HEPATOCYTE GROWTH-FACTOR IS INVOLVED IN FORMATION OF OSTEOCLAST-LIKE CELLS MEDIATED BY CLONAL STROMAL CELLS (MC3T3-G2 PA6)/

Osteoclast formation from hemopoietic precursors has been shown to req uire the support of stromal cells in bone tissue. In this study, we de monstrated that hepatocyte growth factor (HGF) is one of the stromal c ell-derived molecules responsible for osteoclast-like cell formation. For our experiments, we used a coculture system for osteoclastic cell formation and activation in which hemopoietic blast cells are cocultur ed with calvaria-derived stromal MC3T3-G2/PA6 (PA6) cells on dentine s lices in the presence of 1,25-dihydroxyvitamin D-3 [1,25(OH)(2)D-3]. A ddition of anti-HGF neutralizing Ige to the cocultures inhibited the f ormation of osteoclastic cells and their dentine-resorbing activity. W e detected a single 6.0-kb transcript for HGF in PA6 cells, and also r ecognized immunoreactive Mr 81,000 and 88,000 forms of HGF in conditio ned medium (CM) from PA6 cell cultures, the level of which reached 6 n g/ml. Both the CM and HGF stimulated the proliferation of blast cells synergistically with granulocyte-macrophage colony-stimulating factor, resulting in an increased number of osteoclast precursors that respon d to 1,25(OH)(2)D-3 that are tartrate-resistant acid phosphatase-posit ive multinucleate cells in stromal cell-free blast cell cultures in pl astic wells. The effect of the CM was diminished by the addition of an ti-HGF IgG. However, neither the CM nor HGF stimulated the formation o f osteoclastic cells and pits on dentine slices in the absence of PA6 cells. These results suggest that although HGF cannot completely repla ce stromal cells, it is one of the paracrine mediators produced by str omal cells that act on proliferation of osteoclastic cell precursors. (C) 1995 Wiley-Liss, Inc.