Involvement of insulin-like growth factor-1 and its binding proteins in proliferation and differentiation of murine bone marrow-derived macrophage precursors

Abstract

Insulin-like growth factor 1 (IGF-1) and its binding proteins (IGFBPs) are involved in proliferation and differentiation of many cell types. In the present study, the involvement of IGF-1 and IGFBPs in proliferation and differentiation of murine bone marrow-derived macrophages (BMDM) was investigated. L929-conditioned media (LCM) containing abundant macrophage colony-stimulating factor CSF-1 were used to stimulate BMDM development from their bone marrow precursors. The alteration of IGF-1 and IGFBPs during LCM-induced BMDM proliferation and differentiation was first studied. The cells were cultured in RPMI complete media containing 20% LCM for different time periods and then incubated in serum-free media for 24 h. The supernatants were collected for Western ligand blotting and immunoblotting analyses, and the cell pellets for Northern blotting analyses. The mRNA level of IGF-1 increased in a time-dependent manner. An increase of IGFBP-4 accumulation in the conditioned media was also observed during this process. However the mRNA expression of IGFBP-4 remained constant, indicating a posttranscriptional regulation of IGFBP-4 secretion and/or stability. The effects of exogenous recombinant human IGF-1 (rhIGF-1) on BMDM proliferation and differentiation were further studied. Two IGF-1 analogs (long R3 IGF-1 and des [1-3] IGF-1) were also used in parallel with regular IGF-1 to indicate the involvement of IGFBPs in BMDM development. Cells were cultured in complete media containing 20% LCM for different time periods, and then incubated in serum-free media in the presence of rhIGF-1 or its analogs for 24 h. These three forms of IGF-1 all potentiated the proliferation of freshly isolated BMDM precursors (d 0). rhIGF-1 and long R3 IGF-1, but not des (1-3) IGF-1, continued to stimulate the cell proliferation on d 1. The effects of these three forms of IGF-1 on BMDM differentiation were investigated using mannose receptor expression as a marker. Long R3 IGF-1 and des (1-3) IGF-1, but not rhIGF-1, enhanced BMDM differentiation on d 4. The different effects of rhIGF-1 and its analogs on BMDM differentiation suggest that the accumulation of IGFBP-4 in BMDM development might have an inhibitory effect on IGF-1 actions by sequestering free IGF-1.