CYCLOSPORINE-A AND ITS NONIMMUNOSUPPRESSIVE DERIVATIVE EXHIBIT A DIFFERENTIAL EFFECT ON CELL-MEDIATED MINERALIZATION IN CULTURE

Chronic immunosuppressive treatment with cyclosporin A (CsA) is associ ated with decreased bone density. However, in culture, CsA inhibits os teoclast differentiation and bone resorption. This raises the question as to whether CsA also affects osteoblast function. Immunophilin, one of the CsA-binding cyclophilins that is implicated in the immunosuppr essive action of CsA via calcineurin, is a peptidyl prolyl cis-trans i somerase (PPI). CsA also binds a mitochondrial membrane PPI which is i mplicated in controlling permeability transition pores. Therefore, in the present study we tested the effect of CsA on cell mediated mineral ization in parallel with mitochondrial rhodamine retention as an indic ator of mitochondrial membrane potential. Rat marrow stromal cells wer e grown in dexamethasone (DEX) medium to stimulate mineralization in c ulture, and CsA was added to various cultures using different treatmen t schedules. Low dose (0.1 mu M) CsA inhibited mineralization, compare d to controls, when present in the cultures during days 3-11 of DEX st imulation. Contrarily, high dose CsA (1.0 mu M) resisted the inhibitor y effect of the low dose. SDZ 220-384 (SDZ), a non-immunosuppressive d erivative of CsA which is known, like CsA, to bind to mitochondrial cy clophilin but does not inhibit calcineurin, was also tested. Both high and low doses of SDZ decreased mineralization when present in the cul tures from day 3 or from day 0. The similar effect of the low CsA dose and SDZ on mineralization is in accord with their ability to block pe rmeability transition pores. The differential effect, on day 21 minera lization, between high CsA dose and SDZ took place in parallel to thei r opposing effects on mitochondrial membrane potential. On days 4-8, m itochondrial rhodamine retention was higher under CsA than under SDZ. Under these conditions there was no significant difference between the effects of these drugs on cell proliferation measured on day 11, ther e was a minor decrease in specific alkaline phosphatase activity by SD Z, too small to explain the extent of mineralization inhibition by SDZ . These results suggest that permeability transition pores might be in volved in controlling mineralization. Unlike SDZ, CsA exhibits an addi tional effect on the mitochondrial membrane potential and on mineraliz ation when applied at a high dose on day 3. Therefore identifying the additional activity of high dose CsA, which is missing in SDZ, may be beneficial. Such activity is expected to resist changes in rhodamine r etention and decreased mineralization induced by SDZ, and yet enable p reservation of immunosuppressive activity of CsA. (C) 1997 Wiley-Liss, Inc.