Fluid flow-induced tyrosine phosphorylation and participation of growth factor signaling pathway in osteoblast-like cells

To investigate how mechanical loading stimulates bone cells, we subjected m urine osteoblast-like cells, MC3T3E1, to fluid flow generated by shaking cu lture dishes. Since we had previously found that egr-1 mRNA is up-regulated by the flow, and that the regulation involves tyrosine kinase, we examined which proteins are tyrosine-phosphorylated by flow. Western blotting and i mmunoprecipitation of cell lysates showed tyrosine phosphorylation enhancem ent of many proteins, including ERK2 and Shc, and activation of ERK1/2. Alt hough these responses did not occur in serum-free media, addition of EGF or bFGF recovered the responses. AC1478, an inhibitor of ECF receptor kinase activity, abolished tyrosine phosphorylation enhancement, ERK1/2 activation , and egr-1 mRNA accumulation induced by the flow of EGF-containing serum-f ree media. These results suggest that growth factor signaling pathways are involved in these responses. Repetition of fluid flow induced repeatedly up -regulation of egr-1 mRNA. Such events may also occur in bone under mechani cal loading. (C) 2000 Wiley-Liss, Inc.