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Thromb Haemost 2008; 100(05): 821-828
DOI: 10.1160/TH08-07-0496
Platelets and Blood Cells
Schattauer GmbH

Phorbol 12-myristate 13-acetate (PMA) responsive sequence in Gαq promoter during megakaryocytic differentiation

Regulation by EGR-1 and MAP kinase pathway
Gauthami Jalagadugula
1   Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania, USA
,
Danny N. Dhanasekaran
2   Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania, USA
4   Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania, USA
,
A. Koneti Rao
1   Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania, USA
3   Departments of Medicine, Temple University School of Medicine, Philadelphia, Pennsylvania, USA
› Author AffiliationsFinancial support: This work was supported by grant NIH R01 HL56724 (AKR).
Further Information

Publication History

Received 31 July 2008

Accepted 10 August 2008

Publication Date:
22 November 2017 (online)

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Summary

Gαq plays a major role in platelet signal transduction, but little is known regarding its transcriptional regulation. We have reported that Gαq is upregulated during phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic transformation of human erythroleukemia (HEL) cells and regulated by EGR-1, an early growth transcription factor. These studies focused on the initial 238 bp of the 5’ upstream region of the Gαq gene. In the present studies we characterize a minimal region -1042/-1037 bp from ATG in the 5’ upstream of the Gαq promoter that is associated with PMA responsiveness. In luciferase reporter gene studies in HEL cells, Gαq 5’ upstream promoter sequence -1042/-1 showed an about four-fold increased activity in PMA-treated compared to untreated cells. Deletion of 6-nt-1042/-1037 eliminated the difference. Gel-shift studies on Gαq probe (-1042/-1012 bp) revealed binding of EGR-1 with PMA-treated but not untreated nuclear extracts, and this was dependent on the sequence –1042/-1037.Silencing of endogenous EGR-1 inhibited Gαq induction by PMA. MEK/ERK inhibitor U0126 blocked PMA effect on promoter activity of the -1042/-1 construct. In conclusion, EGR-1 binding to sequence –1042/-1037 bp in Gαq promoter mediates the induction of Gαq gene by PMA via the MEK/ERK signaling pathway. These studies provide the first evidence of a PMA-responsive element in Gαq promoter, and new insights into regulation of Gαq gene by EGR-1.

Keywords

EGR-1 - Gαq - MAP kinase - megakaryocytic differentiation - PMA responsive sequence
 

  • References

  • 1 Brass LF, Manning DR, Cichowski K. et al. Signaling through G proteins in platelets: to the integrins and beyond. Thromb Haemost 1997; 78: 581-589.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 2 Offermanns S. Activation of platelet function through G protein-coupled receptors. Circ Res 2006; 99: 1293-1304.
    CrossrefPubMedGoogle Scholar
  • 3 Lee SB, Rao AK, Lee K-H. et al. Decreased expression of phospholipase C-β2 isozyme in human platelets with impaired function. Blood 1996; 88: 1684-1691.
    PubMedGoogle Scholar
  • 4 Offermanns S, Toombs CF, Hu YH. et al. Defective platelet activation in Gαq-deficient mice. Nature 1997; 389: 183-186.
    CrossrefPubMedGoogle Scholar
  • 5 Gabbeta J, Yang X, Kowalska MA. et al. Platelet signal transduction defect with Gα subunit dysfunction and diminished Gαq in a patient with abnormal platelet responses. Proc Natl Acad Sci USA 1997; 94: 8750-8755.
    CrossrefPubMedGoogle Scholar
  • 6 Gabbeta J, Vaidyula VR, Dhanasekaran DN. et al. Human platelet Gαq deficiency is associated with decreased Gαq gene expression in platelets but not neutrophils. Thromb Haemost 2002; 87: 129-133.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 7 Jalagadugula G, Dhanasekaran DN, Kim S. et al. Early growth response transcription factor EGR-1 regulates Galphaq gene in megakaryocytic cells. J Thromb Haemost 2006; 04: 2678-2686.
    CrossrefPubMedGoogle Scholar
  • 8 Yan SF, Pinsky DJ, Mackman N. et al. Egr-1: is it always immediate and early?. J Clin Invest 2000; 105: 553-554.
    CrossrefPubMedGoogle Scholar
  • 9 Silverman ES, Collins T. Pathways of Egr-1-mediated gene transcription in vascular biology. Am J Pathol 1999; 154: 665-670.
    CrossrefPubMedGoogle Scholar
  • 10 McCaffrey TA, Fu C, Du B. et al. High-level expression of Egr-1 and Egr-1-inducible genes in mouse and human atherosclerosis. J Clin Invest 2000; 105: 653-662.
    CrossrefPubMedGoogle Scholar
  • 11 Fu M, Zhu X, Zhang J. et al. Egr-1 target genes in human endothelial cells identified by microarray analysis. Gene 2003; 315: 33-41.
    CrossrefPubMedGoogle Scholar
  • 12 Cao XM, Koski RA, Gashler A. et al. Identification and characterization of the Egr-1 gene product, a DNA-binding zinc finger protein induced by differentiation and growth signals. Mol Cell Biol 1990; 10: 1931-1939.
    CrossrefPubMedGoogle Scholar
  • 13 Nishizuka Y. Protein kinase C and lipid signaling for sustained cellular responses. Faseb J 1995; 09: 484-496.
    CrossrefPubMedGoogle Scholar
  • 14 Jaken S. Protein kinase C isozymes and substrates. Curr Opin Cell Biol 1996; 08: 168-173.
    CrossrefPubMedGoogle Scholar
  • 15 Parekh DB, Ziegler W, Parker PJ. Multiple pathways control protein kinase C phosphorylation. Embo J 2000; 19: 496-503.
    CrossrefPubMedGoogle Scholar
  • 16 Long MW, Heffner CH, Williams JL. et al. Regulation of megakaryocyte phenotype in human erythroleukemia cells. J Clin Invest 1990; 85: 1072-1084.
    CrossrefPubMedGoogle Scholar
  • 17 Hong Y, Martin JF, Vainchenker W. et al. Inhibition of protein kinase C suppresses megakaryocytic differentiation and stimulates erythroid differentiation in HEL cells. Blood 1996; 87: 123-131.
    PubMedGoogle Scholar
  • 18 Cheng T, Wang Y, Dai W. Transcription factor egr-1 is involved in phorbol 12-myristate 13-acetate-induced megakaryocytic differentiation of K562 cells. J Biol Chem 1994; 269: 30848-30853.
    PubMedGoogle Scholar
  • 19 Simmons DL, Levy DB, Yannoni Y. et al. Identification of a phorbol ester-repressible v-src-inducible gene. Proc Natl Acad Sci USA 1989; 86: 1178-1182.
    CrossrefPubMedGoogle Scholar
  • 20 Irving SG, June CH, Zipfel PF. et al. Mitogen-induced genes are subject to multiple pathways of regulation in the initial stages of T-cell activation. Mol Cell Biol 1989; 09: 1034-1040.
    CrossrefPubMedGoogle Scholar
  • 21 Angel P, Imagawa M, Chiu R. et al. Phorbol esterinducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell 1987; 49: 729-739.
    CrossrefPubMedGoogle Scholar
  • 22 Keen JC, Cianferoni A, Florio G. et al. Characterization of a novel PMA-inducible pathway of interleukin-13 gene expression in T cells. Immunology 2006; 117: 29-37.
    CrossrefPubMedGoogle Scholar
  • 23 Malakooti J, Sandoval R, Memark VC. et al. Zinc finger transcription factor Egr-1 is involved in stimulation of NHE2 gene expression by phorbol 12-myristate 13-acetate. Am J Physiol Gastrointest Liver Physiol 2005; 289: G653-663.
    CrossrefPubMedGoogle Scholar
  • 24 D’Angelo DD, Oliver BG, Davis MG. et al. Novel role for Sp1 in phorbol ester enhancement of human platelet thromboxane receptor gene expression. J Biol Chem 1996; 271: 19696-19704.
    CrossrefPubMedGoogle Scholar
  • 25 Cupit LD, Schmidt VA, Gnatenko DV. et al. Expression of protease activated receptor 3 (PAR3) is upregulated by induction of megakaryocyte phenotype in human erythroleukemia (HEL) cells. Exp Hematol 2004; 32: 991-999.
    CrossrefPubMedGoogle Scholar
  • 26 Dignam JD, Lebovitz RM, Roeder RG. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 1983; 11: 1475-1489.
    CrossrefPubMedGoogle Scholar
  • 27 Yang J, Kawai Y, Hanson RW. et al. Sodium butyrate induces transcription from the G alpha(i2) gene promoter through multiple Sp1 sites in the promoter and by activating the MEK-ERK signal transduction pathway. J Biol Chem 2001; 276: 25742-25752.
    CrossrefPubMedGoogle Scholar
  • 28 Ueda Y, Hirai S, Osada S. et al. Protein kinase C activates the MEK-ERK pathway in a manner independent of Ras and dependent on Raf. J Biol Chem 1996; 271: 23512-23519.
    CrossrefPubMedGoogle Scholar
  • 29 Schonwasser DC, Marais RM, Marshall CJ. et al. Activation of the mitogen-activated protein kinase/ extracellular signal-regulated kinase pathway by conventional, novel, and atypical protein kinase C isotypes. Mol Cell Biol 1998; 18: 790-798.
    CrossrefPubMedGoogle Scholar
  • 30 Eyries M, Agrapart M, Alonso A. et al. Phorbol ester induction of angiotensin-converting enzyme transcription is mediated by Egr-1 and AP-1 in human endothelial cells via ERK1/2 pathway. Circ Res 2002; 91: 899-906.
    CrossrefPubMedGoogle Scholar
  • 31 Blenis J. Signal transduction via the MAP kinases: proceed at your own RSK. Proc Natl Acad Sci USA 1993; 90: 5889-5892.
    CrossrefPubMedGoogle Scholar
  • 32 Pearson G, Robinson F, Beers Gibson T. et al. Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev 2001; 22: 153-183.
    PubMedGoogle Scholar
  • 33 Schramek H. MAP kinases: from intracellular signals to physiology and disease. News Physiol Sci 2002; 17: 62-67.
    PubMedGoogle Scholar
  • 34 Racke FK, Lewandowska K, Goueli S. et al. Sustained activation of the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway is required for megakaryocytic differentiation of K562 cells. J Biol Chem 1997; 272: 23366-23370.
    CrossrefPubMedGoogle Scholar
  • 35 Kim KW, Kim SH, Lee EY. et al. Extracellular signal-regulated kinase/90-KDA ribosomal S6 kinase/nuclear factor-kappa B pathway mediates phorbol 12-myristate 13-acetate-induced megakaryocytic differentiation of K562 cells. J Biol Chem 2001; 276: 13186-13191.
    CrossrefPubMedGoogle Scholar
  • 36 Dieckgraefe BK, Weems DM. Epithelial injury induces egr-1 and fos expression by a pathway involving protein kinase C and ERK. Am J Physiol 1999; 276: G322-330.
    PubMedGoogle Scholar
  • 37 Guillemot L, Levy A, Raymondjean M. et al. Angiotensin II-induced transcriptional activation of the cyclin D1 gene is mediated by Egr-1 in CHO-AT(1A) cells. J Biol Chem 2001; 276: 39394-39403.
    CrossrefPubMedGoogle Scholar
  • 38 Khachigian LM, Lindner V, Williams AJ. et al. Egr-1-induced endothelial gene expression:a common theme in vascular injury. Science 1996; 271: 1427-1431.
    CrossrefPubMedGoogle Scholar
  • 39 Santiago FS, Lowe HC, Kavurma MM. et al. New DNA enzyme targeting Egr-1 mRNA inhibits vascular smooth muscle proliferation and regrowth after injury. Nat Med 1999; 05: 1264-1269.
    CrossrefPubMedGoogle Scholar
  • 40 Cui MZ, Parry GC, Oeth P. et al. Transcriptional regulation of the tissue factor gene in human epithelial cells is mediated by Sp1 and EGR-1. J Biol Chem 1996; 271: 2731-2739.
    CrossrefPubMedGoogle Scholar
  • 41 Silverman ES, Du J, De Sanctis GT. et al. Egr-1 and Sp1 interact functionally with the 5-lipoxygenase promoter and its naturally occurring mutants. Am J Respir Cell Mol Biol 1998; 19: 316-323.
    CrossrefPubMedGoogle Scholar
  • 42 DiVall SA, Radovick S, Wolfe A. Egr-1 binds the GnRH promoter to mediate the increase in gene expression by insulin. Mol Cell Endocrinol 2007; 270: 64-72.
    CrossrefPubMedGoogle Scholar
  • 43 Mechtcheriakova D, Wlachos A, Holzmuller H. et al. Vascular endothelial cell growth factor-induced tissue factor expression in endothelial cells is mediated by EGR-1. Blood 1999; 93: 3811-3823.
    PubMedGoogle Scholar