SRC PROTEIN AND TYROSINE-PHOSPHORYLATED PROTEIN PROFILES IN MARROW STROMA DURING OSTEOGENIC STIMULATION

Src protein is essential for the regulation of bone turnover primarily via bone resorption because it is required in osteoclast differentiat ion and function. We followed temporal changes oi Src protein abundanc e in marrow stromal cells induced to mineralize by dexamethasone (DEX) , growth in cold temperature, or both. Given the tyrosine kinase funct ion of Src and its numerous substrates, profiles of phosphotyrosine-co ntaining proteins were followed as well. On day 11 of stimulation, spe cific alkaline phosphatase (ALP) activity at 30 degrees C decreased un der DEX relative to 37 degrees C cultures, in accord with increased ce ll counts. Mineralization per well under DEX increased by 25% at 37 de grees C, whereas at 30 degrees C it increased by more than threefold r egardless of the DEX stimulation. At 30 degrees C, on a per cell basis mineralization increased 2.5 and 3 times with and without DEX, respec tively. Cultures at 37 degrees C showed a general drop per fell of man y phosphotyrosine-containing proteins on day 3 relative to days 1 and 2 in both DEX-stimulated and nonstimulated cultures; several proteins did recover (recuperate) thereafter. On days 1 and 2, the phosphotyros ine signal was higher in several proteins under DEX stimulation; this trend became inverted after day 3. The changes in abundance per cell o f Src protein (pp60src) followed a similar trend, and in addition a tr uncated Src molecule, p54/52src, was detected as a putative cleavage p roduct presumably representing its carboxy terminus. The pp60src was m ost abundant, relative to its truncated product, in day 7 nonstimulate d cultures, whereas under DEX stimulation the truncated species pp54/5 2src showed the highest relative abundance on days 7. At 30 degrees C, DEX stimulation accentuated the increase in Src protein on day 3, sho wed no change on day 7, and returned to increase Src protein on day 10 . Potassium ionophorvalinomycin, considered to select against minerali zing osteoprogenitors at 30 degrees C, showed on day 10 in the absence of DEX a relative increase in truncated Src protein compared to both DEX-stimulated and nonstimulated cultures in the absence of valinomyci n. On day 7 of DEX stimulation, the presence of valinomycin resulted i n low p54/52src. Among phosphotyrosine-containing proteins, a 32-34 kD a band, as yet unidentified, showed the most concordant changes with m ineralization induction. P32-34 decreased by DEX on days 2 and 8 and i ncreased by low temperature alone or combined with DEX on day 3. On da y 7, p32-34 did not change under DEX, but valinomycin selected cells w ith less phoshpotyrosine-containing p32-34. Taken together, high Src a bundance at the start of osteogenic induction followed by a decrease 1 week later is probably related to energy metabolism-dependent inducti on of mineralization. This is in temporal accord with the increase in Src truncation and fluctuation in mitochondrial membrane potential (wh ich affects mineralization). The reported binding of amino-terminal Sr c oligopeptide to p32 ADP/ATP carrier in the mitochondrial inner membr ane raises the question of its possible involvement in mitochondria-re gulated mineralization. (C) 1998 Wiley-Liss, Inc.