Differential Activation of MAPK Signaling Pathways by TGF-β1 Forms the Molecular Mechanism Behind Its Dose-Dependent Bidirectional Effects on Hematopoiesis

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Abstract
We have earlier reported that transforming growth factor-β1 (TGF-β1), a well-known inhibitor of hematopoiesis, stimulated colony formation from adult human bone marrow mononuclear cells (BM MNC) when used at low concentrations. We examined the possible molecular mechanism behind this bidirectional effect using CD34+ cells isolated from human BM for clonal assays and the KG1a cell line as a model system for analysis of proteins for signaling pathways by immunoblotting. We found that TGF-β1 at low doses (picogram levels) stimulated the colony formation from CD34+ cells, indicating that these progenitors form the direct target of stimulatory action of TGF-β1. CD34+ cells were found to be more sensitive to the TGF-β1 concentration than the total MNC. We used the KG1a cell line as a model system for identification of mitogen-activated protein kinase (MAPK) and AKT signaling pathways involved in the process. Low doses strongly induced p44/42 MAPK phosphorylation, whereas high doses induced p38 activation. Use of specific p44/42 MAPK inhibitor PD 98059 in the colony assay abrogated the stimulatory effect of low TGF-β1. On the other hand, use of p38 MAPK inhibitor SB 203580 along with low TGF-β1 concentrations had a synergistic effect on stimulation of colony formation. Treatment of BM MNC with Anisomycin, which activates stress kinases, resulted in a dose-dependent inhibition of colony formation. This inhibition could not be rescued by stimulatory doses of TGF-β1. Phosphorylation of AKT was found to occur in a dose-dependent way but declined slightly at the highest concentration used (10 ng/ml). Inhibition of the AKT pathway by LY 294002 strongly suppressed colony formation. These data indicate clearly that sustained activation of p44/42 MAPK perhaps forms the stimulatory signal induced by low TGF-β1, whereas activation of p38 forms the inhibitory pathway.