Transforming growth factor-beta(1) interferes with thrombopoietin-induced signal transduction in megakaryoblastic and erythroleukemic cells

Objective. Thrombopoietin (TPO) and transforming growth factor-beta (1) (TG F-beta (1)) have been shown to exert opposite effects on proliferation and megakaryocytic differentiation of hematopoietic cells. To determine whether TGF-beta (1) interferes directly with TPO-induced signal transduction in h ematopoietic cells, we compared the regulatory effects in the TPO-responsiv e cell lines Mo-7e and HEL. Material and Methods. The cells were stimulated by 100 ng/mL TPO and/or 100 ng/mL TGF-beta (1) and analyzed for proliferation (H-3 thymidine incorpora tion), viability (trypan blue exclusion), and protein expression and phosph orylation (Western blot). Results. TPO enhanced the proliferation of Mo-7e cells as determined by H-3 -thymidine incorporation, whereas TGF-beta (1) suppressed baseline cell gro wth and antagonized the proliferative effect of TPO. TPO-induced proliferat ion also was reduced by a specific inhibitor of the mitogen-activated prote in kinase (MAPK) pathway (PD098059), which inhibits activation of the MAPK extracellular signal-regulated kinases (ERK) ERK1 and ERK2, and AG490, an i nhibitor of Janus kinase-2, which completely blocked TPO-induced proliferat ion. As demonstrated by Western blotting, TGF-beta (1) reduced the TPO-stim ulated ERK1/ERK2 and STAT5 phosphorylation in Mo-7e and HEL cells. This eff ect was completely reversed by preincubation with a tyrosine phosphatase in hibitor (Na3VO4), which suggests that TGF-beta (1) activated a phosphatase, Although STAT3 also was activated by TPO, STAT3 activation remained unalte red by TGF-beta (1). Conclusion. Taken together, these data suggest that TGF-beta (1) modulates TPO-mediated effects on megakaryocytic proliferation by interfering with TP O-induced signal transduction, particularly by reducing the activities of M APK ERK1/ERK2 and STAT5, (C) 2001 International Society for Experimental He matology. Published by Elsevier Science Inc.