title: Levels of phospho-Smad2/3 are sensors of the interplay between effects of TGF-beta 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-beta (TGF-beta) receptor serine/threonine kinases, in TGF-beta-dependent differentiation of hematopoietic cells, using as a model the human promyelocytic leukemia cell line, HL-60. TGF-beta-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 D(3) also induced phosphorylation of Smad2/3 and monocytic differentiation; however the effects were indirect, dependent on its ability to induce expression of TGF-beta1. Simultaneous treatment of these cells with TGF-beta1 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-beta1 alone. TGF-beta1 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-beta-induced phospho-Smad2 and shifts the differentiation toward monocytic end points. In HL-60R mutant cells, which harbor a defective retinoic acid receptor-alpha (RAR-alpha), ATRA is unable to reduce levels of TGF-beta-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-beta, whereby a putative RAR-alpha-dependent phosphatase activity limits the levels of phospho-Smad2/3 induced by TGF-beta, ultimately reducing the levels of nuclear Smad complexes mediating the TGF-beta-dependent differentiation of the cells to monocytic end points.
authors.affiliation: Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, Bethesda, MD 20892, USA.
authors.forename: Zhouhong
authors.initials: Z
authors.lastname: Cao
authors.forename: Kathleen C
authors.initials: KC
authors.lastname: Flanders
authors.forename: Daniel
authors.initials: D
authors.lastname: Bertolette
authors.forename: Lyudmila A
authors.initials: LA
authors.lastname: Lyakh
authors.forename: Jens U
authors.initials: JU
authors.lastname: Wurthner
authors.forename: W Tony
authors.initials: WT
authors.lastname: Parks
authors.forename: John J
authors.initials: JJ
authors.lastname: Letterio
authors.forename: Francis W
authors.initials: FW
authors.lastname: Ruscetti
authors.forename: Anita B
authors.initials: AB
authors.lastname: Roberts
mesh_terms: D002454:Cell Differentiation / Q000187:drug effects; D002762:Cholecalciferol / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D004347:Drug Interactions; D006098:Granulocytes / Q000166:cytology* / Q000187:drug effects; D018922:HL-60 Cells; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009000:Monocytes / Q000166:cytology* / Q000187:drug effects; D010766:Phosphorylation / Q000187:drug effects; D015398:Signal Transduction; D051899:Smad2 Protein; D051900:Smad3 Protein; D015534:Trans-Activators / Q000378:metabolism / Q000502:physiology*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1; D014212:Tretinoin / Q000494:pharmacology*
chemical_list: D004268:DNA-Binding Proteins; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; D051899:Smad2 Protein; D051900:Smad3 Protein; C508960:TGFB1 protein, human; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D002762:Cholecalciferol; D014212:Tretinoin; D020928:Mitogen-Activated Protein Kinases