SYNERGISTIC EFFECTS OF PARATHYROID-HORMONE AND 1,25-DIHYDROXYVITAMIN-D-3 ON PROLIFERATION AND VITAMIN-D-RECEPTOR EXPRESSION OF RAT GROWTH CARTILAGE CELLS

We investigated possible interaction of 1,25-dihydroxyvitamin D-3 [1,25-(OH)(2)D-3] and PTH on: 1) proliferation (monolayer culture) and colony formation (agarose stabilized suspension cultures); 2) expression of 1,25-(OH)(2)D-3 receptor (VDR); and 3) cAMP response to PTH, using primary cultures of chondrocytes from rat tibia proximal epiphysis. 1 alpha,25-(OH)(2)D-3 stereospecifically stimulated DNA synthesis, cell counts, and colony formation at low concentration (10(-12) M). Within 6 h bovine PTH (bPTH)(1-34), human PTH (hPTH)(28-48) (10(-10) M), (Bu)(2)cAMP (1-2 mM), and 12-O-tetradecanoyl-13-acetate (10(-8) M) increased [H-3]thymidine incorporation in the absence and presence of 1,25-(OH)(2)D-3. Both PTH fragments also stimulated chondrocyte growth and colony formation in a Ca-dependent fashion. Prolonged exposure to bPTH(1-34) or hPTH(28-48) did not affect baseline DNA synthesis but increased the stimulatory effect of 1,25-(OH)(2)D-3. This increase was inhibited in the presence of H7 (inhibition of PKC) or the monoclonal hPTH(1-38) antibody A1-70. In subconfluent chondrocyte cultures VDR was up-regulated by bPTH(1-34) and hPTH(28-48) (10(-10) M) or activators of protein kinase C (PKC), but not by (Bu)(2)cAMP. It was blocked by cycloheximide and actinomycin D and persisted in the presence of Ca-channel blockers. Inhibition of PKC by H7 also blocked the effect of bPTH(1-34) on VDR. The cAMP response to bPTH(1-34) was not affected by 1,25-(OH)(2)D-3. We conclude that: 1) DNA synthesis, cell proliferation, and colony formation in chondrocyte monolayer or suspension cultures is increased by aminoterminal and midregional PTH fragments and by cAMP analogs in a Ca- dependent fashion; 2) bPTH(1-34) and hPTH(28-48) up-regulate VDR by cAMP-independent, PKC-dependent steps requiring transcriptional and translational processes; both PTH fragments also amplify the effect of 1,25-(OH)(2)D-3 on DNA synthesis; and 3) no difference is found between the bPTH(1-34) and hPTH(28-48) fragments with respect to chondrocyte proliferation and VDR up-regulation, although the two differ with respect to stimulation of cAMP production.