title: The HIV-1 Tat protein enhances megakaryocytic commitment of K562 cells by facilitating CREB transcription factor coactivation by CBP.
abstract: The human immunodeficiency virus type 1 (HIV-1) Tat protein regulates transcription factor functions and alters cellular gene expression. Because hematopoietic progenitor cell (HPC) differentiation requires activation of lineage-specific transcription factors, Tat may affect hematopoiesis in HIV-1-infected micro-environments. We have monitored the molecular effects of Tat on megakaryocytic differentiation in the HPC line, K562. Flow cytometry analysis of CD61 indicated that phorbol myristate acetate (PMA) (16 nM) stimulated megakaryocytic commitment of K562 cells was increased (3- to 4-fold) following exposure to Tat (1-100 ng/ml). Activation of the megakaryocytic transcription factor cAMP regulatory element binding protein (CREB) and its coactivation by the CREB binding protein (CBP) was subsequently monitored. CREB phosphorylation and DNA binding were measured by Western immunodetection and electrophoretic mobility shift assays (EMSA), respectively. Within 2 hrs after stimulation, Tat increased both CREB phosphorylation and DNA binding by 7- to 10-fold. Transient cotransfection with CREB reporter and CBP expression plasmids demonstrated that Tat treatment increases (3- to 4-fold) both PMA-stimulated and CBP-mediated transcription via the cAMP regulatory element. Histone acetyl transferase (HAT) activity was increased (8- to 10-fold) in Tat-stimulated cells, which suggested increased chromosomal accessibility of transcription factors. Two-hybrid cotransfection assays using reporter plasmid containing the GAL4 DNA-binding domain and expression plasmid coding for the GAL4-CBP fusion protein, showed that Tat increases (2-fold) CBP-mediated coactivation of CREB. Both reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis showed that Tat treatment increases CBP gene expression (7- to 9-fold) and protein levels (5- to 7-fold) within 6-12 hrs after stimulation. Our findings indicated that Tat treatment increases both CREB function and CREB coactivation by CBP, which may facilitate megakaryocytic commitment of K562 cells. Induction of this molecular signaling by HIV-1 Tat protein may have relevance in understanding the HIV-induced hematologic manifestations and possibly in regulation of viral infectivity parameters in progenitor cell reservoirs.
authors.affiliation: Department of Pharmacology, Tulane University Health Sciences Center, New Orleans, LA 70112, USA.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Williams
authors.forename: Debasis
authors.initials: D
authors.lastname: Mondal
authors.forename: Krishna C
authors.initials: KC
authors.lastname: Agrawal
mesh_terms: D050882:CREB-Binding Protein / Q000378:metabolism*; D002273:Carcinogens / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D015696:Gene Products, tat / Q000378:metabolism* / Q000494:pharmacology; D015658:HIV Infections / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D039661:Integrin beta3 / Q000096:biosynthesis; D020014:K562 Cells; D008533:Megakaryocytes / Q000378:metabolism*; D010766:Phosphorylation; D020218:Response Elements / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D015854:Up-Regulation / Q000187:drug effects; D054322:tat Gene Products, Human Immunodeficiency Virus
chemical_list: C115163:CREB1 protein, human; D002273:Carcinogens; D017362:Cyclic AMP Response Element-Binding Protein; D015696:Gene Products, tat; C061248:ITGB3 protein, human; D039661:Integrin beta3; D054322:tat Gene Products, Human Immunodeficiency Virus; D050882:CREB-Binding Protein; C494363:CREBBP protein, human; D013755:Tetradecanoylphorbol Acetate