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A Sp1 Binding Site of the Tumor Necrosis Factor α Promoter Functions as a Nitric Oxide Response Element

1999; Elsevier BV; Volume: 274; Issue: 47 Linguagem: Inglês

10.1074/jbc.274.47.33190

ISSN

1083-351X

Autores

Shuibang Wang, Weihan Wang, Robert Wesley, Robert L. Danner,

Tópico(s)

Immune Response and Inflammation

Resumo

Regulation of gene transcription is an incompletely understood function of nitric oxide (NO). Human leukocytes produce increased amounts of tumor necrosis factor α (TNF-α) in response to NO. This effect is associated with decreases in intracellular cAMP, suggesting that NO might regulate gene transcription through promoter sequences sensitive to cAMP such as cAMP response elements (CRE) and Sp1 binding sites. Here we report that a Sp1 binding site in the TNF-α promoter conveys NO responsiveness. Human U937 cells were differentiated for TNF-α production with phorbol 12-myristate 13-acetate. NO donors and H89, an inhibitor of cAMP-dependent protein kinase increased, while dibutyryl cAMP (Bt2cAMP) decreased TNF-α promoter activity. Deletion or mutation of the proximal Sp1 site, but not the CRE site, abolished the activating effects of NO donors and H89. Further, NO- and H89-mediated increases in TNF-α promoter activity were associated with decreased Sp1 binding. The insertion of Sp1 sites into a minimal cytomegalovirus promoter conferred NO responsiveness, an effect blocked by Bt2cAMP. Mutation of these inserted Sp1 sites prevented this heterologous promoter from responding to NO, H89 and Bt2cAMP. These results identify the Sp1 binding site as a promoter motif that allows NO to control gene transcription. Regulation of gene transcription is an incompletely understood function of nitric oxide (NO). Human leukocytes produce increased amounts of tumor necrosis factor α (TNF-α) in response to NO. This effect is associated with decreases in intracellular cAMP, suggesting that NO might regulate gene transcription through promoter sequences sensitive to cAMP such as cAMP response elements (CRE) and Sp1 binding sites. Here we report that a Sp1 binding site in the TNF-α promoter conveys NO responsiveness. Human U937 cells were differentiated for TNF-α production with phorbol 12-myristate 13-acetate. NO donors and H89, an inhibitor of cAMP-dependent protein kinase increased, while dibutyryl cAMP (Bt2cAMP) decreased TNF-α promoter activity. Deletion or mutation of the proximal Sp1 site, but not the CRE site, abolished the activating effects of NO donors and H89. Further, NO- and H89-mediated increases in TNF-α promoter activity were associated with decreased Sp1 binding. The insertion of Sp1 sites into a minimal cytomegalovirus promoter conferred NO responsiveness, an effect blocked by Bt2cAMP. Mutation of these inserted Sp1 sites prevented this heterologous promoter from responding to NO, H89 and Bt2cAMP. These results identify the Sp1 binding site as a promoter motif that allows NO to control gene transcription. nitric oxide tumor necrosis factor α phorbol 12-myristate 13-acetate dibutyryl cAMP cAMP-dependent protein kinase cAMP response element S-nitroso-N-acetylpenicillamine S-nitrosoglutathione polymerase chain reaction tetracyclin tetracyclin-responsive transcriptional activator chloramphenicol acetyltransferase cytomegalovirus electrophoretic mobility shift assay base pair(s) Nitric oxide (NO)1regulates vascular tone (1Huang P.L. Huang Z. Mashimo H. Bloch K.D. Moskowitz M.A. Bevan J.A. Fishman M.C. Nature. 1995; 377: 239-242Crossref PubMed Scopus (1768) Google Scholar), inflammatory responses (2Van Dervort A.L. Yan L. Madara P.J. Cobb J.P. Wesley R.A. Corriveau C.C. Tropea M.M. Danner R.L. J. Immunol. 1994; 152: 4102-4109PubMed Google Scholar, 3Lander H.M. Sehajpal P. Levine D.M. Novogrodsky A. J. Immunol. 1993; 150: 1509-1516PubMed Google Scholar, 4Corriveau C.C. Madara P.J. Van Dervort A.L. Tropea M.M. Wesley R.A. Danner R.L. J. Infect. Dis. 1998; 177: 116-126Crossref PubMed Scopus (39) Google Scholar), and gene transcription (5Hartsfield C.L. Alam J. Cook J.L. Choi A.M. Am. J. Physiol. 1997; 273: L980-L988PubMed Google Scholar, 6Ichiki T. Usui M. Kato M. Funakoshi Y. Ito K. Egashira K. Takeshita A. Hypertension. 1998; 31: 342-348Crossref PubMed Google Scholar, 7Liu Y. Christou H. Morita T. Laughner E. Semenza G.L. Kourembanas S. J. Biol. Chem. 1998; 273: 15257-15262Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). Although NO regulates vascular tone through soluble guanylate cyclase, some actions of NO utilize alternative signal transduction pathways. Previous experiments have shown that NO donors increase tumor necrosis factor α (TNF-α) synthesis in human neutrophil (2Van Dervort A.L. Yan L. Madara P.J. Cobb J.P. Wesley R.A. Corriveau C.C. Tropea M.M. Danner R.L. J. Immunol. 1994; 152: 4102-4109PubMed Google Scholar) and peripheral blood mononuclear cell preparations (3Lander H.M. Sehajpal P. Levine D.M. Novogrodsky A. J. Immunol. 1993; 150: 1509-1516PubMed Google Scholar) through a cGMP-independent mechanism. Recently, we further demonstrated that endogenously produced NO also up-regulates TNF-α production by a cGMP-independent mechanism in phorbol 12-myristate 13-acetate (PMA)-differentiated U937 cells transfected with murine, inducible NO synthase (8Yan L. Wang S. Rafferty S.P. Wesley R.A. Danner R.L. Blood. 1997; 90: 1160-1167Crossref PubMed Google Scholar).U937 cells lack soluble guanylate cyclase and do not respond to NO with a cGMP signal (8Yan L. Wang S. Rafferty S.P. Wesley R.A. Danner R.L. Blood. 1997; 90: 1160-1167Crossref PubMed Google Scholar, 9Wang S. Yan L. Wesley R.A. Danner R.L. J. Biol. Chem. 1997; 272: 5959-5965Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), suggesting that these cells might be useful for exploring cGMP-independent mechanisms by which NO regulates gene transcription. Experiments with U937 cells have demonstrated that NO augments TNF-α production through a signaling pathway dependent on decreases in intracellular cAMP (9Wang S. Yan L. Wesley R.A. Danner R.L. J. Biol. Chem. 1997; 272: 5959-5965Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). This finding suggests that NO might regulate TNF-α at the level of gene transcription through effects on cAMP sensitive promoter sites.cAMP is known to regulate gene transcription through effects on cAMP response elements (CRE) (10Lee K.A. Masson N. Biochim. Biophys. Acta. 1993; 1174: 221-233Crossref PubMed Scopus (188) Google Scholar). Furthermore, for some genes such as CYP11A and urokinase, cAMP has been shown to alter promoter activity through incompletely defined effects on Sp1 binding sites (11Venepally P. Waterman M.R. J. Biol. Chem. 1995; 270: 25402-25410Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 12Grimaldi P. Piscitelli D. Albanesi C. Blasi F. Geremia R. Rossi P. Mol. Endocrinol. 1993; 7: 1217-1225PubMed Google Scholar). Similar to CRE-binding proteins, cAMP-dependent protein kinase (PKA) phosphorylation of Sp1 has been shown to enhance its DNA binding activity (13Rohlff C. Ahmad S. Borellini F. Lei J. Glazer R. J. Biol. Chem. 1997; 272: 21137-21141Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). Although Sp1 binding can activate transcription (14Cieslik K. Zembowicz A. Tang J.-L. Wu K.K. J. Biol. Chem. 1998; 273: 14885-14890Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 15Pugh B.F. Tjian R. Cell. 1990; 61: 1187-1197Abstract Full Text PDF PubMed Scopus (733) Google Scholar), for some genes Sp1 functions as a repressor (16Roman D.G. Toledano M.B. Leonard W.J. New Biol. 1990; 2: 642-647PubMed Google Scholar, 17Madsen C.S. Hershey J.C. Hautmann M.B. White S.L. Owens G.K. J. Biol. Chem. 1997; 272: 6332-6340Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The promoter motifs or cell characteristics that determine these divergent effects of Sp1 binding are not fully understood. Therefore, CRE or Sp1 binding sites in the TNF-α promoter (18Leitman D.C. Mackow E.R. Williams T. Baxter J.D. West B.L. Mol. Cell. Biol. 1992; 12: 1352-1356Crossref PubMed Google Scholar, 19Leitman D.C. Ribeiro R.C. Mackow E.R. Baxter J.D. West B.L. J. Biol. Chem. 1991; 266: 9343-9346Abstract Full Text PDF PubMed Google Scholar, 20Kramer B. Meichle A. Hensel G. Charnay P. Kronke M. Biochim. Biophys. Acta. 1994; 1219: 413-421Crossref PubMed Scopus (117) Google Scholar) may be important in mediating cAMP-dependent effects of NO on TNF-α transcription. To investigate this question, we examined the effects of NO on the human TNF-α promoter in differentiated U937 cells. Nitric oxide (NO)1regulates vascular tone (1Huang P.L. Huang Z. Mashimo H. Bloch K.D. Moskowitz M.A. Bevan J.A. Fishman M.C. Nature. 1995; 377: 239-242Crossref PubMed Scopus (1768) Google Scholar), inflammatory responses (2Van Dervort A.L. Yan L. Madara P.J. Cobb J.P. Wesley R.A. Corriveau C.C. Tropea M.M. Danner R.L. J. Immunol. 1994; 152: 4102-4109PubMed Google Scholar, 3Lander H.M. Sehajpal P. Levine D.M. Novogrodsky A. J. Immunol. 1993; 150: 1509-1516PubMed Google Scholar, 4Corriveau C.C. Madara P.J. Van Dervort A.L. Tropea M.M. Wesley R.A. Danner R.L. J. Infect. Dis. 1998; 177: 116-126Crossref PubMed Scopus (39) Google Scholar), and gene transcription (5Hartsfield C.L. Alam J. Cook J.L. Choi A.M. Am. J. Physiol. 1997; 273: L980-L988PubMed Google Scholar, 6Ichiki T. Usui M. Kato M. Funakoshi Y. Ito K. Egashira K. Takeshita A. Hypertension. 1998; 31: 342-348Crossref PubMed Google Scholar, 7Liu Y. Christou H. Morita T. Laughner E. Semenza G.L. Kourembanas S. J. Biol. Chem. 1998; 273: 15257-15262Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). Although NO regulates vascular tone through soluble guanylate cyclase, some actions of NO utilize alternative signal transduction pathways. Previous experiments have shown that NO donors increase tumor necrosis factor α (TNF-α) synthesis in human neutrophil (2Van Dervort A.L. Yan L. Madara P.J. Cobb J.P. Wesley R.A. Corriveau C.C. Tropea M.M. Danner R.L. J. Immunol. 1994; 152: 4102-4109PubMed Google Scholar) and peripheral blood mononuclear cell preparations (3Lander H.M. Sehajpal P. Levine D.M. Novogrodsky A. J. Immunol. 1993; 150: 1509-1516PubMed Google Scholar) through a cGMP-independent mechanism. Recently, we further demonstrated that endogenously produced NO also up-regulates TNF-α production by a cGMP-independent mechanism in phorbol 12-myristate 13-acetate (PMA)-differentiated U937 cells transfected with murine, inducible NO synthase (8Yan L. Wang S. Rafferty S.P. Wesley R.A. Danner R.L. Blood. 1997; 90: 1160-1167Crossref PubMed Google Scholar). U937 cells lack soluble guanylate cyclase and do not respond to NO with a cGMP signal (8Yan L. Wang S. Rafferty S.P. Wesley R.A. Danner R.L. Blood. 1997; 90: 1160-1167Crossref PubMed Google Scholar, 9Wang S. Yan L. Wesley R.A. Danner R.L. J. Biol. Chem. 1997; 272: 5959-5965Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), suggesting that these cells might be useful for exploring cGMP-independent mechanisms by which NO regulates gene transcription. Experiments with U937 cells have demonstrated that NO augments TNF-α production through a signaling pathway dependent on decreases in intracellular cAMP (9Wang S. Yan L. Wesley R.A. Danner R.L. J. Biol. Chem. 1997; 272: 5959-5965Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). This finding suggests that NO might regulate TNF-α at the level of gene transcription through effects on cAMP sensitive promoter sites. cAMP is known to regulate gene transcription through effects on cAMP response elements (CRE) (10Lee K.A. Masson N. Biochim. Biophys. Acta. 1993; 1174: 221-233Crossref PubMed Scopus (188) Google Scholar). Furthermore, for some genes such as CYP11A and urokinase, cAMP has been shown to alter promoter activity through incompletely defined effects on Sp1 binding sites (11Venepally P. Waterman M.R. J. Biol. Chem. 1995; 270: 25402-25410Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 12Grimaldi P. Piscitelli D. Albanesi C. Blasi F. Geremia R. Rossi P. Mol. Endocrinol. 1993; 7: 1217-1225PubMed Google Scholar). Similar to CRE-binding proteins, cAMP-dependent protein kinase (PKA) phosphorylation of Sp1 has been shown to enhance its DNA binding activity (13Rohlff C. Ahmad S. Borellini F. Lei J. Glazer R. J. Biol. Chem. 1997; 272: 21137-21141Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). Although Sp1 binding can activate transcription (14Cieslik K. Zembowicz A. Tang J.-L. Wu K.K. J. Biol. Chem. 1998; 273: 14885-14890Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 15Pugh B.F. Tjian R. Cell. 1990; 61: 1187-1197Abstract Full Text PDF PubMed Scopus (733) Google Scholar), for some genes Sp1 functions as a repressor (16Roman D.G. Toledano M.B. Leonard W.J. New Biol. 1990; 2: 642-647PubMed Google Scholar, 17Madsen C.S. Hershey J.C. Hautmann M.B. White S.L. Owens G.K. J. Biol. Chem. 1997; 272: 6332-6340Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The promoter motifs or cell characteristics that determine these divergent effects of Sp1 binding are not fully understood. Therefore, CRE or Sp1 binding sites in the TNF-α promoter (18Leitman D.C. Mackow E.R. Williams T. Baxter J.D. West B.L. Mol. Cell. Biol. 1992; 12: 1352-1356Crossref PubMed Google Scholar, 19Leitman D.C. Ribeiro R.C. Mackow E.R. Baxter J.D. West B.L. J. Biol. Chem. 1991; 266: 9343-9346Abstract Full Text PDF PubMed Google Scholar, 20Kramer B. Meichle A. Hensel G. Charnay P. Kronke M. Biochim. Biophys. Acta. 1994; 1219: 413-421Crossref PubMed Scopus (117) Google Scholar) may be important in mediating cAMP-dependent effects of NO on TNF-α transcription. To investigate this question, we examined the effects of NO on the human TNF-α promoter in differentiated U937 cells. We thank Dr. James S. Economou from UCLA School of Medicine for plasmid pXP1(−1311TNF) and Dr. Rob Hooft van Huijsduijnen from Geneva Biomedical Research Institute for plasmid pUHG10.3CAT.

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