Figure 3

Induction of matrix metalloproteinases by relaxin but not by estrogen is accompanied by loss of glycosaminoglycans (GAGs). (a) Disc explants were cultured for 48 hours in basal control medium (Ct), β-estradiol (Es, 20 ng/ml), or relaxin (R, 0.1 ng/ml) or in β-estradiol plus relaxin (Es+R), then sectioned and stained with Safranin-O for GAGs. The percentage area staining positive for GAGs was determined histomorphometrically and plotted. Values are means ± SD. (b) Hormone-mediated changes in GAG synthesis were assessed by ³⁵S-labeling of fibrocartilaginous disc explants. The explants were washed and digested with papain, and the radioactivity was measured. Fold changes (means ± SD) in ³⁵S incorporated into the explants incubated with hormones relative to that in control discs were determined and plotted. (c) To evaluate the modulation of tissue inhibitor of metalloproteinases-1 (TIMP-1) by hormones, the conditioned medium, standardized dry tissue weight (mg), was resolved electrophoretically and transferred to nitrocellulose membranes, and the membranes were probed with anti-TIMP-1 antibody. (d) The bands were quantified by videodensitometry, and the fold induction (mean ± SD) of TIMP-1 by various hormone treatments relative to untreated control explants was plotted. T-1, TIMP-1. * P < 0.05, ** P < 0.01 by Fisher's test.