SYNTHESIS OF MEMBRANE-BOUND AND MATRIX-BOUND COLONY-STIMULATING FACTOR-I BY CULTURED OSTEOBLASTS

Colony-stimulating factor-1 (CSF-1) is synthesized as a secreted or me mbrane-bound molecule. We investigated whether osteoblastic cells prod uce these forms of CSF-1. Glutaraldehyde-fixed cell layers supported p roliferation of the macrophage cell line BAC1.2F5, suggesting the pres ence of membrane- or/and matrix-associated CSF-1. Furthermore, CSF-1 a ctivity could be either extracted from the matrix or released from the cell membrane. A neutralizing antiserum against CSF-1 inhibited these activities. After labeling the cellular proteins with [S-35] met/cys or [S-35] SO42- CSF-1 was immunoprecipitated and analyzed by SDS-PAGE. Under nonreducing conditions, bands with MW more than 200, 200, 100, and 50 kd were detected. These bands shifted to lower MW under reducin g conditions. Treatment with chondroitin lyase ABC decreased the MW of the 200 kd monomer, proving the proteoglycan structure. Much smaller quantities of CSF-1 were found in the matrix extract than in the condi tioned medium. Transforming growth factor beta (TGF-beta) increased bo th the synthesis of CSF-1 and its accumulation in the matrix. CSF-1 re leased with trypsin from the membrane fraction yielded on SDS-PAGE a b and with MW of 60 and 30 kd under nonreducing and reducing conditions, respectively. Transcripts encoding both the secreted and the membrane -associated forms of the cytokine were detected in osteoblasts by reve rse transcription polymerase chain reaction. These data indicate that osteoblastic cells produce the secreted forms, either remaining in the culture supernatant, or being associated to the matrix, and the membr ane associated form of CSF-1. (C) 1996 Wiley-Liss, Inc.