LONG-ACTING CAMP ANALOGS ENHANCE SULFATE INCORPORATION INTO MATRIX PROTEOGLYCANS AND SUPPRESS CELL-DIVISION OF FETAL RAT CHONDROCYTES IN MONOLAYER-CULTURE

The relationship between replication and the synthesis of matrix sulfated proteoglycans was investigated with fetal rat chondrocytes grown in monolayer culture. The effect of N6 O2'' dibutyryl cyclic[c]AMP [DBcAMP], cAMP, 8 Bromo cyclic[c]AMP [8 Br cAMP], sodium butyrate and hydroxyurea was examined. Between 0.05 and 0.5 mM DBcAMP, a dose related inhibition of cell division and stimulation of 35SO42- incorporation into matrix proteoglycans was demonstrated. At the higher concentrations of DBcAMP, cell division was completely inhibited and the enhancement of 35SO42- incorporation into matrix proteoglycans ranged between 40 and 120% (P < 0.01). Utilizing 14C-glucosamine and photometric determination of proteoglycans with Alcian Blue, it was demonstrated that the increased sulfate incorporation reflected enhanced extracellular matrix accumulation. The DBcAMP effects were mimicked by 8 Br-cAMP, suggesting they were mediated by the adenylate cyclase system. cAMP (0.05-0.5 mM), sodium butyrate (0.1-0.5 mM) and hydroxyurea (0.5-5 mM) partially or fully inhibited cell division, but failed or only slightly enhanced sulfate incorporation. The enhanced sulfated proteoglycan deposition promoted by DBcAMP began 8-12 h after serum stimulation, its onset occurred prior to thymidine incorporation and the effect persisted for 28 h. Cell volume determination demonstrated an increase in size of DBcAMP treated chondrocytes between 8 and 12 h, coincident with the onset of increased sulfate incorporation. These results are consistent with a model where matrix sulfated proteoglycan deposition by chondrocytes is mediated by intracellular cAMP levels and occurs in the G1 phase of the cell cycle.