenylmercuric acetate (APMA; final concentration 1 mM) for 30 min at 37 °C before the addition of rAgg1mut and further incubation at 37 °C for 24 h. 2-fold serial dilutions of this digest (equivalent to 250, 125, 62.5, 31.3, 15.6, and 7.8 l of conditioned medium) were separated on 10% SDS-polyacrylamide gels and immunoblotted with monoclonal antibody BC-3 Conditioned medium from porcine chondrocyte cultures treated with IL-1␣ was incubated with a range of specific metalloproteinase inhibitors over the dose ranges indicated: 1,10-phenanthroline dissolved in Me2SO (dose range 2– 0.1 mM), EDTA dissolved in DMEM (10 – 0.1 mM), actinonin dissolved in distilled water (500 –1 M), TIMP-1 dissolved in distilled water (255 nM), and TIMP-2 dissolved in distilled water (285 nM). The immunoblots were incubated with substrate for 5–15 min at room temperature to achieve optimum color development
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