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Transcriptional Regulation of the Transforming Growth Factor-β2 Promoter by cAMP-responsive Element-binding Protein (CREB) and Activating Transcription Factor-1 (ATF-1) Is Modulated by Protein Kinases and the Coactivators p300 and CREB-binding Protein

1999; Elsevier BV; Volume: 274; Issue: 48 Linguagem: Inglês

10.1074/jbc.274.48.34020

ISSN

1083-351X

Autores

Michelle L. Kingsley-Kallesen, David L. Kelly, Angie Rizzino,

Tópico(s)

Kruppel-like factors research

Resumo

Transcription of the transforming growth factor-β2 (TGF-β2) gene is dependent on a cAMP-response element/activating transcription factor (CRE/ATF) site that is bound by CREB and ATF-1 as well as an E-box motif that is bound by upstream stimulatory factors 1 and 2 (USF1 and USF2). To identify additional factors involved in the expression of the TGF-β2 gene, we employed F9 embryonal carcinoma (EC) cells, which express TGF-β2 only after the cells differentiate. We show that overexpression of the transcription factors, CREB, ATF-1, USF1, and USF2 dramatically increases TGF-β2 promoter activity in F9-differentiated cells. We further show that the coactivators p300 and CBP up-regulate the TGF-β2 promoter when CREB and ATF-1 are expressed in conjunction with protein kinases that phosphorylate CREB on serine 133 and ATF-1 on serine 63. Importantly, we identify the presence of serine 133-phosphorylated CREB in the nucleus of F9-differentiated cells but not in the nucleus of F9 EC cells. This phosphorylated form is present in whole cell extracts of both the parental and differentiated cells, suggesting that nuclear accumulation of serine 133-phosphorylated CREB is regulated during differentiation of F9 EC cells and is likely to play an important role in the activation of the TGF-β2 gene. Transcription of the transforming growth factor-β2 (TGF-β2) gene is dependent on a cAMP-response element/activating transcription factor (CRE/ATF) site that is bound by CREB and ATF-1 as well as an E-box motif that is bound by upstream stimulatory factors 1 and 2 (USF1 and USF2). To identify additional factors involved in the expression of the TGF-β2 gene, we employed F9 embryonal carcinoma (EC) cells, which express TGF-β2 only after the cells differentiate. We show that overexpression of the transcription factors, CREB, ATF-1, USF1, and USF2 dramatically increases TGF-β2 promoter activity in F9-differentiated cells. We further show that the coactivators p300 and CBP up-regulate the TGF-β2 promoter when CREB and ATF-1 are expressed in conjunction with protein kinases that phosphorylate CREB on serine 133 and ATF-1 on serine 63. Importantly, we identify the presence of serine 133-phosphorylated CREB in the nucleus of F9-differentiated cells but not in the nucleus of F9 EC cells. This phosphorylated form is present in whole cell extracts of both the parental and differentiated cells, suggesting that nuclear accumulation of serine 133-phosphorylated CREB is regulated during differentiation of F9 EC cells and is likely to play an important role in the activation of the TGF-β2 gene. transforming growth factor-β2 embryonal carcinoma retinoic acid cAMP-response element activating transcription factor CRE-binding protein upstream stimulatory factor CREB binding protein calmodulin kinase II and IV, respectively protein kinase A Rous sarcoma virus chloramphenicol acetyltransferase hemagglutinin Tris-buffered saline catalytic subunit of PKA serine 133 to alanine 133 mutant of CREB Transforming growth factor-β2 (TGF-β2)1 is a member of the TGF-β superfamily, which represents a growing number of structurally related yet functionally distinct polypeptides involved in the regulation of a broad range of cellular events, including cell growth and differentiation as well as tissue morphogenesis (reviewed in Refs. 1Roberts A.B. 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These cells resemble biochemically and morphologically the inner cell mass of the early mouse embryo and under normal culture conditions exhibit very limited spontaneous differentiation (19Bernstine E.G. Hooper M.L. Grandchamp S. Ephrussi B. Proc. Natl. Acad. Sci. U. S. A. 1973; 70: 3899-3903Crossref PubMed Scopus (517) Google Scholar). Treatment of F9 EC cells with retinoic acid (RA) induces differentiation toward an extraembryonic endoderm-like phenotype (20Strickland S. Mahdavi B. Cell. 1978; 15: 393-403Abstract Full Text PDF PubMed Scopus (1132) Google Scholar). Utilizing the F9 EC model system, previous work by this laboratory and others have demonstrated that TGF-β2 is activated at both the RNA and protein levels when F9 cells are induced to differentiate with RA (21Kelly D. Campbell J. Tiesman J. Rizzino A. Cytotechnology. 1990; 4: 227-242Crossref PubMed Scopus (31) Google Scholar,22Mummery C.L. Slager H. Kruijer W. Feijen A. Freund E. 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Chem. 1992; 267: 19938-19943Abstract Full Text PDF PubMed Google Scholar) and an E-box motif located between −50 and −45 (25Scholtz B. Kingsley-Kallesen M. Rizzino A. J. Biol. Chem. 1996; 271: 32375-32380Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The nucleotide sequence of and spacing between both of these sites are evolutionary conserved in the human, chicken, and mouse promoters (16Noma T. Glick A.B. Geiser A.G. O'Reilly M.A. Miller J. Roberts A.B. Sporn M.B. Growth Factors. 1991; 4: 247-255Crossref PubMed Scopus (87) Google Scholar,26Burt D.W. Patton I.R. DNA Cell Biol. 1991; 10: 723-734Crossref PubMed Scopus (52) Google Scholar). 2P. Wilder and A. Rizzino, unpublished observations. 2P. Wilder and A. Rizzino, unpublished observations. Mutation of either site reduces the expression of TGF-β2 promoter/reporter gene constructs approximately 60–80% (23Kelly D. O'Reilly M. Rizzino A. Dev. 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Previous studies have demonstrated that these transcription factors are present in nuclear extracts prepared from both F9 EC cells and F9-differentiated cells and can bind to their respective cis-regulatory elements in vitro (25Scholtz B. Kingsley-Kallesen M. Rizzino A. J. Biol. Chem. 1996; 271: 32375-32380Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 27Kelly D. Scholtz B. Orten D.J. Hinrichs S.H. Rizzino A. Mol. Reprod. Dev. 1995; 40: 135-145Crossref PubMed Scopus (28) Google Scholar, 28Scholtz B. Kelly D. Rizzino A. Mol. Reprod. Dev. 1995; 41: 140-148Crossref PubMed Scopus (12) Google Scholar). Expression of a dominant-negative USF expression plasmid reduces the expression of a TGF-β2 promoter/reporter construct by approximately 80% (25Scholtz B. Kingsley-Kallesen M. Rizzino A. J. Biol. Chem. 1996; 271: 32375-32380Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). 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Nature. 1993; 365: 855-859Crossref PubMed Scopus (1763) Google Scholar, 42Kwok R.P.S. Lundblad J.R. Chrivia J.C. Richards J.P. Bächinger H.P. Brennan R.G. Roberts S.G.E. Green M.R. Goodman R.H. Nature. 1994; 370: 223-226Crossref PubMed Scopus (1280) Google Scholar, 43Lee J.S. Zhang X. Shi Y. J. Biol. Chem. 1996; 271: 17666-17674Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 44Shimomura A. Ogawa Y. Kitani T. Fujisawa H. Hagiwara M. J. Biol. Chem. 1996; 271: 17957-17960Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), we examined the ability of p300 and CBP to modulate TGF-β2 promoter activity. We determined that p300 and CBP augment TGF-β2 promoter activity when expressed in conjunction with CREB or ATF-1 and the catalytic subunit of PKA or constitutively active CaMKIV. This activation is dependent on an intact CRE/ATF site but not an intact E-box. We also show that the serine 133-phosphorylated form of CREB is present in the nucleus of F9-differentiated cells but not in the nucleus of F9 EC cells. Importantly, this phosphorylated form is present in whole cell extracts of both the parental and differentiated cells, suggesting that nuclear accumulation of serine 133-phosphorylated CREB is regulated during differentiation of F9 EC cells. In view of the findings presented here, we propose that accumulation of serine 133-phosphorylated CREB in the nucleus of F9-differentiated cells allows for recruitment of p300/CBP to the TGF-β2 promoter, and this is likely to play an important role in the activation of the TGF-β2 gene when EC cells differentiate. Dulbecco's modified Eagle's medium HG-21 and Ham's F-12 were purchased from Life Technologies, Inc. Fetal bovine serum was obtained from HyClone (Logan, UT). All-trans-RA was purchased from Acros Organics, a division of Fisher. All other chemicals, including protease and phosphatase inhibitors, gelatin, and dibutyryl cAMP were purchased from Sigma, unless otherwise indicated. F9 EC cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and grown on tissue culture dishes coated with 0.1% gelatin. When cellular extracts were prepared, differentiation of F9 EC cells was induced by a 4-day treatment with 5 μm RA. When differentiated cells were used in transfection studies, they were treated for 3 days with 5 μm RA before transfection. Stock cultures and all experimental cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2. F9-differentiated cells were transfected in monolayer by the calcium phosphate precipitation method as modified by our laboratory (23Kelly D. O'Reilly M. Rizzino A. Dev. Biol. 1992; 153: 172-175Crossref PubMed Scopus (21) Google Scholar). The plasmids utilized in each study are described in the figure legends. In each experiment, 2 μg of either pCH110 (Amersham Pharmacia Biotech) or pCH111 (obtained from Dr. Ron Hines, Medical College of Wisconsin, Milwaukee, WI) was co-transfected to normalize for transfection efficiency. pCH110 contains the β-galactosidase reporter gene under the control of the SV40 promoter and enhancer. The pCH111 plasmid contains the β-galactosidase reporter under the control of the RSV long terminal repeat. After an overnight incubation with the DNA-calcium phosphate precipitate, the cells were washed twice with Dulbecco's modified Eagle's medium/F-12 medium (1:1) and refed with Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (and 5 μm RA for F9-differentiated cells). Chloramphenicol acetyltransferase (CAT) activities were determined 48 h after transfection by the method of Seed and Sheen (72Seed B. Sheen J.Y. 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Chem. 1996; 271: 32375-32380Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Unless otherwise noted, the null vector, pUC-19 (Invitrogen, Carlsbad, CA) was used to keep the total DNA concentration the same in each experiment. The eukaryotic expression plasmids psvUSF1-pN3, containing the full-length cDNA encoding human USF1, and psvUSF2-pN4, containing the full-length cDNA encoding murine USF2, were obtained from Dr. Michèle Sawadogo (75Meier J.L. Luo X. Sawadogo M. Straus S.E. Mol. Cell. Biol. 1994; 14: 6896-6907Crossref PubMed Scopus (103) Google Scholar). pECEATF-1 and pECEATF-2 expression plasmids were obtained from Dr. Michael O'Reilly and contain the human cDNAs for ATF-1 and ATF-2 under the control of the SV40 promoter and enhancer (76Ellis L. Clauser E. Morgan D.O. Edery M. Roth R. Rutter W.J. Cell. 1986; 45: 721-732Abstract Full Text PDF PubMed Scopus (696) Google Scholar). The expression plasmid, pRc/RSV-mCBP.HA.RK contains the full-length mouse CBP cDNA with a hemagglutinin (HA) tag (42Kwok R.P.S. Lundblad J.R. Chrivia J.C. Richards J.P. Bächinger H.P. Brennan R.G. Roberts S.G.E. Green M.R. Goodman R.H. Nature. 1994; 370: 223-226Crossref PubMed Scopus (1280) Google Scholar), and the expression plasmid, pRc/RSV-CREB 341, contains the cDNA for CREB 341 (77Walton K.M. Rehfuss R.P. Chrivia J.C. Lockner J.E. Goodman R.H. Mol. Endocrinol. 1992; 6: 647-655PubMed Google Scholar). CBP and CREB expression plasmids were provided by Dr. Richard Goodman. The expression plasmid for p300, pCMVβp300-CHA, was obtained from Dr. David Livingston and contains a p300 cDNA insert from nucleotides 1134–8329 with a C-terminal HA tag cloned into the pCI vector (78Eckner R. Ewen M.E. Newsome D. Gerdes M. DeCaprio J.A. Lawrence J.B. Livingston D.M. Genes Dev. 1994; 8: 869-884Crossref PubMed Scopus (921) Google Scholar). The pSKG4 plasmid was provided by Dr. Steven K. Hanks and contains the cDNA for the human PKA type α catalytic subunit driven by the CMV promoter in the pcD vector (79Maldonado F. Hanks S.K. Nucleic Acids Res. 1988; 16: 8189-8190Crossref PubMed Scopus (60) Google Scholar). pRSV-CaMKII-(1–290) contains a cDNA insert that contains residues 1–290 of CaMKII and codes for a constitutively active form of the kinase (80Sun P. Enselen H. Myung P.S. Maurer R.A. Genes Dev. 1994; 8: 2527-2539Crossref PubMed Scopus (638) Google Scholar). pRSV-Mouse CaMKIV-(1–313) contains residues 1–313 of the mouse cDNA of CaMKIV and produces a constitutively active form of the kinase (80Sun P. Enselen H. Myung P.S. Maurer R.A. Genes Dev. 1994; 8: 2527-2539Crossref PubMed Scopus (638) Google Scholar). The CaM kinase expression plasmids were provided by Dr. Richard Maurer. All plasmids were purified by Qiagen (Chatsworth, CA) tip-500 columns. Nuclear extracts of F9 EC cells and F9-differentiated cells (4-day treatment with RA) were prepared as described previously (25Scholtz B. Kingsley-Kallesen M. Rizzino A. J. Biol. Chem. 1996; 271: 32375-32380Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) with minor modifications described here. All buffers were supplemented with the following protease and phosphatase inhibitors: 2.5 kallikrein-inactivating units/ml aprotinin, 0.2 mmphenylmethylsu

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