THE EFFECTS OF NATURAL AND SYNTHETIC RETINOIDS ON THE DIFFERENTIATIONOF RCJ C5.18 CHONDROGENIC CELLS

RCJ C 5.18 (C 5.18) is a chondrogenic clonal cell line which, under st andard culture conditions, develops chondroblastic features including the production of a cartilagenous matrix. Retinoic acid (RA) is known to inhibit the chondrogenic differentiation of C 5.18 cells and this m ay parallel the teratogenic effects of retinoids in vivo; however, the question as to which of the 3 retinoic acid receptors (RAR alpha, bet a, gamma) or the 3 retinoid X receptors (RXR alpha, beta, gamma) media te this RA-induced inhibition remains unanswered. We tested several re tinoids with different receptor binding characteristics. Cartilage for mation in C 5.18 cultures was evaluated by counting the number of cart ilage nodules formed, and by quantitating the glycosaminoglycan conten t of the cultures using alcian blue staining. All of the retinoids pre vented cartilage formation in a dose-dependent manner. Treatment with the retinoids did not affect cell number, thereby ruling out any toxic effects. RA, which binds to all 3 RARs with similar affinity, produce d a 50% inhibition (IC50) of cartilage formation at 4 x 10(-10) M. We also tested Ch55, which also binds to all 3 RARs, but with higher affi nity than RA. This compound was approximately 10 times more potent tha n RA (IC50 2 x 10(-11)M). 9-cis RA, which binds to the 3 RARs with aff inities similar to RA and also binds to the 3 RXRs, was less active (I C50 8 x 10(-9) M), suggesting that RXR binding interferes with the inh ibitory effect of ligand-activated RARs. 9-cis retinal, for which the binding characteristics are unknown, had the same effect as 9-cis RA. The synthetic retinoids Am80 (IC50 6 X10(-11) M) and Am580 (IC50 4 X 1 0(-11) M) were more potent than RA in inhibiting cartilage formation. Since these compounds bind only to RAR alpha and beta, and with higher affinity than RA, the observed inhibition of cartilage formation sugg ests that interaction of the ligand with RAR alpha and/or beta is suff icient to induce the effects on cartilage development. If reports sugg esting that Am580 binds exclusively to RAR alpha are correct, these fi ndings could imply that RAR alpha alone is sufficient to mediate the i nhibitory effect of the retinoids. Surprisingly, Re80, which binds to RAR alpha and beta with a binding affinity similar to RA (and does not bind significantly to RAR gamma or the RXRs), was the most potent ret inoid in our system (7 x 10(-13) M). This finding supports mediation o f the retinoid effect through alpha and beta receptors. Our results su ggest that the retinoids act via RAR alpha and/or beta to inhibit chon droblast differentiation. (C) 1994 Wiley-Liss, Inc.