Levels of Smad7 regulate Smad and mitogen activated kinases (MAPKs) signaling and controls erythroid and megakaryocytic differentiation of erythroleukemia cells.

Smad and MAPK signaling cascades are involved in erythroid and megakaryocytic differentiation. The inhibitory Smad for TGF-beta/activin signaling, Smad7, may directly or indirectly affect these signaling pathways. By modulating Smad7 expression, we attempted to delineate the relevance of Smad7 during erythro-megakaryocytic (E/M) differentiation of human erythroleukemia cells. Smad7 transcripts were detected at low levels in different erythroleukemia cell lines (TF-1, HEL and K562). Reduction of expression of endogenous Smad7 by RNA interference enhanced erythroid differentiation of K562 cells in response to physiological doses of activin-A/TGF-beta1. Stable over-expression of Smad7 in K562 cells (K562/7) prevented activation of Smad2/3 and MAPK (ERK1/2, p38 and JNK1/2) proteins by activin-A/TGF-beta1 and subsequent induction of erythroid differentiation. High levels of Smad7 also interfered with hydroxyurea- and butyrate-, but not hemin-induced erythroid differentiation. Interestingly, K562/7 cells were found to harbor a significant proportion (about 35%) of large ploy nucleated cells compared to fewer than 12% in control cells. K562/7 cells treated with phorbol 12-myristate 13-acetate (PMA), showed a great shift in ploidy towards high ploidy classes (> or =8N) accompanied with an increase in the expression of the maturation marker CD42b. We showed here that: (a) low levels of endogenous Smad7 in erythroleukemia cells are physiologically relevant, and (b) high levels of Smad7 interferes with TGF-beta/activin-induced Smad/MAPK signaling and erythro-differentiation and promotes megakaryocytic differentiation, possibly by blocking autocrine TGF-beta.