Levels of phospho-Smad2/3 are sensors of the interplay between effects of TGF-β and retinoic acid on monocytic and granulocytic differentiation of HL-60 cells

Abstract

We have investigated the role of Smad family proteins, known to be important cytoplasmic mediators of signals from the transforming growth factor–β (TGF-β) receptor serine/threonine kinases, in TGF-β–dependent differentiation of hematopoietic cells, using as a model the human promyelocytic leukemia cell line, HL-60. TGF-β–dependent differentiation of these cells to monocytes, but not retinoic acid–dependent differentiation to granulocytes, was accompanied by rapid phosphorylation and nuclear translocation of Smad2 and Smad3. Vitamin D3 also induced phosphorylation of Smad2/3 and monocytic differentiation; however the effects were indirect, dependent on its ability to induce expression of TGF-β1. Simultaneous treatment of these cells with TGF-β1 and all-trans-retinoic acid (ATRA), which leads to almost equal numbers of granulocytes and monocytes, significantly reduced the level of phospho–Smad2/3 and its nuclear accumulation, compared with that in cells treated with TGF-β1 alone. TGF-β1 and ATRA activate P42/44 mitogen-activated protein (MAP) kinase with nearly identical kinetics, ruling out its involvement in these effects on Smad phosphorylation. Addition of the inhibitor-of-protein serine/threonine phosphatases, okadaic acid, blocks the ATRA-mediated reduction in TGF-β–induced phospho-Smad2 and shifts the differentiation toward monocytic end points. In HL-60R mutant cells, which harbor a defective retinoic acid receptor–α (RAR-α), ATRA is unable to reduce levels of TGF-β–induced phospho-Smad2/3, coincident with its inability to differentiate these cells along granulocytic pathways. Together, these data suggest a new level of cross-talk between ATRA and TGF-β, whereby a putative RAR-α–dependent phosphatase activity limits the levels of phospho-Smad2/3 induced by TGF-β, ultimately reducing the levels of nuclear Smad complexes mediating the TGF-β–dependent differentiation of the cells to monocytic end points.

Targets of the transcriptional repressor oncoprotein Gfi-1

“…4B) and expression (Fig. 4C) of target genes during granulocytic maturation of HL-60 cells, induced to differentiate by treatment with ATRA (34)(35)(36) over the course of 4 days. Note that Gfi-1 is also expressed in HL-60 cells (Fig.…”

Retinoic acid induces TGFβ-dependent autocrine fibroblast growth

“…Similarly, T-RA increases expression of TGFb1 and the TGFb receptor in HL60 cells leading to an autocrine antiproliferative growth loop (Falk et al, 1991;Nunes et al, 1996). However, it has been shown that T-RA reduces SMAD phosphorylation in HL60 cells, thus negatively regulating TGFb-signalling and differentiation into monocytes (Cao et al, 2003). T-RA has also been shown to regulate TGFb expression and proliferation in embryonic palate mesenchymal cells (Nugent et al, 1998) and more recently, to regulate TGFb in the embryonal yolk sac to ensure visceral endoderm survival (Bohnsack et al, 2004).…”

Signal transduction pathways that contribute to myeloid differentiation

“…263,264 The differential effect of TGFb on monocytic versus granulocytic differentiation may be explained by the enhancement of Smad2/3 phosphorylation and nuclear translocation during vitamin D3-induced monocytic differentiation, and the reduction of Smad2/3 phosphorylation and nuclear accumulation during ATRA-induced granulocytic differentiation. [265][266][267] Conclusions/future perspectives Emerging evidence indicates that multiple signaling pathways are activated during cytokine-induced myeloid differentiation and that these pathways may play critical roles in facilitating the differentiation process. Moreover, the impact that activation of a given pathway has on normal myeloid differentiation may be markedly different from the impact that is seen when that pathway is activated in a differentiation-defective leukemia.…”