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.