Transcriptional Regulation of bcl-2 Mediated by the Sonic Hedgehog Signaling Pathway through gli-1
2004; Elsevier BV; Volume: 279; Issue: 2 Linguagem: Inglês
10.1074/jbc.m310589200
ISSN1083-351X
AutoresRebecca L. Bigelow, Nikhil Chari, Anne Birgitte Undén, Kevin B. Spurgers, Sangjun Lee, Dennis R. Roop, Rune Toftgård, Timothy J. McDonnell,
Tópico(s)Nonmelanoma Skin Cancer Studies
ResumoBasal cell carcinomas (BCCs) express high levels of the antiapoptotic proto-oncogene, bcl-2, and we have shown that bcl-2 contributes to the malignant phenotype in a transgenic mouse model. The basis of bcl-2 transcriptional regulation in keratinocytes is unknown. The sonic hedgehog (SHH) signaling pathway is frequently altered in BCCs. Mediators of shh signaling include the downstream transactivator, gli-1, and transrepressor, gli-3. Seven candidate gli binding sites were identified in the bcl-2 promoter. Cotransfection of increasing amounts of gli-1 in keratinoycytes resulted in a corresponding dose-dependent increase in bcl-2 promoter luciferase activity. Gli-1 was also able to up-regulate endogenous bcl-2. Gli-3 cotransfection resulted in no significant changes in bcl-2 promoter activity compared with control. Gli-3 has been demonstrated to be proteolytically processed into an N-terminal repressive form that can inhibit downstream transactivation by gli-1. Gli-3 mutants possessing only the N-terminal region or the C-terminal region were made and used in luciferase assays. The N terminus of gli-3 inhibited gli-1 transactivation of the bcl-2 promoter. Gel shift analysis and luciferase assays demonstrated that gli binding site 4 (-428 to -420), is important for gli transcriptional regulation. Skin samples from transgenic mice expressing an RU486 gli-1 transgene exhibited significantly higher levels of endogenous bcl-2 protein in epidermal keratinocytes as assessed by immunoblotting and immunohistochemistry. Together, these findings provide consistent evidence that gli proteins can transcriptionally regulate the bcl-2 promoter and that gli-3 can inhibit transactivation by gli-1. These studies further suggest that one consequence of the deregulation of shh signaling in BCC is the up-regulation of bcl-2. Basal cell carcinomas (BCCs) express high levels of the antiapoptotic proto-oncogene, bcl-2, and we have shown that bcl-2 contributes to the malignant phenotype in a transgenic mouse model. The basis of bcl-2 transcriptional regulation in keratinocytes is unknown. The sonic hedgehog (SHH) signaling pathway is frequently altered in BCCs. Mediators of shh signaling include the downstream transactivator, gli-1, and transrepressor, gli-3. Seven candidate gli binding sites were identified in the bcl-2 promoter. Cotransfection of increasing amounts of gli-1 in keratinoycytes resulted in a corresponding dose-dependent increase in bcl-2 promoter luciferase activity. Gli-1 was also able to up-regulate endogenous bcl-2. Gli-3 cotransfection resulted in no significant changes in bcl-2 promoter activity compared with control. Gli-3 has been demonstrated to be proteolytically processed into an N-terminal repressive form that can inhibit downstream transactivation by gli-1. Gli-3 mutants possessing only the N-terminal region or the C-terminal region were made and used in luciferase assays. The N terminus of gli-3 inhibited gli-1 transactivation of the bcl-2 promoter. Gel shift analysis and luciferase assays demonstrated that gli binding site 4 (-428 to -420), is important for gli transcriptional regulation. Skin samples from transgenic mice expressing an RU486 gli-1 transgene exhibited significantly higher levels of endogenous bcl-2 protein in epidermal keratinocytes as assessed by immunoblotting and immunohistochemistry. Together, these findings provide consistent evidence that gli proteins can transcriptionally regulate the bcl-2 promoter and that gli-3 can inhibit transactivation by gli-1. These studies further suggest that one consequence of the deregulation of shh signaling in BCC is the up-regulation of bcl-2. Non-melanoma skin cancer is the most frequently diagnosed form of cancer in the United States and basal cell carcinomas (BCC) 1The abbreviations used are: BCC, basal cell carcinomas; shh, sonic hedgehog; ptc, patched; smo, smoothened; GST, glutathione S-transferase. 1The abbreviations used are: BCC, basal cell carcinomas; shh, sonic hedgehog; ptc, patched; smo, smoothened; GST, glutathione S-transferase. constitutes more than 75% of these neoplasms (1.Miller S.J. J. Am. Acad. Dermatol. 1991; 24: 1-13Abstract Full Text PDF PubMed Scopus (371) Google Scholar). Recent evidence has established the importance of the shh pathway in the development of BCC (2.Gailani M.R. Bale A.E. J. Natl. Cancer Inst. 1997; 89: 1103-1109Crossref PubMed Scopus (102) Google Scholar, 3.Ruiz i Altaba A. Trends Genet. 1999; 15: 418-425Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 4.Wicking C. Shanley S. Smyth I. Gillies S. Negus K. Graham S. Suthers G. Haites N. Edwards M. Wainwright B. Chenevix-Trench G. Am. J. Hum. Genet. 1997; 60: 21-26PubMed Google Scholar, 5.Kallassy M. Toftgard R. Ueda M. Nakazawa K. Vorechovsky I. Yamasaki H. Nakazawa H. Cancer Res. 1997; 57: 4731-4735PubMed Google Scholar, 6.Dahmane N. Lee J. Robins P. Heller P. Ruiz i Altaba A. Nature. 1997; 389: 876-881Crossref PubMed Scopus (538) Google Scholar). The sonic hedgehog (shh) pathway was first characterized in normal developmental processes, including the formation of the dorsal to ventral axis of the neural tube, the anterior to posterior axis of the limb bud, and the development of the foregut (7.Chiang C. Litingtung Y. Lee E. Young K.E. Corden J.L. Westphal H. Beachy P.A. Nature. 1996; 383: 407-413Crossref PubMed Scopus (2557) Google Scholar, 8.Litingtung Y. Lei L. Westphal H. Chiang C. Nat. Genet. 1998; 20: 58-61Crossref PubMed Scopus (573) Google Scholar, 9.Weed M. Mundlos S. Olsen B.R. Matrix Biol. 1997; 16: 53-58Crossref PubMed Scopus (39) Google Scholar). Alterations in the sonic hedgehog pathway have been implicated in the development of malignancies other than basal cell carcinoma including medulloblastoma and rhabdomyosarcoma (3.Ruiz i Altaba A. Trends Genet. 1999; 15: 418-425Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). The sonic hedgehog receptor, patched (ptc), normally functions to inhibit shh signaling. Binding of shh to ptc relieves the inhibition, allowing for transduction to continue through smoothened (smo) and a large multiprotein complex in the cytosol (10.Ingham P.W. EMBO J. 1998; 17: 3505-3511Crossref PubMed Scopus (381) Google Scholar). The downstream transcription factors, gli-1, -2, and -3 bind to the same DNA sequence (5′-GACCACCCA-3′) (11.Kinzler K.W. Vogelstein B. Mol. Cell. Biol. 1990; 10: 634-642Crossref PubMed Scopus (416) Google Scholar, 12.Ruppert J.M. Vogelstein B. Arheden K. Kinzler K.W. Mol. Cell. Biol. 1990; 10: 5408-5415Crossref PubMed Scopus (157) Google Scholar). Gli-2 and Gli-3 have both transactivation and repressive domains, whereas gli-1 has been suggested to function only as a transactivator (13.Dai P. Akimaru H. Tanaka Y. Maekawa T. Nakafuku M. Ishii S. J. Biol. Chem. 1999; 274: 8143-8152Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, 14.Sasaki H. Nishizaki Y. Hui C. Nakafuku M. Kondoh H. Development. 1999; 126: 3915-3924Crossref PubMed Google Scholar). Protein kinase A is believed to be the common negative regulator of the pathway (10.Ingham P.W. EMBO J. 1998; 17: 3505-3511Crossref PubMed Scopus (381) Google Scholar). It has been demonstrated that PKA activity indirectly induces cleavage of gli-3 into its repressor form, which can be inhibited by shh (13.Dai P. Akimaru H. Tanaka Y. Maekawa T. Nakafuku M. Ishii S. J. Biol. Chem. 1999; 274: 8143-8152Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar). The pathway is further complicated by the evidence that gli-3 may be required for transcriptional up-regulation of gli-1 (13.Dai P. Akimaru H. Tanaka Y. Maekawa T. Nakafuku M. Ishii S. J. Biol. Chem. 1999; 274: 8143-8152Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar). BCC is associated with mutations in the shh pathway. Loss of heterozygosity is frequently found at the location of patched, 9q22, in patients suffering from nevoid basal cell carcinoma syndrome who are predisposed to developing BCCs, and in sporadic BCC tumors (2.Gailani M.R. Bale A.E. J. Natl. Cancer Inst. 1997; 89: 1103-1109Crossref PubMed Scopus (102) Google Scholar, 15.Gailani M.R. Leffell D.J. Ziegler A. Gross E.G. Brash D.E. Bale A.E. J. Natl. Cancer Inst. 1996; 88: 349-354Crossref PubMed Scopus (163) Google Scholar). Premature protein termination and inactivating mutations of patched leads to increased expression of a mutant patched protein (2.Gailani M.R. Bale A.E. J. Natl. Cancer Inst. 1997; 89: 1103-1109Crossref PubMed Scopus (102) Google Scholar, 4.Wicking C. Shanley S. Smyth I. Gillies S. Negus K. Graham S. Suthers G. Haites N. Edwards M. Wainwright B. Chenevix-Trench G. Am. J. Hum. Genet. 1997; 60: 21-26PubMed Google Scholar). The loss of ptc leads to subsequent increased expression of smoothened, shh, and gli-1, because of the positive feedback loop initiated by gli-1 transactivation (5.Kallassy M. Toftgard R. Ueda M. Nakazawa K. Vorechovsky I. Yamasaki H. Nakazawa H. Cancer Res. 1997; 57: 4731-4735PubMed Google Scholar, 6.Dahmane N. Lee J. Robins P. Heller P. Ruiz i Altaba A. Nature. 1997; 389: 876-881Crossref PubMed Scopus (538) Google Scholar, 16.Green J. Leigh I.M. Poulsom R. Quinn A.G. Br. J. Dermatol. 1998; 139: 911-915Crossref PubMed Scopus (57) Google Scholar). This effect may be augmented by activating missense mutations in smo (17.Xie J. Murone M. Luoh S.M. Ryan A. Gu Q. Zhang C. Bonifas J.M. Lam C.W. Hynes M. Goddard A. Rosenthal A. Epstein Jr., E.H. de Sauvage F.J. Nature. 1998; 391: 90-92Crossref PubMed Scopus (1104) Google Scholar). The mutations in ptc and smo result in 50% of BCCs overexpressing shh and 98% overexpressing gli-1 (6.Dahmane N. Lee J. Robins P. Heller P. Ruiz i Altaba A. Nature. 1997; 389: 876-881Crossref PubMed Scopus (538) Google Scholar). Transgenic mice with shh or gli-1 targeted to the basal layer of the epidermis developed features similar to BCCs (18.Oro A.E. Higgins K.M. Hu Z. Bonifas J.M. Epstein Jr., E.H. Scott M.P. Science. 1997; 276: 817-821Crossref PubMed Scopus (599) Google Scholar, 19.Nilsson M. Unden A.B. Krause D. Malmqwist U. Raza K. Zaphiropoulos P.G. Toftgard R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3438-3443Crossref PubMed Scopus (357) Google Scholar) and ptc-/+ mice developed spontaneous BCCs upon UV radiation (20.Aszterbaum M. Epstein J. Oro A. Douglas V. LeBoit P.E. Scott M.P. Epstein Jr, E.H. Nat. Med. 1999; 5: 1285-1291Crossref PubMed Scopus (345) Google Scholar). The bcl-2 family is important in the regulation of apoptosis (21.McDonnell T.J. Beham A. Sarkiss M. Andersen M.M. Lo P. Experientia (Basel). 1996; 52: 1008-1017Crossref PubMed Scopus (106) Google Scholar). bcl-2 is composed of three exons, with an untranslated first exon and two introns of 220 bp and a large 370-kb intron (22.Seto M. Jaeger U. Hockett R.D. Graninger W. Bennett S. Goldman P. Korsmeyer S.J. EMBO J. 1988; 7: 123-131Crossref PubMed Scopus (456) Google Scholar). Studies show that bcl-2 has two promoter regions. P2 is located immediately 5′ to the open reading frame in exon II and contains both a TATA and CAAT box (22.Seto M. Jaeger U. Hockett R.D. Graninger W. Bennett S. Goldman P. Korsmeyer S.J. EMBO J. 1988; 7: 123-131Crossref PubMed Scopus (456) Google Scholar). This site is responsible for a small percentage of transcripts in cells such as B cells (22.Seto M. Jaeger U. Hockett R.D. Graninger W. Bennett S. Goldman P. Korsmeyer S.J. EMBO J. 1988; 7: 123-131Crossref PubMed Scopus (456) Google Scholar). The second promoter, P1, is located in exon I in a GC-rich region and does not contain a TATA nor a CAAT box (22.Seto M. Jaeger U. Hockett R.D. Graninger W. Bennett S. Goldman P. Korsmeyer S.J. EMBO J. 1988; 7: 123-131Crossref PubMed Scopus (456) Google Scholar). P1 contains several Sp1 binding sites and has multiple transcription initiation sites (22.Seto M. Jaeger U. Hockett R.D. Graninger W. Bennett S. Goldman P. Korsmeyer S.J. EMBO J. 1988; 7: 123-131Crossref PubMed Scopus (456) Google Scholar). Transcription factors previously reported to be involved in the regulation of bcl-2 include: Brn3a in neuronal cells (23.Smith M.D. Ensor E.A. Coffin R.S. Boxer L.M. Latchman D.S. J. Biol. Chem. 1998; 273: 16715-16722Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), WT1 (24.Mayo M.W. Wang C.Y. Drouin S.S. Madrid L.V. Marshall A.F. Reed J.C. Weissman B.E. Baldwin A.S. EMBO J. 1999; 18: 3990-4003Crossref PubMed Scopus (216) Google Scholar, 25.Hewitt S.M. Hamada S. McDonnell T.J. Rauscher III, F.J. Saunders G.F. Cancer Res. 1995; 55: 5386-5389PubMed Google Scholar), Aiolos in T-cells (26.Romero F. Martinez A.C. Camonis J. Rebollo A. EMBO J. 1999; 18: 3419-3430Crossref PubMed Scopus (77) Google Scholar), HIV Tat (27.Wang Z. Morris G.F. Reed J.C. Kelly G.D. Morris C.B. Virology. 1999; 257: 502-510Crossref PubMed Scopus (26) Google Scholar), and PAX8 (28.Hewitt S.M. Hamada S. Monarres A. Kottical L.V. Saunders G.F. McDonnell T.J. Anticancer Res. 1997; 17: 3211-3215PubMed Google Scholar). In addition to these positive regulatory sites, the promoter also contains pie1 binding sites, which serve as negative regulatory regions and are thought to be important in pre-B cell selection (29.Chen H.M. Boxer L.M. Mol. Cell. Biol. 1995; 15: 3840-3847Crossref PubMed Google Scholar, 30.Young R.L. Korsmeyer S.J. Mol. Cell. Biol. 1993; 13: 3686-3697Crossref PubMed Scopus (180) Google Scholar). We have previously demonstrated that the members of the bcl-2 family are expressed in specific regions of the epidermis. Bcl-2 is exclusively expressed in the basal layer of the epidermis and undergoes a 2–3-fold increase in expression in BCCs (31.Rodriguez-Villanueva J. Colome M.I. Brisbay S. McDonnell T.J. Pathol. Res. Pract. 1995; 191: 391-398Crossref PubMed Scopus (54) Google Scholar, 32.Delehedde M. Cho S.H. Sarkiss M. Brisbay S. Davies M. El-Naggar A.K. McDonnell T.J. Cancer. 1999; 85: 1514-1522Crossref PubMed Scopus (63) Google Scholar). In contrast, bcl-2 is undetectable in squamous cell carcinomas (31.Rodriguez-Villanueva J. Colome M.I. Brisbay S. McDonnell T.J. Pathol. Res. Pract. 1995; 191: 391-398Crossref PubMed Scopus (54) Google Scholar, 32.Delehedde M. Cho S.H. Sarkiss M. Brisbay S. Davies M. El-Naggar A.K. McDonnell T.J. Cancer. 1999; 85: 1514-1522Crossref PubMed Scopus (63) Google Scholar). The increased expression of bcl-2 may contribute to the malignant phenotype through inhibition of cell death (33.Rodriguez-Villanueva J. Greenhalgh D. Wang X.J. Bundman D. Cho S. Delehedde M. Roop D. McDonnell T.J. Oncogene. 1998; 16: 853-863Crossref PubMed Scopus (70) Google Scholar, 34.Delehedde M. Cho S.H. Hamm R. Brisbay S. Ananthaswamy H.N. Kripke M. McDonnell T.J. J. Invest. Dermatol. 2001; 116: 366-373Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Transcriptional regulators of bcl-2 expression in keratinocytes have yet to be identified. Here we demonstrate a link between the shh signaling pathway and bcl-2 expression in keratinocytes. Seven candidate gli binding sites were identified in the bcl-2 promoter. Gli binding site 4 was shown to be important in the transcriptional regulation by gli. Additionally it was demonstrated that a balance between gli-1 transactivation and gli-3 repression controls bcl-2 levels in keratinocytes. These observations suggest that the overexpression of gli-1 in basal cell carcinomas may contribute to the development of BCC by transcriptionally up-regulating bcl-2, thus inhibiting cell death. Plasmid Constructs—The pGL3-2.8 bcl-2 promoter construct is illustrated in Fig. 1. The sequence depicted in Fig. 1 can be found in GenBank™, accession numbers X51898 and M13994. A three-step cloning strategy was used. Briefly, a 308-bp product was PCR amplified with primer generated XhoI and HindIII sites from a 7.8-kb genomic HindIII fragment of the Bcl-2 gene. This 308-bp product covers a region just 5′ to the AccI site to just 5′ of the initiation codon. This product was cloned into the XhoI and HindIII sites of the pGL3 basic luciferase vector (Promega, Madison, WI) to produce pGL3-308 bcl-2. Next, a 486-bp XhoI,AccI restriction fragment was cloned into pGL3-308 bcl-2 producing pGL3-748 bcl-2, which lacks gli binding sites 1 through 6. Finally, a 2100-bp XhoI fragment was cloned into pGL3-748 bcl-2 producing pGL3-2.8 bcl-2. The remaining bcl-2 luciferase deletion constructs were made using unique restriction sites in the promoter as observed in Fig. 6 and blunt-end ligation. pGL3–2.8bcl-2Δ1, BbrPI and BlnI; pGL3-2.8 bcl-2Δ2–5, BlnI and StuI; pGL3-2.8 bcl-2Δ5, StuI; pGL3-2.8 bcl-2Δ1–5-luc, BbrPI and StuI. pGL3-2.8 bcl-2Δ1–5/7 was made by cutting pGL3-2.8 bcl-2 with BssHII and isolating the 831-bp fragment and inserting it into MluI cut pGL3 basic vector. All constructs were sequence verified.Fig. 6Bcl-2 luciferase constructs. The pGL3-2.8 bcl-2 luciferase construct was made as described under “Experimental Procedures.” Unique restriction sites were used to make bcl-2 luciferase construct mutants as described. The numbers on the schematic of the bcl-2 gene (numbers 1–7) correspond to the potential gli binding sites. P1 and P2 represent the two transcriptional start sites.View Large Image Figure ViewerDownload Hi-res image Download (PPT) pGL3SV40 bcl-2-4 was made by cutting pGL3-2.8 bcl-2 with XmaI, isolating the 237-bp fragment, and inserting it into XmaI, calf intestinal alkaline phosphatase-treated pGL3SV40 promoter vector (Promega). pGL3SV40 bcl-2mut4 was made by site-directed mutagenesis using the mutagenesis primers: site4mutS, 5′-GGACCCCAGCGATTCTTAAGTCGCACCGG-3′, and site4mutA, 5′-CCGGTGCGACTTAAGAATCGCTGGGGTCC-3′, along with the forward primer RVprimer3 and reverse primer GLprimer2, specific for the pGL3 vectors from Promega. PCR was performed using 96 °C for 4 min followed by 35 cycles or 96 °C for 1 min, 62 °C for 1 min, 72 °C for 1 min, and a final 5-min 72 °C elongation step. The final 237-bp PCR product was cut with XmaI and inserted into XmaI cut pGL3SV40. The constructs were sequence verified. Gli-1 and gli-3 cDNA constructs were generously provided by Dr. Bert Volgelstein. Gli-1 pcDNA was made by inserting the HindIII/XbaI fragment of gli-1 into the HindIII/XbaI site of the pcDNA 3.1 Zeocin (+) vector from Invitrogen (Carlsbad, CA). Gli-3 pcDNA was made by inserting the EcoRI fragment of gli-3 that cuts at bp 105 in the cDNA and on the 3′ side of gli-3 in the pBlueScript vector into the EcoRI site of pcDNA 3.1 His B (Invitrogen) in-frame with a 5′ His tag. Gli-3ΔN pcDNA was made by cutting gli-3 pcDNA with BamHI, which cuts at 1238 in the cDNA upstream of the zinc fingers and with EcoRI. The 3817-bp C-terminal fragment was isolated and ligated to BamHI/EcoRI-cut pcDNA 3.1 His A (Invitrogen) in-frame with the 5′ terminal His tag. Gli-3ΔC pcDNA was cloned by cutting gli-3 pcDNA with ClaI, which cuts at 2285 in the cDNA downstream of the zinc fingers and with EcoRV, which cuts 3′ to the cDNA insertion site. The 10-kb fragment was isolated, klenowed, and ligated. All plasmids were sequence verified. Cell Culture—Primary keratinocytes were obtained from human neonatal foreskin and co-cultured with a feeder layer of 3T3 J2 fibroblasts (∼1 × 106) treated with mitomycin C (4 μg/ml) for 2 h. The tissue samples were washed in sterile, serum-free Dulbecco's modified Eagle's medium. Red blood cells and stroma were removed. The tissue was minced with sterile curved iris scissors and transferred to a sterile spinner bottle with 10 ml of 0.1% trypsin for 45 min at room temperature. After 45 min cells were pelleted by centrifugation at 1000 rpm for 5 min. The trypsin was aspirated and the cells were washed in 10 ml of serum-containing keratinocyte media (3 parts Ham's F-12, 1 part Dulbecco's modified Eagle's medium, 5% fetal bovine serum, 0.4 μg/ml hydrocortisone, 8.4 ng/ml cholera toxin, 5 μg/ml insulin, 24 μg/ml adenine, 1× penicillin/streptomycin) without epidermal growth factor and replated in a 0.5–1.0-ml volume on a feeder layer of 3T3 J2 fibroblasts. The trypsinization step was repeated for an additional 45 min for 4 more rounds. After 24 h the medium was changed and epidermal growth factor (10 ng/ml) was added to the media. After 5–7 days the feeder layer was removed with 0.02% EDTA in phosphate-buffered saline (pH 7.35) and a fresh feeder layer was added. Fibroblasts were maintained in Dulbecco's modified Eagle's medium with 5% calf serum and 1× penicillin/streptomycin. Media was changed every other day. SDS-PAGE—The TnT coupled reticulocyte lysate system kit from Promega was used to make in vitro transcribed/translated protein according to the manufacturer's protocol with gli-1 pcDNA and gli-3 pcDNA expression constructs. 5 μl of [35S]methionine-labeled in vitro transcribed/translated protein was boiled for 5 min in 4× sample loading buffer (200 mm Tris, 400 mm β-mercaptoethanol, 8% SDS, 0.4% bromphenol blue, and 40% glycerol) and run on a 7.5% SDS-acrylamide gel, dried, and autoradiographed. Electrophoretic Mobility Shift Assays—Single stranded oligonucleotides were obtained from Sigma and annealed at a final concentration of 50 ng/μl in 10× annealing buffer (100 mm Tris, pH 7.5, 60 mm MgCl2, 500 mm NaCl, 60 mm β-mercaptoethanol, and 2 mg/ml gelatin) by boiling for 5 min and cooled to room temperature overnight. Oligonucleotides were end labeled using the forward labeling reaction incubating 1 μl of double stranded DNA, 5 μl of 5× forward labeling buffer, 1 μl of T4 kinase, 2.5 μl of [32P]ATP, and 15.5 μl of water at 37 °C for 10 min and heat inactivating for 10 min at 65 °C. 25 μl of water was added to equal 1 ng/μl. The labeled DNA was purified on a G-25 column according to the manufacturers protocol. The binding reaction was performed by incubating 10 μl of binding buffer (10 mm Tris-HCl, pH 7.5, 50 mm NaCl, 5 mm MgCl2, 0.5 mm dithiothreitol and 4% glycerol), 1 μl of poly(DI-DC) (1 μg/μl), 3 μl of unlabeled TnT reaction protein with 1 μl of cold competitor oligonucleotide for 30 min on ice. 1 μl of the hot probe was added and incubated for 30 min on ice. The reactions were run on a 4% acrylamide gel in 0.5× TBE at 100 V that was prerun for 1 h. The gel was dried and autoradiographed. Oligonucleotides used in the analysis include: gli positive control, 5′-GATCTAAGAGCTCCCGAAGACCACCCACAATGATGGTTGTATGT-3′; gli mutant, 5′-GATCTAAGAGCTCCCGAAGACTATTTGCAATGATGGTTGTATGT-3′; bcl-2-1, 5′-TGGAGACCTTTAGGAGCCCACCCACCCCAGCGTTAGGACGGTGG-3′; bcl-2-2, 5′-GTCCAGGCGACACACACACTCCCACATACACGGCCAGAAAAGGT-3′; bcl-2-3, 5′-CGTGCGATTCCCCGGGAGCCCCCACCCCGTCGGACCCCAGCG-3′; bcl-2-4, 5′-GTCGCGGACCCCAGCGACCACCAAGTCGCACCGGCCTCCGCAGG-3′; bcl-2-5, 5′-AGCAGAAGGCCCCGCGCACACCCACGCGCGGGGCCCGCGGGGAG-3′; bcl-2-6, 5′-CCCCACCCCTCGCCGCACCACACACAGCGCGGGCTTCTAGCGCT-3′; bcl-2-7, 5′-GTCCAAGAATGCAAAGCACATCCAATAAAATAGCTGGATTATAA-3′. These were annealed to their corresponding 3′ to 5′ oligonucleotides. Luciferase Assays—The dual luciferase reporter kit from Promega was used for luciferase assays. Primary keratinocytes were plated in 12-well plates on a feeder layer of mitomycin C-treated 3T3 J2 fibroblasts. When the cells reached 60% confluency, the feeder layer was removed with 0.02% EDTA in phosphate-buffered saline, pH 7.35. 1, 3, or 5 μg of gli-1 pcDNA or gli-3 pcDNA were transfected with nonspecific DNA to equal a total of 5 μg with LipofectAMINE (Invitrogen) according to the manufacturer's protocol. 2 μg of the pGL3-2.8 bcl-2 reporter construct and 0.5 μg of the Renilla luciferase construct under control of the cytomegalovirus promoter (Promega) to normalize for transfection efficiency were also transfected. To assess the effect of gli-3 and gli-3 mutants on gli-1 transactivation, the experiment was performed as stated above except, 2 μg of each cDNA construct was used and nonspecific DNA was used to normalize to 4 μg. In addition 2 μg of the pGL3-2.9 bcl-2 and 0.5 μg of Renilla were used. Deletion construct luciferase assays were performed by transfection of 2 μg of gli-1 pcDNA or vector control, 2 μg of the corresponding bcl-2 luciferase construct, and 0.5 μg of Renilla luciferase. In all experiments, protein lysates were taken 24 h after transfection according to the protocol from the dual-luciferase reporter kit and read on a luminometer. Each experiment was repeated at least 3 times, each time in triplicate. Analysis of Skin from gli-1 Transgenic Mice—Skin samples were obtained from K14p65GL control mice and K14p65GL/gli-1 bitransgenic mice (19.Nilsson M. Unden A.B. Krause D. Malmqwist U. Raza K. Zaphiropoulos P.G. Toftgard R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3438-3443Crossref PubMed Scopus (357) Google Scholar). The construct used to generate these mice targets a gli-1 transgene specifically to the epidermis using an RU486 inducible keratin 14 promoter. The bigenic mice were exposed to the inducer RU486 for 8 weeks by subcutaneous implantation of a pellet containing 0.55 mg of RU486 with a release time of 60 days (Innovation Research). Tissues used for immunohistochemistry were formalin-fixed and -paraffin embedded. Preceding incubation in 1% H2O2 in methanol for 30 min, sections were dewaxed, rehydrated, and submerged in 10 mm citrate buffer while being heated in a 97 °C water bath for 20 min. Sections were incubated overnight at 4 °C with a 1:200 dilution of a mouse monoclonal antibody against bcl-2 from Transduction Laboratories (Lexington, KY) diluted in phosphate-buffered saline containing 0.1% bovine serum albumin. The immunoreaction was visualized using an avidin-biotin complex (Dako, Carpinteria, CA) with 0.004% hydrogen peroxidase as substrate and diaminobenzidine as a chromogen. Counterstaining was performed with Mayer's hematoxylin. Consecutive serial sections were used as controls with omission of primary antibody. Additionally, 2-mm mouse ear biopsies were ground in a solution containing SDS buffer (5% SDS, 20% β-mercaptoethanol in Tris, pH 6.8) with a pestle for immunoblot analysis. This was then boiled for 10 min and centrifuged at 4 °C for 30 min. Supernatant was used for SDS-PAGE analysis with loading buffers as described previously and run on a 12.5% SDS-acrylamide gel. Protein expression was assessed using mouse bcl-2 antibody from BD Pharmingen (San Diego, CA) and α-actin, Sigma. RNA was isolated from skin biopsies using the RNeasy Mini kit from Qiagen (Valencia, CA) as per the supplier's protocol. 1 μg was treated with RQ1 RNase-free DNase (Promega) according to the supplier's protocol. Synthesis of cDNA was done using Superscript RNase H-reverse transcriptase (Invitrogen), 50 ng of oligo(dT)15 primer (Promega), PCR nucleotide mixture, and recombinant RNasin ribonuclease inhibitor (Promega). 5 μl of cDNA was used for the PCR using Taq DNA polymerase (Promega) and oligonucleotide primers specific for gli-1 for 40 cycles. Reaction products were electrophoresed on a 2% SeKem LE-agarose gel. Identification of Candidate Gli Binding Sites in the bcl-2 Promoter—The observation that both gli-1 and bcl-2 are overexpressed in basal cell carcinomas prompted us to search the bcl-2 promoter for potential gli binding sites. Gli-1, -2, and -3 bind to the DNA consensus sequence 5′-GACCACCCA-3′ (11.Kinzler K.W. Vogelstein B. Mol. Cell. Biol. 1990; 10: 634-642Crossref PubMed Scopus (416) Google Scholar, 12.Ruppert J.M. Vogelstein B. Arheden K. Kinzler K.W. Mol. Cell. Biol. 1990; 10: 5408-5415Crossref PubMed Scopus (157) Google Scholar). PatSearch version 1.1 from Transfac was used to locate potential regions of gli binding. Data base analysis revealed seven potential sites (Fig. 1). Sites 1 through 5 are located upstream of the P1 transcriptional start site, whereas site 6 is located downstream of P1 in exon 2 and site 7 is located slightly upstream of the P2 transcriptional start site. The homology of each gli binding site to the canonical consensus sequence 5′-GACCACCCA-3′ varied from 55% (site 2) to 89% (sites 1 and 4). Gli-1 Positively Regulates bcl-2 Transcription—Gli-1 has been shown to contain only a transactivation domain and is considered the primary regulator of shh-induced transactivation (13.Dai P. Akimaru H. Tanaka Y. Maekawa T. Nakafuku M. Ishii S. J. Biol. Chem. 1999; 274: 8143-8152Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, 14.Sasaki H. Nishizaki Y. Hui C. Nakafuku M. Kondoh H. Development. 1999; 126: 3915-3924Crossref PubMed Google Scholar). Gli-3, however, contains both a transactivation domain and a repression domain (13.Dai P. Akimaru H. Tanaka Y. Maekawa T. Nakafuku M. Ishii S. J. Biol. Chem. 1999; 274: 8143-8152Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, 14.Sasaki H. Nishizaki Y. Hui C. Nakafuku M. Kondoh H. Development. 1999; 126: 3915-3924Crossref PubMed Google Scholar). Luciferase assays were used to determine whether gli-1 and gli-3 are able to regulate bcl-2 transcription. A bcl-2 reporter construct (pGL3–2.8 bcl-2) was made by ligating a 2.8-kb fragment of the bcl-2 promoter to the firefly luciferase gene. Assays were done in primary keratinocyte cultures obtained from neonatal human foreskin. Increasing concentrations of gli-1 pcDNA transfected with the pGL3–2.8 bcl-2 promoter construct resulted in a dose-dependent increase in promoter activity. In contrast, increasing concentrations of gli-3 pcDNA resulted in no apparent change in steady-state promoter activity (Fig. 2). Reporter luciferase values were normalized for transfection efficiency using values obtained with the Renilla luciferase construct. Similar results were obtained using NIH 3T3 fibroblasts (data not shown). The N-terminal Domain of Gli-3 Inhibits Gli-1 Transactivation—To determine whether gli-3 is able to inhibit gli-1 transactivation of the bcl-2 promoter, luciferase assays were done in primary ke
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