Phorbol Ester-induced Transcription of a Fibroblast Growth Factor-binding Protein Is Modulated by a Complex Interplay of Positive and Negative Regulatory Promoter Elements

Earlier studies from our laboratory showed that a secreted binding protein for fibroblast growth factors (FGF-BP) is expressed at high levels in squamous cell carcinoma (SCC) cell lines. Overexpression studies or conversely reduced expression of FGF-BP by ribozyme targeting have elucidated a direct role of this protein in angiogenesis during tumor development. We have also observed a significant up-regulation of FGF-BP during TPA (12-O-tetradecanoylphorbol-13-acetate) promotion of skin cancer. Here we investigate the mechanism of TPA induction of FGF-BP gene expression in the human ME-180 SCC cell line. We found that TPA increased FGF-BP mRNA levels in a time- and dose-dependent manner mediated via the protein kinase C signal transduction pathway. Results from actinomycin D and cycloheximide experiments as well as nuclear transcription assays revealed that TPA up-regulated the steady-state levels of FGF-BP mRNA by increasing its rate of gene transcription independently ofde novo protein synthesis. We isolated the human FGF-BP promoter and determined by deletion analysis that TPA regulatory elements were all contained in the first 118 base pairs upstream of the transcription start site. Further mutational analysis revealed that full TPA induction required interplay between several regulatory elements with homology to Ets, AP-1, and CAATT/enhancer binding protein C/EBP sites. In addition, deletion or mutation of a 10-base pair region juxtaposed to the AP-1 site dramatically increased TPA induced FGF-BP gene expression. This region represses the extent of the FGF-BP promoter response to TPA and contained sequences recognized by the family of E box helix-loop-helix transcription factors. Gel shift analysis showed specific and TPA-inducible protein binding to the Ets, AP-1, and C/EBP sites. Furthermore, distinct, specific, and TPA-inducible binding to the imperfect E box repressor element was also apparent. Overall, our data indicate that TPA effects on FGF-BP gene transcription are tightly controlled by a complex interplay of positive elements and a novel negative regulatory element. Earlier studies from our laboratory showed that a secreted binding protein for fibroblast growth factors (FGF-BP) is expressed at high levels in squamous cell carcinoma (SCC) cell lines. Overexpression studies or conversely reduced expression of FGF-BP by ribozyme targeting have elucidated a direct role of this protein in angiogenesis during tumor development. We have also observed a significant up-regulation of FGF-BP during TPA (12-O-tetradecanoylphorbol-13-acetate) promotion of skin cancer. Here we investigate the mechanism of TPA induction of FGF-BP gene expression in the human ME-180 SCC cell line. We found that TPA increased FGF-BP mRNA levels in a time- and dose-dependent manner mediated via the protein kinase C signal transduction pathway. Results from actinomycin D and cycloheximide experiments as well as nuclear transcription assays revealed that TPA up-regulated the steady-state levels of FGF-BP mRNA by increasing its rate of gene transcription independently ofde novo protein synthesis. We isolated the human FGF-BP promoter and determined by deletion analysis that TPA regulatory elements were all contained in the first 118 base pairs upstream of the transcription start site. Further mutational analysis revealed that full TPA induction required interplay between several regulatory elements with homology to Ets, AP-1, and CAATT/enhancer binding protein C/EBP sites. In addition, deletion or mutation of a 10-base pair region juxtaposed to the AP-1 site dramatically increased TPA induced FGF-BP gene expression. This region represses the extent of the FGF-BP promoter response to TPA and contained sequences recognized by the family of E box helix-loop-helix transcription factors. Gel shift analysis showed specific and TPA-inducible protein binding to the Ets, AP-1, and C/EBP sites. Furthermore, distinct, specific, and TPA-inducible binding to the imperfect E box repressor element was also apparent. Overall, our data indicate that TPA effects on FGF-BP gene transcription are tightly controlled by a complex interplay of positive elements and a novel negative regulatory element. FGF-BP 1The abbreviations used are: FGF-BP, fibroblast growth factor-binding protein; bFGF, basic FGF; TPA, 12-O-tetradecanoylphorbol-13-acetate; SCC, squamous cell carcinoma; PKC, protein kinase C; C/EBP, CAATT/enhancer binding protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IMEM, improved minimum essential medium; kb, kilobase pair(s); DTT, dithiothreitol; PCR, polymerase chain reaction; UTR, untranslated region; CMV, cytomegalovirus. is a secreted protein that binds to acidic FGF and basic FGF in a non-covalent reversible manner (1Wu D. Kan M. Sato G.H. Okamoto T. Sato J.D. J. Biol. Chem. 1991; 266: 16778-16785Abstract Full Text PDF PubMed Google Scholar). FGF-BP mRNA has been found to be up-regulated in squamous cell carcinoma (SCC) cell lines of different origin, in SCC tumor samples from the head and neck, and in some colon cancer cell lines (1Wu D. Kan M. Sato G.H. Okamoto T. Sato J.D. J. Biol. Chem. 1991; 266: 16778-16785Abstract Full Text PDF PubMed Google Scholar, 2Czubayko F. Smith R.V. Chung H.C. Wellstein A. J. Biol. Chem. 1994; 269: 28243-28248Abstract Full Text PDF PubMed Google Scholar). More recently, developmental expression of the mouse FGF-BP gene was found to be prominent in the skin and intestine during the perinatal phase and is down-regulated in adult mice (3Kurtz A. Wang H.L. Darwiche N. Harris V. Wellstein A. Oncogene. 1997; 14: 2671-2681Crossref PubMed Scopus (56) Google Scholar). We previously described that expression of FGF-BP in a non-tumorigenic human cell line (SW-13) which expresses bFGF leads to a tumorigenic and angiogenic phenotype (2Czubayko F. Smith R.V. Chung H.C. Wellstein A. J. Biol. Chem. 1994; 269: 28243-28248Abstract Full Text PDF PubMed Google Scholar). Expression of FGF-BP in these cells solubilizes their endogenous bFGF from its extracellular storage and allows it to reach its receptor, suggesting that FGF-BP serves as an extracellular carrier molecule for bFGF (2Czubayko F. Smith R.V. Chung H.C. Wellstein A. J. Biol. Chem. 1994; 269: 28243-28248Abstract Full Text PDF PubMed Google Scholar, 4Rak J. Kerbel R.S. Nat. Med. 1997; 3: 1083-1084Crossref PubMed Scopus (66) Google Scholar). Expression of FGF-BP under the control of a tetracycline-responsive promoter system in SW-13 cells revealed its role during the early phase of tumor growth (5Liaudet-Coopman E.D.E. Schulte A.M. Cardillo M. Wellstein A. Biochem. Biophys. Res. Commun. 1996; 229: 930-937Crossref PubMed Scopus (17) Google Scholar). To assess the significance of FGF-BP endogenously expressed in tumors, we depleted human SCC (ME-180) and colon carcinoma (LS174T) cell lines of their FGF-BP by targeting with specific ribozymes (6Czubayko F. Liaudet-Coopman E.D.E. Aigner A. Tuveson A.T. Berchem G. Wellstein A. Nat. Med. 1997; 3: 1137-1140Crossref PubMed Scopus (218) Google Scholar). This study showed that the reduction of FGF-BP reduced the release of biologically active bFGF from cells in culture. In addition, the growth and angiogenesis of xenografted tumors in mice was decreased in parallel with the reduction of FGF-BP, suggesting that some human tumors can utilize FGF-BP as an angiogenic switch molecule. The fact that FGF-BP has been detected in only a few types of tumors, where it seems to play a crucial role in angiogenesis, led us to investigate the mechanisms responsible for turning its expression on or the of FGF-BP in SCC cell we showed that used as a FGF-BP gene expression in by and mechanisms E.D.E. Wellstein A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). In FGF-BP expression in SCC and their tumor growth and angiogenesis E.D.E. Berchem Wellstein A. Res. 1997; 3: Google Scholar). the FGF-BP mRNA expression in the adult mouse skin was found to be dramatically increased during the early of mouse skin (3Kurtz A. Wang H.L. Darwiche N. Harris V. Wellstein A. Oncogene. 1997; 14: 2671-2681Crossref PubMed Scopus (56) Google as well as in human skin Aigner and A. FGF-BP expression in was up-regulated in cell lines an the in the of FGF-BP (3Kurtz A. Wang H.L. Darwiche N. Harris V. Wellstein A. Oncogene. 1997; 14: 2671-2681Crossref PubMed Scopus (56) Google Scholar). In this and the fact that FGF-BP play a role in the of human skin we to investigate the effects of the tumor promoter TPA on FGF-BP gene that FGF-BP mRNA expression is up-regulated by TPA in the ME-180 SCC cell line and that this induction is mediated by direct of the human FGF-BP promoter that the TPA induction is mediated by of several regulatory Furthermore, the induction of gene expression by TPA can be by a repressor element juxtaposed to the AP-1 site which sequences recognized by E box element factors. The ME-180 squamous cell carcinoma cell line was from were in improved minimum essential with ME-180 cells were to on in IMEM, and with in was isolated with the and of were by in and The were in for at was at in the were with and for at and with and for at was at were by for in and were by of from human FGF-BP (2Czubayko F. Smith R.V. Chung H.C. Wellstein A. J. Biol. Chem. 1994; 269: 28243-28248Abstract Full Text PDF PubMed Google and human The of the was of mRNA levels was a ME-180 cells were to on were in and with TPA in for from cells for were isolated in as described E.D.E. Wellstein A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). transcription assays were with as described E.D.E. Wellstein A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). of were to of for at the were with for at and for at in A. The were for in at The of in was determined a and were for with was from the region of the human FGF-BP from the FGF-BP and a control for the of was with kinase and of for and were a from ME-180 cells was isolated as described for ME-180 mRNA was from an of was with or of ME-180 mRNA or control with or of control in the of DTT, and to for at were in the of of and of for at were on a with and for of upstream of the human FGF-BP gene was isolated from a human the to the polymerase from the of the human FGF-BP were to for the of and to for from a and were a by and to This has been to were the Google Scholar). and were from in the site and the and site of to and contained site from the and of the FGF-BP and of the The promoter deletion and were by from upstream to to to to to and to all a site. was from the gene was were with and the site. and were by chain with a promoter were by specific promoter were as site deletion from to was to and to AP-1 site deletion from to was to and to site deletion from to was to and to site deletion from to was to and to and deletion from to was to and to The was by a C to mutation at from to were with to upstream or the The were from and and to of the with was by in the of by to and in a of were with and the site. and were by chain with a To control for TPA effects on sequences to the FGF-BP promoter we a of the for their response to TPA under the used in our as in of of the promoter A.T. J. 1991; PubMed Scopus Google the kinase promoter Google or the promoter which were these an induction TPA ME-180 cells were in in IMEM, at a of and of were in of IMEM, and were to at for was increased to with IMEM, to and for at were and in for and for with or for was determined by with of a and found to be the for all to a TPA induction of were for protein and for were by of and cell was by of was for the system was in a are expressed of of cell was determined by ME-180 cells were to on for and with or TPA for were to J.D. Res. PubMed Scopus Google with the cells were in of DTT, J. PubMed Scopus Google with and on for were at and in of C DTT, and at for for at was used in binding assays and at of was with kinase and of for and were a of or of ME-180 nuclear binding and of and or of and for on were and for of were on and by in at V. The AP-1 was We have previously detected an up-regulation of FGF-BP mRNA TPA of mouse skin during the of skin tumors (3Kurtz A. Wang H.L. Darwiche N. Harris V. Wellstein A. Oncogene. 1997; 14: 2671-2681Crossref PubMed Scopus (56) Google and also in human skin data that the control of this angiogenic switch play an role in skin To this we the of the tumor promoter TPA on FGF-BP gene expression in ME-180 cells which high levels of the FGF-BP E.D.E. Wellstein A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). were with TPA from to which in an in the steady-state levels of FGF-BP mRNA analysis showed that the induction was by mRNA by TPA as to the of and was used to FGF-BP The of TPA induction of FGF-BP mRNA in ME-180 is in We the as The of TPA on FGF-BP mRNA was also observed in SCC cell and that TPA induction of FGF-BP mRNA is in SCC cell lines. To TPA induction of FGF-BP mRNA was mediated a ME-180 cells were or for with specific C M. T. Biochem. Biophys. Res. Commun. PubMed Scopus Google and with or TPA for can be in of the cells with C to TPA the TPA that the induction of FGF-BP by TPA is mediated via a In addition, it is that TPA an up-regulation of by of In C to be to protein kinase it early induction of the effects of TPA on A. M. Biol. PubMed Scopus Google Scholar). the fact that C induction of FGF-BP mRNA and the TPA that induction of FGF-BP mRNA is an up-regulation of a of We have previously that FGF-BP gene expression can be and mechanisms E.D.E. Wellstein A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). we to the TPA induction of FGF-BP mRNA was at the or We first TPA the of the FGF-BP were to of of transcription or the TPA induction of FGF-BP D or cycloheximide were with or TPA and FGF-BP mRNA levels were determined in of TPA and actinomycin D the TPA of TPA and cycloheximide data that TPA increased the rate of FGF-BP gene transcription independently of novo protein and the of the FGF-BP To that the of the FGF-BP was by TPA ME-180 cells were for with TPA and actinomycin D was to in of cells with TPA the of the FGF-BP mRNA that the of the FGF-BP is by To that TPA the rate of transcription of the FGF-BP we nuclear transcription ME-180 cells were with or TPA for were from isolated ME-180 cell and to a in TPA increased FGF-BP levels of and to showed that FGF-BP transcription was up-regulated by and and of was used as a control transcription of this gene are with the steady-state mRNA and the effects of actinomycin D and cycloheximide the induction of FGF-BP mRNA by TPA in ME-180 cells is to a up-regulation of In to the of the human FGF-BP of upstream to the of human FGF-BP was isolated from a human and The transcription start site of the human gene was determined analysis with from The start site with the is in between the human and mouse FGF-BP promoter which we previously (3Kurtz A. Wang H.L. Darwiche N. Harris V. Wellstein A. Oncogene. 1997; 14: 2671-2681Crossref PubMed Scopus (56) Google revealed a region of high homology with the first upstream from the transcription start homology in upstream suggesting that the of the promoter be for of FGF-BP in and homology of the human FGF-BP the of the human FGF-BP promoter from to is homology between promoter sequences of human and mouse start are by an between the mouse and lines in the transcription binding are on the to the of the human FGF-BP promoter and the of the used to the promoter in analysis of the promoter the of transcription binding that were between mouse and human FGF-BP and that have in FGF-BP in a box is at base pairs upstream from the transcription start for and of the human FGF-BP promoter we found a binding site for a of the binding protein family of transcription factors which play a role in the phase response and in a of cell A. PubMed Scopus Google Scholar, T. PubMed Scopus Google Scholar, 1991; PubMed Scopus Google Scholar). AP-1 binding site to juxtaposed to a with homology to an binding to suggesting to the juxtaposed site found in the and in the promoter A. J. PubMed Scopus Google Scholar, A. J. PubMed Scopus Google Scholar). In addition, a binding site to an binding to and a site to are in the region of the promoter and play a role in of FGF-BP as To the promoter elements in FGF-BP gene by TPA, deletion were on the of binding on the were ME-180 and their was in the or of TPA The of is in the of and The in the or of TPA we a TPA induction of the several kinase the and the were with TPA ME-180 cells control induction by TPA of a of has been described previously and is mediated in the J.D. F. 1994; Google Scholar). The TPA induction to the FGF-BP promoter was to be that observed the control The FGF-BP promoter from to was induced control in the of TPA and showed the TPA as the promoter from to which base pairs of promoter the on TPA induction and was also induced deletion from to which of the full TPA of the binding site from to on TPA induction of the FGF-BP the deletion to an in of the promoter suggesting that the site is a of promoter of FGF-BP is required for TPA TPA induction was reduced deletion of the binding site from to and is reduced deletion of the AP-1 site from to that of these to some in TPA The of these was at of the deletion from to which sequences homology to a TPA induction to the control The of this was that of the In to the of binding site to TPA promoter were and for TPA the of the promoter from to of the site to or deletion of the AP-1 site to reduced TPA induction to the promoter of and AP-1 to in a significant in and in TPA that in for full promoter of the juxtaposed site TPA suggesting that are also The of the binding to TPA induction of the FGF-BP promoter was determined by an deletion from to with the deletion which contained only the site and some TPA an deletion of this site showed a significant in TPA of has been to and is in induction of as T. T. M. T. A. PubMed Scopus Google Scholar, M. T. M. J. 1994; PubMed Scopus Google Scholar, D. 1997; Google Scholar). seems to play a role in TPA induction of the FGF-BP promoter deletion of this site the induction by the AP-1 site and the site a region of homology between the human and mouse promoter this region was to have on TPA an deletion this region to was as a in the TPA induction of the FGF-BP promoter increased from to suggesting the of a repressor which with this site. The of between the human and mouse in this region a in the of FGF-BP between the The to deletion an to which is juxtaposed to the of the AP-1 site and which some to the E box element recognized by a of helix-loop-helix factors T. Google Scholar). To this imperfect E box for repressor a C to mutation at was the promoter This showed a in TPA induction to the mutation is the this promoter also showed increased induction by TPA, suggesting that mediated by this site is on the site. data that the mutation at as well as the deletion from to of the FGF-BP promoter which the response to In to that TPA induction of FGF-BP was to direct by transcription we analysis to transcription binding to FGF-BP promoter promoter from to the site as a the binding of specific protein in the of ME-180 nuclear was detected binding to all was increased in the of TPA and was in the of Further analysis showed that the factors binding to the element were only by elements from the promoter D. T. 1997; PubMed Scopus Google and A. D. D. PubMed Scopus Google in to for binding has previously been described that specific the of the site are required for high binding of family J. Res. PubMed Scopus Google Scholar, A. Biophys. PubMed Scopus Google Scholar, A. J. 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Med. 1997; 3: PubMed Scopus Google for the specific complex was only at high that the FGF-BP element a different To investigate the of the FGF-BP promoter by AP-1 and the of the E box repressor shift experiments were the promoter the juxtaposed element as a In the of ME-180 nuclear the element an complex and a The binding of all was induced by TPA and was by of the To the specific of these were in the AP-1 site or the repressor site and for their to for with the AP-1 site in a of only the and for the suggesting that the to factors to the AP-1 site. was with the repressor binding of the on the binding to the AP-1 site is reduced that the protein binding to the repressor element. Furthermore, was with an AP-1 which an AP-1 site by sequences which are to the FGF-BP for only the AP-1 complex was observed and that the AP-1 site and the repressor site of the FGF-BP promoter and specific transcription that are induced in the of our data that sequences between and of the FGF-BP promoter a novel TPA regulatory of positive and negative control In this we that TPA induction of FGF-BP mRNA levels is of gene This is in to the of FGF-BP gene expression which we have previously is mediated and mechanisms E.D.E. Wellstein A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). In at at early the mechanism which is on protein the of the FGF-BP mRNA is E.D.E. Wellstein A. J. Biol. 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Google Scholar). the which of the helix-loop-helix family with this site. binding to an element has been described in to a of the nuclear helix-loop-helix J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and nuclear cells have a angiogenesis D. 1997; PubMed Scopus Google Scholar). it is nuclear with in helix-loop-helix factors are to as as the family of that E box sequences during PubMed Scopus Google Scholar, J. 14: PubMed Scopus Google and the to a and 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). we binding to the AP-1 site and to the E box repressor site. binding to of these is increased with transcription binding to E box elements has been described for a of different Google Scholar, J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, J. PubMed Scopus (17) Google Scholar). The that TPA factors which and induction of FGF-BP a mechanism by which transcription of the FGF-BP gene be tightly and a of expression of this Overall, our data that the TPA induction of the FGF-BP promoter is induced site and a site and that the extent of induction is by factors that to an E box repressor element which to the AP-1 site. is that TPA also the expression of in of the extracellular as and F. M. Res. PubMed Scopus Google Scholar, G. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Biol. PubMed Scopus Google Scholar). these are by transcription factors as which we are in FGF-BP promoter and our data the that a specific of transcription factors be induced to a of in angiogenesis, and during skin tumor development. We Aigner and for data and all of the and Wellstein for