TRANSFORMING GROWTH-FACTOR-BETA - IS IT A DOWNREGULATOR OF STEM-CELL INHIBITION BY MACROPHAGE INFLAMMATORY PROTEIN 1-ALPHA

Transforming growth factor beta1 (TGF-beta1) and macrophage inflammatory protein 1alpha (MIP-1alpha) have recently been identified as potent inhibitors of hemopoietic stem cell proliferation. From previous studies, these molecules appear to have similar functions in the control of stem cell proliferation. This study was designed to investigate the relationship, if any, between these two negative regulators in an attempt to elucidate possible distinctive roles for each within the hemopoietic system. We report here that both MIP-1alpha and TGF-beta are capable of inhibiting the same stem cell population (colony-forming unit [CFU]-A/CFU-S) with similar potencies. We further show that TGF-beta potently inhibits MIP-1alpha gene expression in bone marrow-derived macrophages, the presumed source of MIP-1alpha in the bone marrow. This inhibition is not specific to MIP-1alpha in that expression of MIP-1beta, a related molecule that does not exhibit potent stem cell inhibitory properties, is inhibited in a similar manner. The inhibition of MIP-1alpha gene expression is also seen as a reduction in MIP-1alpha protein production, which markedly decreases 24 h after treating RAW 264.7 cells, a murine macrophage cell line, with TGF-beta. These in vitro results suggest that in the presence of active TGF-beta in vivo, and in the absence of upregulators of MIP-1alpha transcription, very little MIP-1alpha will be produced. To address how MIP-1alpha's target cells, the stem cells, would respond to TGF-beta, and the consequently low levels of MIP-1alpha produced, we analyzed the effect of TGF-beta on MIP-1alpha receptor levels on FDCP-MIX cells, a murine stem cell line. We show that TGF-beta (100 pM) reversibly downregulates MIP-1alpha receptor levels on these cells to a maximum of 50-70% after 24 h. This level of downregulation does not change upon increasing the concentration of TGF-beta or the length of exposure of the cells to TGF-beta. Scatchard analysis shows that TGF-beta downregulates MIP-1alpha receptor numbers with no change in affinity of the remaining receptors. These results suggest that TGF-beta may be capable of interfering with MIP-1alpha's role as a stem cell inhibitor. Indeed, they suggest that in the presence of active TGF-beta in vivo, MIP-1alpha is at best a weak contributor to the overall physiological inhibition of stem cells.