Matrix metalloproteinase-3 in articular cartilage is upregulated by joint immobilization and suppressed by passive joint motion

Abstract

Both underloading and overloading of joints can lead to articular cartilage degradation, a process mediated in part by matrix metalloproteinases (MMPs). Here we examine the effects of reduced loading of rat hindlimbs on articular cartilage expression of MMP-3, which not only digests matrix components but also activates other proteolytic enzymes. We show that hindlimb immobilization resulted in elevated MMP-3 mRNA expression at 6h that was sustained throughout the 21day immobilization period. MMP-3 upregulation was higher in the medial condyle than the lateral, and was greatest in the superficial cartilage zone, followed by middle and deep zones. These areas also showed decreases in safranin O staining, consistent with reduced cartilage proteoglycan content, as early as 7days after immobilization. One hour of daily moderate mechanical loading, applied as passive joint motion, reduced the MMP-3 and ADAMTS-5 increases that resulted from immobilization, and also prevented changes in safranin O staining. Intra-articular injections of an MMP-3 inhibitor, N-isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid (NNGH), dampened the catabolic effects of a 7day immobilization period, indicating a likely requirement for MMP-3 in the regulation of proteoglycan levels through ADAMTS-5. These results suggest that biomechanical forces have the potential to combat cartilage destruction and can be critical in developing effective therapeutic strategies.

Figure 1

MMP-3 mRNA and activity changes in articular cartilage during immobilization. Levels of MMP-3 mRNA by RT-PCR (A) and total MMP-3 activity (B) expressed relative to levels in unimmobilized controls. Whole condyles, rather than tissue sections, were used as tissue sources for MMP-3 activity assays. Data show mean ± SEM (n=5). * = P < 0.05 versus unimmobilized control.

Figure 2

MMP-3 expression and activity in medial and lateral condyles. (A) Sites of tissue analysis by laser capture microdissection. Sagittal sections were taken for LCM from the mid-regions of each condyle as indicated by lines. (B) MMP-3 mRNA levels expressed relative to levels in the lateral condyle of non-immobilized controls. (C) MMP-3 total activity expressed relative to activity in lateral condyles of non-immobilized controls. Data in show mean ± SEM (n = 5). * = P < 0.05 versus unimmobilized control or indicated comparison.

Figure 3

Zonal analysis of MMP-3 mRNA expression following 21 days of immobilization. (A) Safranin O stained section of cartilage indicating zones selected for analysis by Laser Capture Microdissection. (B) MMP-3 mRNA levels (expressed as copy number/ng RNA). Data show mean and SEM (n = 5). * = P < 0.05.

Figure 4

MMP-3 protein expression in medial and lateral condyles following immobilization. (A). Immunohistochemistry of MMP-3 and (B) Safranin O-fast green staining of medial and lateral condyles. Bar = 100μm.

Figure 5

Changes in cartilage mRNA, enzyme activity and histology due to immobilization and motion loading. (A) Relative MMP-3 mRNA gene expression after 6 and 24 hours, and 7 days after immobilization, with and without motion loading. (B) MMP-3 activity after 7 days immobilization with and without motion loading. (C) Immunohistochemistry of MMP-3, ADAMTS-5 and Safranin-O staining of control, 7day immobilized, immobilized + passive motion loaded cartilage, and NNGH treated cartilage from medial condyles. Data show mean + SEM (n = 5). * = P < 0.05 versus unimmobilized control; + = P < 0.05 versus immobilization. Bar = 100μm.