Figure 4.

Bone-resorbing activity and expression of RANKL and MMPs in coculture of mouse calvaria and MDA-231 cells. (A) Illustrations of the experimental procedure of coculture of mouse calvaria and MDA-231 cells. Mouse calvariae were cocultured for 5 days with MDA-231 cells. As a positive control of bone resorption, calvariae were cultured for 5 days with 2 ng ml⁻¹ of IL-1. MDA-231 cells (1 × 10⁵ cells) were cultured for 5 days as MDA-231 control without calvaria. (B) Conditioned media were collected, and the concentration of calcium in the medium was measured to monitor the bone-resorbing activity. Data are expressed as means±s.e.m. of six cultures. Significantly different from the control (^*P<0.001). (C) Total RNA was extracted from calvaria-excluded MDA-231 cells, and Northern blotting was performed using ³²P-labelled cDNA probes for RANKL as described in Materials and Methods. (D) Conditioned media were collected and gelatin zymography (upper panel) and Western blotting (lower panel) were performed as described in Materials and Methods. In the upper panel, gelatinase activities corresponding to pro-MMP-2, active-MMP-2 and pro-MMP-9 are indicated by arrows. In the lower panel, MMP-13 was detected using anti-mouse MMP-13 antibody. Note that the marked expression of RANKL, MMP-2 and MMP-13 was detected in the calvaria cocultured with MDA-231 cells, and that the levels were similar to those indued by IL-1.