OPPOSING EFFECTS OF CYCLOSPORINE-A AND TYRPHOSTIN AG-1478 INDICATE A ROLE FOR SRC PROTEIN IN THE CELLULAR CONTROL OF MINERALIZATION

Cyclosporin A (CsA) induces osteoporosis but not through direct activa tion of osteoclasts. CsA also inhibits cell-mediated mineralization in marrow stromal cell culture, whereas the tyrphostin AG-1478 increases mineralization. These antagonistic effects on mineralization were use d to discern molecules that underwent phosphorylation changes in assoc iation with their opposing effects on mineralization. In parallel, qua ntitative changes in Src protein were followed. Multiple dexamethasone (DEX)-stimulated stromal cell cultures were grown with and without a mineralization-inhibiting dose (0.1 mu M) of CsA and were harvested on different days of DEX stimulation. Immunoblots of gel-fractionated ce ll extracts showed that the most noticeable changes in tyrosine phosph orylated proteins (TPP) were seen on day 8 of DEX stimulation. At leas t 15 TPP bands, mostly smaller than 53 kDa, were more prominent in CsA -treated cultures on day 8. Under CsA, Src protein quantity decreased on day 8, but its cleavage product (52/54 kDa) was sixfold more abunda nt then on day 7. Day 8 was chosen to test the effect of AG-1478 on th e CsA-induced TPP changes. Dimethyl sulfoxide (DMSO) alone, the solven t of AG-1478, increased mineralization in CsA-treated versus CsA-untre ated cultures and slightly decreased Src and its cleavage product. AG- 1478 at 5 mu M, in CsA cultures increased the specific alkaline phosph atase activity threefold, with a slight change in mineralization relat ive to controls grown with DMSO alone. This was accompanied by decreas ed intensity of several TPP bands smaller than 36 kDa. In contrast, tr eatment with 50 mu M of AG-1478 increased the intensity of TPP bands a t the same molecular size range. This high AG-1478 dose decreased cell counts selecting mineralizing cells. The results indicate that increa sed Src protein cleavage product on day 8 by CsA is associated with mi neralization inhibition, which is opposed by DMSO and 50 mu M AG-1478, thus antagonizing the effect of CsA on mineralization. Direct or indi rect interaction between Src and TPP, antagonistically affected by CsA and AG-1478, is likely to underlay cellular control of mineralization . Changes in p19 and p29 intensity showed association with mineralizat ion that was reflected by a significant direct and inverse correlation , respectively, with calcium precipitation per cell. (C) 1998 Wiley-Li ss, Inc.