The Effects of Transforming Growth Factor-B1 on IL-3 Induced Phosphorylation in Murine Primary Myeloid Cells and Cell Lines

Abstract

Studies of hematopoiesis have led to the observation that the regulation of this process is influenced by many growth factors which can have both positive and negative effects. Transforming growth factor-β (TGF-βl) is a member of a family of polypeptides which regulates cell growth and differentiation. It was the purpose of this study to investigate the possible mechanisms for the effects of TGF-β on hematopoietic progenitor cell proliferation. TGF-β is a direct, bidirectional modulator of colony stimulating factor (CSF) -induced hematopoietic progenitor cell growth (Keller and Ruscetti, 1991). These effects have been shown to correlate with TGF-β-induced modulation of CSF receptor expression (Jacobsen et al., 1991, Jacobsen et al., 1992). Since interleukin-3 (IL-3) and other CSF's have been shown to rapidly induce phosphorylation (5-10 min) of a variety of proteins in cell lines, the effects TGF-β would have on these early IL-3 induced signals in both a cell line and normal bone marrow were examined. The findings were then compared with CSF receptor down modulation on these cells. IL-3 induced the tyrosine phosphorylation of a variety of proteins in both normal murine bone marrow cells and in a cloned hematopoietic cell line, 32DCL-23, in a time-dependent manner. TGF-β inhibited IL-3 induced tyrosine phosphorylation that was detectable following 3 to 6 hrs of TGF-β pre-incubation, and this inhibition was maximal by 24 hrs. TGF-β alone had no significant effect on phosphorylation prior to 24 hrs. There were common proteins phosphorylated in response to IL-3 in normal bone marrow and in 32DCL-23, including a 140 kd protein, thought to be the receptor complex (Sorensen, et al. ,1989). The decrease in phosphorylation observed was a generalized decrease, affecting all but one of the proteins induced by IL-3 and the decrease in phosphorylation ranged from 2 to 3 fold. TGF-β did not affect early IL-3 induced phosphorylation. However, TGF-β did inhibit phosphorylation after 3 to 6 hrs of pre-incubation. This delayed effect correlated temporally with depressed CSF receptor expression on these cells, suggesting that receptor down modulation might represent one mechanism for the TGF-β induced inhibition of IL-3 stimulated phosphorylation.