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Induction of MMP-3 by Hyaluronan Oligosaccharides in Temporomandibular Joint Chondrocytes

¹ Department of Biochemistry, Rush Medical College, Rush University Medical Center, 1653 W. Congress Parkway, Chicago, IL 60612, USA; and
² Department of Orthodontics and Craniofacial Developmental Biology, Division of Cervico-Gnathostomatology, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan;

View larger version (113K): [in a new window]   Figure 1. Distribution of HA in TMJ condyle cartilage. Cryostat sections of TMJ articular cartilage were incubated with B-HABP for the visualization of HA distribution (B,E) or BSA (control staining; C,F). Rhodamine-red fluorescence reveals HA deposition; nuclei are stained with DAPI. Panels A and D show brightfield views of sections depicted in panels B and E, respectively. SL, superficial layer; FZ, fibrous zone; PZ, proliferative zone; TZ, transitional zone; HZ, hypertrophic zone; bars, 70 µm.

View larger version (85K): [in a new window]   Figure 2. Expression of CD44 in TMJ condyle cartilage. Cryostat sections of TMJ articular cartilage were incubated with biotinylated anti-CD44 antibody (C,D,G,H) or biotinylated-rat IgG2b (E,F), with rhodamine-red detection and DAPI nuclear stain. Panels A and B show brightfield views of sections depicted in panels C and D, respectively. Regions identified by the rectangles in C and D are shown at higher magnification in panels G and H. Bars = 70 µm in panels A-F and 20 µm in panels G and H.

View larger version (105K): [in a new window]   Figure 3. Effect of HAoligos on cartilage matrix loss in the TMJ condyle. (A) TMJ articular cartilages were incubated in serum-free medium without or with HAoligos for 4 days. Cryostat sections of articular cartilage were stained with Safranin-O/fast green. Magnified views (x4) of the surface layer are shown in the insets. Bars = 100 µm. (B) Caseinolytic activity in the conditioned medium from cartilage explants treated with (+) or without (–) HAoligos for 2 or 4 days was assessed by casein zymography; clear bands represent enzymatic degradation of Coomassie-blue-stainable casein substrate. FZ, fibrous zone; CZ, cartilaginous zone.

View larger version (24K): [in a new window]   Figure 4. Effect of HAoligos on MMP-3 expression. TMJ chondrocytes exhibit dose-dependent (A) and time-course-dependent (B) effects of HAoligos on MMP-3 mRNA. Shown are calculated ratios of the treated samples (black bars) to untreated controls (white bars). Caseinolytic activity in the conditioned medium of TMJ chondrocytes treated for 24 hrs with increasing concentrations of HAoligos (C) or ± 250 µg/mL HAoligos for 12 or 24 hrs (D) was assessed by casein zymography. Panel E illustrates the reversibility of these HAoligos effects following wash-out of HAoligos or wash-out in the presence of HMW-HA. Real-time PCR was performed, and changes in MMP-3 mRNA expression are depicted as fold-increase ratios of treated samples to untreated controls. Data represent the mean ± SD of three experiments. *p < 0.05; **p < 0.01.