title: Cytokine signals through STAT3 promote expression of granulocyte secondary granule proteins in 32D cells.
abstract: OBJECTIVE
  In a previous study, we showed that activation of a transfected human erythropoietin receptor (EPOR) in the murine myeloid cell line 32D resulted in the development of morphologic features of granulocytic differentiation and expression of the neutrophil primary granule protein myeloperoxidase. We now studied if EPOR signaling could also mediate secondary granule protein gene expression and investigated the signal transduction requirements for induction of secondary granule gene expression in 32D cells.
  
  
  METHODS
  Wild-type and variant 32D cells expressing normal or chimeric EPORs or receptors for granulocyte colony-stimulating factor (G-CSFRs) were stimulated with EPO or G-CSF and the expression of granulocyte-specific genes was analyzed by Northern blot analysis. To determine the signaling mechanisms required for secondary granule protein gene induction, the activation of STAT pathways following growth factor stimulation was studied by Western blot analysis.
  
  
  RESULTS
  We found that EPO treatment of 32D cells engineered to express EPOR did not result in induction of the secondary granule protein genes encoding lactoferrin and 24p3 lipocalin, the mouse homolog of human N-Gal, or the myeloid transcription factor C/EBPepsilon. Replacement of the intracellular domain of EPOR with the intracellular domain of G-CSFR in a chimeric receptor was associated with EPO-mediated induction of lactoferrin, 24p3 lipocalin, and C/EBPepsilon genes. We found that STAT3 phosphorylation was mediated by the intracellular domain of G-CSFR, but not EPOR. Replacement of one or two of the STAT5 binding sites in the intracytoplasmic domain of the EPOR with STAT3 binding sites resulted in EPO-mediated STAT3 activation and a marked increase in the expression of the 24p3 lipocalin gene. Knockdown of STAT3 protein levels with siRNA caused significant decrease in 24p3 lipocalin gene induction.
  
  
  CONCLUSIONS
  These results indicate that EPOR signaling cannot substitute for G-CSFR signaling to stimulate secondary granule protein gene expression in 32D cells. In addition, STAT3 is a critical mediator of 24p3 lipocalin gene expression in these cells.
authors.affiliation: Department of Medicine, Yale University School of Medicine, New Haven, CT 06520-8021, USA.
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.forename: Murat O
authors.initials: MO
authors.lastname: Arcasoy
authors.forename: Stephanie S
authors.initials: SS
authors.lastname: Watowich
authors.forename: Bernard G
authors.initials: BG
authors.lastname: Forget
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D003594:Cytoplasmic Granules / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004921:Erythropoietin / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D006098:Granulocytes / Q000502:physiology*; D006801:Humans; D051379:Mice; D014176:Protein Biosynthesis / Q000187:drug effects* / Q000502:physiology; D017467:Receptors, Erythropoietin / Q000235:genetics / Q000378:metabolism; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins; D050796:STAT3 Transcription Factor; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D015534:Trans-Activators / Q000378:metabolism*; D015533:Transcriptional Activation
chemical_list: D004268:DNA-Binding Proteins; D017467:Receptors, Erythropoietin; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011994:Recombinant Proteins; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; C494087:Stat3 protein, mouse; D015534:Trans-Activators; D004921:Erythropoietin
references.citation: Jpn J Cancer Res. 1989 Nov;80(11):1077-82
references.pmid: 2481663
references.citation: J Endocrinol. 1999 Jul;162(1):11-9
references.pmid: 10396016
references.citation: Proc Natl Acad Sci U S A. 1992 Mar 15;89(6):2140-4
references.pmid: 1312714
references.citation: Chin Med J (Engl). 1992 Sep;105(9):707-12
references.pmid: 1283843
references.citation: Blood. 1993 May 1;81(9):2223-36
references.pmid: 8481505
references.citation: Cell. 1993 Jul 30;74(2):227-36
references.pmid: 8343951
references.citation: Blood. 1994 Feb 1;83(3):799-807
references.pmid: 8298140
references.citation: Intern Med. 1993 Aug;32(8):648-50
references.pmid: 7508772
references.citation: Blood. 1994 Sep 15;84(6):1737-46
references.pmid: 7521686
references.citation: Blood. 1994 Sep 15;84(6):1760-4
references.pmid: 7521688
references.citation: Blood. 1995 Feb 1;85(3):812-7
references.pmid: 7833481
references.citation: Curr Opin Cell Biol. 1994 Dec;6(6):804-8
references.pmid: 7880526
references.citation: Immunity. 1999 Aug;11(2):153-61
references.pmid: 10485650
references.citation: Science. 1995 Aug 25;269(5227):1108-12
references.pmid: 7652557
references.citation: Nature. 1995 Oct 19;377(6550):591-4
references.pmid: 7566171
references.citation: EMBO J. 1995 Nov 15;14(22):5557-68
references.pmid: 8521813
references.citation: EMBO J. 1996 May 15;15(10):2434-41
references.pmid: 8665851
references.citation: Gene. 1996 May 8;170(2):173-80
references.pmid: 8666241
references.citation: Blood. 1996 Aug 1;88(3):761-77
references.pmid: 8704229
references.citation: Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8324-8
references.pmid: 8710869
references.citation: J Biol Chem. 1996 Oct 4;271(40):24753-60
references.pmid: 8798745
references.citation: Blood. 1996 Oct 1;88(7):2442-9
references.pmid: 8839834
references.citation: Immunity. 1996 Nov;5(5):491-501
references.pmid: 8934575
references.citation: Proc Natl Acad Sci U S A. 1997 Jan 21;94(2):569-74
references.pmid: 9012825
references.citation: Blood. 1997 Jun 15;89(12):4628-35
references.pmid: 9192789
references.citation: J Leukoc Biol. 1999 Nov;66(5):727-32
references.pmid: 10577501
references.citation: Oncogene. 2000 May 15;19(21):2612-8
references.pmid: 10851060
references.citation: J Biol Chem. 2000 Aug 25;275(34):26566-75
references.pmid: 10858439
references.citation: Biochim Biophys Acta. 2000 Oct 18;1482(1-2):272-83
references.pmid: 11058768
references.citation: Curr Opin Hematol. 2001 Jan;8(1):23-7
references.pmid: 11138622
references.citation: Immunity. 2001 Feb;14(2):193-204
references.pmid: 11239451
references.citation: Curr Opin Cell Biol. 2001 Apr;13(2):211-7
references.pmid: 11248555
references.citation: Proc Natl Acad Sci U S A. 2001 Jul 3;98(14):8000-5
references.pmid: 11438745
references.citation: Blood. 2001 Aug 15;98(4):897-905
references.pmid: 11493431
references.citation: Oncogene. 2001 Oct 29;20(49):7178-85
references.pmid: 11704846
references.citation: Eur J Haematol. 2001 Aug;67(2):77-87
references.pmid: 11722594
references.citation: Oncogene. 2002 May 13;21(21):3295-313
references.pmid: 12032771
references.citation: Immunity. 2002 Jul;17(1):63-72
references.pmid: 12150892
references.citation: Mol Cell. 2002 Nov;10(5):1033-43
references.pmid: 12453412
references.citation: Blood. 2003 Apr 15;101(8):2990-5
references.pmid: 12672695
references.citation: J Biol Regul Homeost Agents. 2003 Jan-Mar;17(1):46-65
references.pmid: 12757021
references.citation: Exp Hematol. 2003 Dec;31(12):1310-6
references.pmid: 14662339
references.citation: Blood. 2004 Mar 1;103(5):1693-701
references.pmid: 14604978
references.citation: Cell. 1989 Apr 21;57(2):277-85
references.pmid: 2539263
references.citation: Blood. 1997 Jul 15;90(2):489-519
references.pmid: 9226149
references.citation: Science. 1997 Sep 12;277(5332):1630-5
references.pmid: 9287210
references.citation: Blood. 1997 Oct 1;90(7):2784-95
references.pmid: 9326246
references.citation: Proc Natl Acad Sci U S A. 1997 Nov 25;94(24):13187-92
references.pmid: 9371821
references.citation: Proc Natl Acad Sci U S A. 1998 Jun 9;95(12):6573-5
references.pmid: 9618452
references.citation: Blood. 1998 Jul 15;92(2):345-7; discussion 352
references.pmid: 9657727
references.citation: Blood. 1998 Jul 15;92(2):348-51; discussion 352
references.pmid: 9657728
references.citation: Blood. 1998 Jul 15;92(2):353-61
references.pmid: 9657731
references.citation: Blood. 1998 Aug 15;92(4):1219-24
references.pmid: 9694710
references.citation: Proc Natl Acad Sci U S A. 1999 Jan 19;96(2):698-702
references.pmid: 9892696
references.citation: J Biol Chem. 1999 May 21;274(21):14956-62
references.pmid: 10329697
references.citation: Blood. 1999 Jun 15;93(12):4154-66
references.pmid: 10361113
references.citation: Blood. 1999 Jul 1;94(1):39-45
references.pmid: 10381496
references.citation: Blood. 1991 Feb 1;77(3):419-34
references.pmid: 1991159