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Identification of Nerve Growth Factor-responsive Element of the TCL1 Promoter as a Novel Negative Regulatory Element

2006; Elsevier BV; Volume: 281; Issue: 38 Linguagem: Inglês

10.1074/jbc.m602420200

ISSN

1083-351X

Autores

Makoto Hiromura, Futoshi Suizu, Masumi Narita, Keiichi Kinowaki, Masayuki Noguchi,

Tópico(s)

Macrophage Migration Inhibitory Factor

Resumo

The serine/threonine kinase, Akt (protein kinase B) plays a central role in the regulation of intracellular cell survival. Recently, we demonstrated that the proto-oncogene TCL1, overexpressed in human T-cell prolymphocytic leukemia, is an Akt kinase co-activator. Tightly restricted TCL1 gene expression in early developmental cells suggested that the TCL1 gene is regulated at a transcriptional level. To characterize how TCL1 gene expression is regulated, we cloned the 5′-promoter of the TCL1 gene located at human chromosome 14q32. The 5′-TCL1 promoter region contains a TATA box with cis-regulatory elements for Nur77/NGFI-B (nerve growth factor-responsive element (NBRE), CCAAGGTCA), NFκB, and fork head transcription factor. Nur77/NGFI-B, an orphan receptor superfamily transcription factor implicated in T-cell apoptosis, is a substrate for Akt. We hypothesized that TCL1 transactivity is regulated through Akt-induced phosphorylation of Nur77/NGFI-B in vivo. In an electrophoretic mobility shift assay with chromosomal immunoprecipitation assays, wild-type Nur77, but not S350A mutant Nur77, could specifically bind to TCL1-NBRE. A luciferase assay demonstrated that TCL1-NBRE is required for inhibition of TCL1 transactivity upon nerve growth factor/platelet-derived growth factor stimulation, which activates Akt and phosphorylates Nur77. Using a chromosomal immunoprecipitation assay with reverse transcription-PCR, nerve growth factor stimulation inhibited binding of endogenous Nur77 to TCL1-NBRE, in turn, suppressing TCL1 gene expression. The results together establish that TCL1-NBRE is a novel negative regulatory element of Nur77 (NGFI-B). To the best of our knowledge, TCL1-NBRE is the first direct target of Nur77 involving the regulation of intracellular cell death survival. This Akt-induced inhibitory mechanism of TCL1 should play an important role in immunological and/or neuronal development in vivo. The serine/threonine kinase, Akt (protein kinase B) plays a central role in the regulation of intracellular cell survival. Recently, we demonstrated that the proto-oncogene TCL1, overexpressed in human T-cell prolymphocytic leukemia, is an Akt kinase co-activator. Tightly restricted TCL1 gene expression in early developmental cells suggested that the TCL1 gene is regulated at a transcriptional level. To characterize how TCL1 gene expression is regulated, we cloned the 5′-promoter of the TCL1 gene located at human chromosome 14q32. The 5′-TCL1 promoter region contains a TATA box with cis-regulatory elements for Nur77/NGFI-B (nerve growth factor-responsive element (NBRE), CCAAGGTCA), NFκB, and fork head transcription factor. Nur77/NGFI-B, an orphan receptor superfamily transcription factor implicated in T-cell apoptosis, is a substrate for Akt. We hypothesized that TCL1 transactivity is regulated through Akt-induced phosphorylation of Nur77/NGFI-B in vivo. In an electrophoretic mobility shift assay with chromosomal immunoprecipitation assays, wild-type Nur77, but not S350A mutant Nur77, could specifically bind to TCL1-NBRE. A luciferase assay demonstrated that TCL1-NBRE is required for inhibition of TCL1 transactivity upon nerve growth factor/platelet-derived growth factor stimulation, which activates Akt and phosphorylates Nur77. Using a chromosomal immunoprecipitation assay with reverse transcription-PCR, nerve growth factor stimulation inhibited binding of endogenous Nur77 to TCL1-NBRE, in turn, suppressing TCL1 gene expression. The results together establish that TCL1-NBRE is a novel negative regulatory element of Nur77 (NGFI-B). To the best of our knowledge, TCL1-NBRE is the first direct target of Nur77 involving the regulation of intracellular cell death survival. This Akt-induced inhibitory mechanism of TCL1 should play an important role in immunological and/or neuronal development in vivo. Serine threonine kinase Akt, 3The abbreviations used are: Akt, protein kinase B; NBRE, nerve growth factor response element; NGFI-B, nerve growth factor-induced B; NGF, nerve growth factor; Nurr1, Nur-related factor 1; FBS, fetal bovine serum; PDGF, platelet-derived growth factor; DBD, DNA-binding domain; GST, glutathione S-transferase; EMSA, electrophoretic mobility shift assay; CMV, cytomegalovirus; ChIP, chromatin immunoprecipitation assay; PIPES, 1,4-piperazinediethanesulfonic acid; RT, reverse transcription; Ab, antibody; FKHRL, fork head transcription factor; WT, wild type; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase. viral homologue of v-Akt, is an important intracellular regulatory molecule that controls apoptosis (1Brazil D.P. Park J. Hemmings B.A. Cell. 2002; 111: 293-303Abstract Full Text Full Text PDF PubMed Scopus (488) Google Scholar, 2Cantley L.C. Science. 2002; 296: 1655-1657Crossref PubMed Scopus (4681) Google Scholar, 3Du K. Tsichlis P.N. Oncogene. 2005; 24: 7401-7409Crossref PubMed Scopus (117) Google Scholar). Recently, we demonstrated that the proto-oncogene TCL1, whose function was previously unknown, is an Akt kinase co-activator that physically binds and, hence, activates Akt (4Laine J. Künstle G. Obata T. Sha M. Noguchi M. Mol. Cell. 2000; 6: 395-407Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, 5Pekarsky Y. Koval A. Hallas C. Bichi R. Tresini M. Malstrom S. Russo G. Tsichlis P. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3028-3033Crossref PubMed Scopus (314) Google Scholar). These studies together provided not only a molecular basis but also therapeutic implications for human T-cell prolymphocytic leukemia in which the TCL1 oncogene is overexpressed due to a chromosomal translocation (6Laine J. Künstle G. Obata T. Noguchi M. J. Biol. Chem. 2002; 277: 3743-3751Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 7Künstle G. Laine J. Pierron G. Kagami S. Nakajima H. Hoh F. Roumenstand C. Stern M.-H. Noguchi M. Mol. Cell. Biol. 2002; 22: 1513-1525Crossref PubMed Scopus (83) Google Scholar, 8Hiromura M. Okada F. Obata T. Auguin D. Shibata T. Roumestand C. Noguchi M. J. Biol. Chem. 2004; 279: 53407-53418Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 9Lock R.B. Int. J. Biochem. Cell Biol. 2003; 35: 1614-1618Crossref PubMed Scopus (11) Google Scholar, 10Teitell M.A. Nat. Rev. Cancer. 2005; 5: 640-648Crossref PubMed Scopus (85) Google Scholar). In T-cell prolymphocytic leukemia, both TCL1 and TCL1b genes on human chromosome 14q23.1 are activated by translocations and inversions at 14q32.1, juxtaposing them to regulatory elements of T-cell receptor genes. In addition to T-cell prolymphocytic leukemia, in human diseases, TCL1 is overexpressed in Epstein-Barr virus-infected B cell lymphoma, ataxia-telangiectasia, teratoma, and/or AIDS-related lymphoma (10Teitell M.A. Nat. Rev. Cancer. 2005; 5: 640-648Crossref PubMed Scopus (85) Google Scholar, 11Teitell M. Damore M.A. Sulur G.G. Turner D.E. Stern M.H. Said J.W. Denny C.T. Wall R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9809-9814Crossref PubMed Scopus (71) Google Scholar, 12Hoyer K.K. French S.W. Turner D.E. Nguyen M.T. Renard M. Malone C.S. Knoetig S. Qi C.F. Su T.T. Cheroutre H. Wall R. Rawlings D.J. Morse 3rd, H.C. Teitell M.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14392-14397Crossref PubMed Scopus (100) Google Scholar, 13Pekarsky Y. Hallas C. Croce C.M. Oncogene. 2001; 20: 5638-5643Crossref PubMed Scopus (81) Google Scholar). In physiological conditions, however, TCL1 expression is tightly restricted to early developmental T cells (CD3-negative) as well as early B cells with negative IgM expression (14Virgilio L. Narducci M.G. Isobe M. Billips L.G. Cooper M.D. Croce C.M. Russo G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12530-12534Crossref PubMed Scopus (230) Google Scholar, 15Hoyer K.K. Herling M. Bagrintseva K. Dawson D.W. French S.W. Renard M. Weinger J.G. Jones D. Teitell M.A. J. Immunol. 2005; 175: 864-873Crossref PubMed Scopus (30) Google Scholar, 16Kang S.M. Narducci M.G. Lazzeri C. Mongiovi A.M. Caprini E. Bresin A. Martelli F. Rothstein J. Croce C.M. Cooper M.D. Russo G. Blood. 2005; 105: 1288-1294Crossref PubMed Scopus (33) Google Scholar). Three major isoforms of the human TCL1 oncogene are present in both the human and mouse genome, namely TCL1, TCL1b, and MTCP1 (9Lock R.B. Int. J. Biochem. Cell Biol. 2003; 35: 1614-1618Crossref PubMed Scopus (11) Google Scholar, 13Pekarsky Y. Hallas C. Croce C.M. Oncogene. 2001; 20: 5638-5643Crossref PubMed Scopus (81) Google Scholar). Under physiological conditions, TCL1 and TCL1b are highly expressed at early developmental stages in several fetal tissues, including thymus, kidney, lung (TCL1), and spleen (TCL1b). After birth, expression of all TCL1 family members is mainly restricted to lymphoid tissues, although TCL1b mRNA is also found in the placenta. It is notable that in mice both TCL1b and TCL1 mRNAs are abundant in oocytes and two-cell embryos, but rare in various adult tissues and lymphoid cell lines (14Virgilio L. Narducci M.G. Isobe M. Billips L.G. Cooper M.D. Croce C.M. Russo G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12530-12534Crossref PubMed Scopus (230) Google Scholar, 17Hallas C. Pekarsky Y. Itoyama T. Varnum J. Bichi R. Rothstein J.L. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14418-14423Crossref PubMed Scopus (50) Google Scholar, 18Virgilio L. Lazzeri C. Bichi R. Nibu K. Narducci M.G. Russo G. Rothstein J.L. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3885-3889Crossref PubMed Scopus (131) Google Scholar). Tightly restricted physiological expression of TCL1 family proteins suggested that TCL1 gene expression is regulated at a transcriptional level (14Virgilio L. Narducci M.G. Isobe M. Billips L.G. Cooper M.D. Croce C.M. Russo G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12530-12534Crossref PubMed Scopus (230) Google Scholar, 16Kang S.M. Narducci M.G. Lazzeri C. Mongiovi A.M. Caprini E. Bresin A. Martelli F. Rothstein J. Croce C.M. Cooper M.D. Russo G. Blood. 2005; 105: 1288-1294Crossref PubMed Scopus (33) Google Scholar, 19French S.W. Malone C.S. Shen R.R. Renard M. Henson S.E. Miner M.D. Wall R. Teitell M.A. J. Biol. Chem. 2003; 278: 948-955Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). To clarify molecular regulation of TCL1 gene expression, we cloned 1123 bp of the 5′-promoter sequence of TCL1 (including 931 from base pairs of the 5′-TCL1 promoter region) from human chromosome 14q32. Nucleotide sequence analysis of TCL1 promoter region revealed that a Nur77 (NGFI-B) binding site (nerve growth factor response element (NBRE), CCAAGGTCA (30Woronicz J.D. Lina A. Calnan B.J. Szychowski S. Cheng L. Winoto A. Mol. Cell. Biol. 1995; 15: 6364-6376Crossref PubMed Scopus (193) Google Scholar)) is located within the proximal promoter. Nur77 (NGFI-B, nerve growth factor-induced B or TR3) was originally identified as a NGF-induced ligand-dependent transcriptional activator from PC12 cells (rat pheochromocytoma cells) (20Milbrandt J. Neuron. 1988; 1: 183-188Abstract Full Text PDF PubMed Scopus (531) Google Scholar). Unlike other orphan receptor superfamily transcription factors, it is believed that ligands were not required for transactivation through Nur77/NGFI-B (21Winoto A. Littman D.R. Cell. 2002; 109: S57-S66Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). Additional subfamilies of NGFI-B transcription factor have been identified from neuronal cells as Nurr1 (Nur-related factor 1), and neuron-derived orphan receptor 1 (22Law S.W. Conneely O.M. DeMayo F.J. O'Malley B.W. Mol. Endocrinol. 1992; 6: 2129-2135Crossref PubMed Scopus (279) Google Scholar, 23Maruyama K. Tsukada T. Bandoh S. Sasaki K. Ohkura N. Yamaguchi K. Neuroendocrinology. 1997; 65: 2-8Crossref PubMed Scopus (38) Google Scholar, 24Maruyama K. Tsukada T. Ohkura N. Bandoh S. Hosono T. Yamaguchi K. Int. J. Oncol. 1998; 12: 1237-1243PubMed Google Scholar). Subsequent studies demonstrated that Nur77 plays a pivotal role in T-cell apoptosis in vivo (25Liu Z.G. Smith S.W. McLaughlin K.A. Schwartz L.M. Osborne B.A. Nature. 1994; 367: 281-284Crossref PubMed Scopus (505) Google Scholar, 26Woronicz J.D. Calnan B. Ngo V. Winoto A. Nature. 1994; 367: 277-281Crossref PubMed Scopus (508) Google Scholar). Recent studies have further demonstrated that Akt interacts with Nur77 and hence induces phosphorylation of Nur77 (27Matsuyama N. Onishi K. Mori Y. Ueno T. Takayama Y. Gotoh Y. J. Biol. Chem. 2001; 274: 32799-32805Abstract Full Text Full Text PDF Scopus (137) Google Scholar, 28Pekarsky Y. Hallas C. Palamarchuk A. Koval A. Bullrich F. Hirata Y. Bichi R. Letofsky J. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3690-3694Crossref PubMed Scopus (153) Google Scholar). Despite intensive studies, a direct gene target of Nur77, which regulates the cell death survival machinery, is yet to be determined (21Winoto A. Littman D.R. Cell. 2002; 109: S57-S66Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). Therefore, it is noteworthy that the cis-regulatory element of Nur77 (NBRE) is well conserved within the TCL1 5′-proximal promoter region of human, mouse, and Rattus (21Winoto A. Littman D.R. Cell. 2002; 109: S57-S66Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). These observations lead us to hypothesize that the TCL1 gene can be regulated through NBRE by Akt-induced phosphorylation of Nur77/NGFI-B in vivo. By EMSA and luciferase reporter assays, we demonstrated that Nur77 (NGFI-B family) bound to the NBRE site of the TCL1 promoter (TCL1-NBRE, AAGGTCA), and negatively regulated TCL1 gene expression by Akt stimulation. In PC12 cells, NGF stimulation induced endogenous Nur77 phosphorylation, diminished binding of Nur77 with the TCL1-NBRE, and, in turn, suppressed TCL1 transactivation. The results together demonstrated that TCL1-NBRE is a novel functional target of Nur77 (NGFI), which could play an important role for the restricted expression of the TCL1 gene in vivo. Cloning and Construction of 5′-TCL1 Promoter Reporter Constructs—Using the following primers for PCR (forward primer: 5′-ATCATCGAGCTCCAGGCTGGAGCTGGTTTCCATG-3′; reverse primer: 5′-ATCATCAGATCTCGTCCAAATACACGAAC TTCTCCC-3′), we amplified 1123 bp (–931 to +192 of TCL1 proximal promoter region) from human 293 kidney fibroblasts (ATCC). The resultant TCL1 promoter fragment was cloned into SacI and BglII sites of luciferase reporter construct PGL3 (Promega, Madison, WI), and designated as pGL3-TCL1. NBRE mutant was generated by digesting with BstEII (within the binding sequence of NBRE of TCL1 promoter), treated with mung bean nuclease (New England Biolabs), and ligated back into PGL3 (PGL3-mt-NBRE or pGL3-mt-NBRE-470). The resultant nucleotide sequences were mutated from AAGGTCA to AAGCCC at the TCL1-NBRE site. Mutation of cis-regulatory element of FKHRL (pGL3-mt-FKHRL) was introduced by QuikChange (Stratagene) site-directed mutagenesis using pairs of primers (forward primer: 5′-GTTACTGCAAAGCGAAGTGAAATTG-3′ and reverse primer: 5′-CAAT TTCACTTCGCTTTGCAGTAAC-3′). Truncation mutant of TCL1 promoter (PGL3-TCL1-470) was generated by amplifying by the pairs of primers (forward primer: 5′-CGGGGTACCTGATCCCATAAGATGAGAG-3′, reverse primer: 5′-ATCATCAGATCTCGTCCAAATACACGAACTTCTCCC-3′). The resultant truncated promoter fragment (from –470 to +192) was digested with KpnI and BglII and subcloned into PGL3 Luciferase reporter construct (pGL3-TCL1-470). The nucleotide sequences of all the luciferase reporter constructs were confirmed by using the Big Dye terminator cycle sequencing kit (Applied Biosystems) using an ABI PRISM 310 DNA sequence analyzer (Applied Biosystems). Luciferase Reporter Assays—PC12 cells (rat pheochromocytoma cells, ATCC) or 293T cells (human kidney fibroblasts, ATCC) were cultured in the charcoal-treated 10% FBS in 24-well plates (Corning). One microgram of indicated luciferase reporter constructs was transfected (wild-type or mutant forms of TCL1 promoter as indicated) by FuGENE 6 transfection reagent (Roche Applied Science). Thirty-six hours after transfection, the cells were serum-starved and for additional 12 h, stimulated with NGF (50 ng/ml, murine NGF (7S), 13290-010, Invitrogen) or PDGF (100 ng/ml, PDGF-AB, p3326, Sigma) as indicated. Luciferase reporter assays were performed using Dual Luciferase kit (Promega). Twenty-four hours after transfection, cells were treated with 20 μm wortmannin (Calbiochem, KY-12420) or 20 μm PD98059 (Biomol, EI-360) or Me2SO for 30 min at 37 °C as indicated. Cells were then stimulated with 50 ng/ml PDGF for 15 min, and luciferase reporter activities were measured. Statistical analysis was performed using Student's t test, and p < 0.05 was considered to be significant. Generation of Recombinant Nur77—A GST-Nur77-DBD (DNA-binding domain) fusion construct in pGEX-4T (pGEX-4T-Nur77-DBD) was generated by PCR amplification using pairs of primers (forward primer: 5′-CGGGATCCGCACCCGTAACCTCCAC-3′ and reverse primer: 5′-CGGAATTCTCAGGGTTTTGAAGGTAGC-3′) with wild-type Nur77 cDNA as a template and subcloned into pGEX-4T-2 vector (Amersham Biosciences). The resultant pGEX-4T-Nur77-DBD expression vector was transformed into BL21 cells (DE3, Invitrogen) and the GST-Nur77-DBD recombinant protein was harvested, dialyzed in a dialysis buffer (20 mm Tris-HCl, pH 8.0, 150 mm NaCl, and 10% glycerol). The protein concentrations were determined by Bradford Assay (Bio-Rad). Electric Mobility Band Shift Assays—EMSA (Fig. 2A) was performed as described in a previous study (29Noguchi M. Miyamoto S. Silverman T.A. Safer B. J. Biol. Chem. 1994; 269: 29161-29167Abstract Full Text PDF PubMed Google Scholar) with the following modifications. The 5′-end of the following oligonucleotides (5′-GAAAGGGCCAAGGTCACCCCGGTGC-3′ and 3′-CTTTCCCGGTTCCAGTGGGGCCACG-5′) were labeled with IRDye (IRD700, LI-COR Bioscience, Lincoln, NE) and annealed to generate double-stranded probes for EMSA. 293T cells were cultured in charcoal-treated FBS using 100-mm dish (CellBIND Surface, Corning) transfected with indicated Nur77 expression vectors (pCS-myc-Nur77-WT or pCS-myc-Nur77-S350A in which serine 350 was mutated to Ala (28Pekarsky Y. Hallas C. Palamarchuk A. Koval A. Bullrich F. Hirata Y. Bichi R. Letofsky J. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3690-3694Crossref PubMed Scopus (153) Google Scholar, 43Rajpal A. Cho Y.A. Yelent B. Koza-Taylor P.H. Li D. Chen E. Whang M. Kang C. Turi T.G. Winoto A. EMBO J. 2003; 22: 6526-6536Crossref PubMed Scopus (110) Google Scholar)) using calcium phosphate method (4Laine J. Künstle G. Obata T. Sha M. Noguchi M. Mol. Cell. 2000; 6: 395-407Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). Twenty-four hours after transfection the cells were serum-starved for an additional 16 h, stimulated (or non-stimulated) with PDGF (50 ng/ml, PDGF-AB, p3326, Sigma) for 20 min. Forty-eight hours after transfection, the cells were harvested and lysed with Hepes cell lysis buffer (20 mm Hepes-NaOH, pH 7.4, 1 mm EDTA, 150 mm NaCl, 1 mm dithiothreitol, 10% glycerol, 0.5 mm phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin) by the freeze-thaw method. Twenty micrograms of cellular extracts was incubated with 0.1% Triton X-100, 4% glycerol, 1 mm EDTA, 10 mm β-mercaptoethanol, 10 mm Tris HCl (pH 7.4), 1 μg of poly(dI-dC), and 5 μg of bovine serum albumin in the presence of 20 pmol of IRD700-labeled probes for 30 min at 4 °C. The resultant samples were resolved onto 5% acrylamide-0.5× TBE (Tris borate-EDTA) gels, and DNA-protein complexes were visualized using an infrared imaging system (Odyssey, LI-COR Bioscience). EMSA (Figs. 2B, 2D, 2E, and 4B) was performed essentially described previously (30Woronicz J.D. Lina A. Calnan B.J. Szychowski S. Cheng L. Winoto A. Mol. Cell. Biol. 1995; 15: 6364-6376Crossref PubMed Scopus (193) Google Scholar) using fluorescence-labeled double-stranded TCL1-NBRE probes (5′-CGGCCAAGGTCACCCCGGCCAAGGTCACCCCA-3′) using a 5′ EndTag Labeling Kit (Vector Laboratories Inc., Burlingame, CA). 293T cells were cultured in charcoal-treated FBS, transfected with indicated Nur77 expression vectors (pCMV-FLAG-Nur77-WT or pCMV-FLAG-Nur77-S350A) in which serine 350 was mutated to Ala (28Pekarsky Y. Hallas C. Palamarchuk A. Koval A. Bullrich F. Hirata Y. Bichi R. Letofsky J. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3690-3694Crossref PubMed Scopus (153) Google Scholar, 43Rajpal A. Cho Y.A. Yelent B. Koza-Taylor P.H. Li D. Chen E. Whang M. Kang C. Turi T.G. Winoto A. EMBO J. 2003; 22: 6526-6536Crossref PubMed Scopus (110) Google Scholar) using the calcium phosphate method (4Laine J. Künstle G. Obata T. Sha M. Noguchi M. Mol. Cell. 2000; 6: 395-407Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar), and cultured on a 100-mm dish (CellBIND Surface). Twenty-four hours after transfection, the cells were serum-starved for an additional 16 h and stimulated (or non-stimulated) with PDGF (50 ng/ml, PDGF-AB, p3326, Sigma) for 20 min as indicated. The cells were then harvested and lysed with Hepes cell lysis buffer (20 mm Hepes-NaOH, pH 7.4, 1 mm EDTA, 150 mm NaCl, 1 mm dithiothreitol, 10% glycerol, 0.5 mm phenylmethylsulfonyl fluoride, and 5 μg/ml leupeptin) by the freeze-thaw method. Thirty micrograms of cell lysates were incubated with 20 mm Hepes (pH 7.5), 90 mm NaCl, 1.5 mm MgCl2, 10 μm ZnCl2, 10 mm dithiothreitol, 0.02% Triton X-100, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, 10 mm NaF, 1 mm Na3VO4, 2.5 μg of poly(dI-dC), and 5 μg of bovine serum albumin in the presence of 30 pmol of fluorescein-labeled TCL1-NBRE probes for 30 min at 4 °C. The resultant samples were resolved onto 4.5% acrylamide-0.5× TBE gels, and DNA-protein complexes were visualized by Bio-Rad Imaging system (Bio-Rad VersaDoc 5000). GST antibody (0–5 μg, Clontech) was used for the supershift experiment (Fig. 2E). Eighty pmol of recombinant GST-Nur77-DBD protein were used for the cold competition experiments (Fig. 2D) combined with a 500- to 1000-fold molar excess of unlabeled TCL1-NBRE probes as indicated. Thirty micrograms of PC12 cell-derived cell extract, with 3 μg of anti-Nur77 antibody (M-210, sc5569, Santa Cruz Biotechnology, Santa Cruz, CA), anti-NOR1 (C-19, sc-22519, Santa Cruz Biotechnology), anti-Nurr1 (E-20, sc-990, Santa Cruz Biotechnology), or rabbit normal serum as a control, was used for supershift experiment (Fig. 4B). In Vitro ChIP Assay—293 cells (human kidney fibroblast cell line, ATCC) were transiently transfected with 5 μg of luciferase reporter constructs (pGL3-TCL1 or pGL3-mt-NBRE) with Nur77 (pCS-myc-Nur77-WT or pCS-myc-Nur77-S350A (28Pekarsky Y. Hallas C. Palamarchuk A. Koval A. Bullrich F. Hirata Y. Bichi R. Letofsky J. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3690-3694Crossref PubMed Scopus (153) Google Scholar, 31Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5454) Google Scholar)) by the calcium phosphate method. Forty-eight hours after transfection, cells were fixed with 1% formaldehyde and treated with 0.125 m glycine to terminate the fixation reaction. Cells were lysed with cell lysis buffer (5 mm PIPES, pH 8.0, 85 mm KCl, 0.5% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, and 5 μm Leupeptin) at 4 °C for 20 min, centrifuged at 14,000 rpm for 30 min at 4 °C. The resultant cellular extracts were lysed using nuclear lysis buffer (20 mm Hepes-HCl, pH 8.0, 150 mm NaCl, 1% Triton X-100, and 0.1% SDS), immunoprecipitated with protein-agarose-conjugated anti-Myc antibody (9E10, sc-40, Santa Cruz, Biotechnology) for 16 h at 4 °C. The resultant immune complexes were extracted and used for subsequent PCR reaction with pairs of primers (forward primer: 5′-TCCTGGTGTCGACTGTGAGT-3′, reverse primer: 5′-AGCTCCTGGGAACGCAGAC-3′) to detect fragment of TCL1-NBRE fragment by resolving onto 1.0% TAE (Tris acetate-EDTA) agarose gel. Equal amounts of NBRE expression were verified by PCR using the same sets of primers using 5 μlof cellular extracts as a template from the transfected cells and visualized on 1.0% agarose gel. In Vivo Chromatin Immunoprecipitation Assay—In vivo ChIP assays were performed essentially using a QuikChip kit (IMGENEX) with minor modification using 3 μg of anti-Nur77 antibody (M-210, sc5569, Santa Cruz Biotechnology), anti-NOR1 (C-19, sc-22519, Santa Cruz Biotechnology), anti-Nurr1 (E-20, sc-990, Santa Cruz Biotechnology), or rabbit normal serum as a control. PC12 cells (rat pheochromocytoma cells, ATCC) were cultured on a 100-mm dish (CellBIND Surface) with charcoal-treated 10% FBS and 5% horse serum. The cells were serum-starved for 24 h before NGF stimulation (50 ng/ml, murine NGF (7S), 13290-010, Invitrogen) for an additional 6 h and harvested for the subsequent PCR reaction. The 250-bp genomic TCL1 promoter DNA fragments were detected by two-step PCR using the following primer sets (first primer set, 5′-TGACCTCATGGTAAACATAGC-3′ and 5′-ACATATAGCGAGCGCATCAG-3′; second primer set, 5′-ACCTCATGGTAAACATAGCG-3′ and 5′-GTCTATACACACTGCATATGC-3′). The resultant PCR products were loaded onto 1.5% TBE-agarose gel, and the DNA fragments were visualized. RT-PCR—PC12 cells (ATCC) were cultured on a 100-mm dish (CellBIND Surface) with charcoal-treated 10% FBS and 5% horse serum. The cells were serum-starved for 16 h, then stimulated with NGF (50 ng/ml, murine NGF (7S), 13290-010, Invitrogen) for indicated time periods. Total RNA was isolated with TRIzol reagent (Invitrogen). mRNA was isolated using Oligotex-dT30 (Takara) for generating the first strand cDNA using a First-strand cDNA synthesis kit (Takara). RT-PCR was performed using pairs of primers (TCL1, 5′-TGGGTTCACTGTGAGGGTGTC-3′ and 5′-GTAGAGCTGCCACATGAGAGG-3′; glyceraldehyde-3-phosphate dehydrogenase, 5′-AAGTATGATGACATCAAGAAGG-3′ and 5′-AGTTACAGGAGACAACCTGG-3′) with EX Taq polymerase (Takara) using a thermal cycler (iCycler, Bio-Rad). Immunoblotting—293 cells (Fig. 2, A and B) were cultured in a 100-mm dish (CellBIND Surface) in charcoal-treated FBS and transfected with 7.5 μg of expression vectors (or sham-transfected) using the calcium phosphate method. Twenty-four hours after transfection, the cells were serum-starved for an additional 16 h, then stimulated with (or without) PDGF (50 ng/ml, PDGF-AB, p3326, Sigma) as indicated for 8 min, and lysed with Brij 97 buffer in the presence of 1 mm Na3VO4 and 10 mm NaF (4Laine J. Künstle G. Obata T. Sha M. Noguchi M. Mol. Cell. 2000; 6: 395-407Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). Twenty micrograms of cell lysate was loaded onto 7.5% SDS-PAGE (Daiichikagaku) and immunoblotted by the indicated antibodies (anti-Akt, 9272, anti-phospho-Ser-473 Akt, 9271, Cell Signaling; anti-c-Myc, 9E10, Santa Cruz Biotechnology) (Fig. 2A), or anti-FLAG M2, Sigma) using ECL (Amersham Biosciences). To detect phosphorylated Nur77 (Fig. 2B), cell lysates were precleaned, immunoprecipitated with anti-FLAG antibody (M2, Sigma) using a ProA/ProG mixture (4Laine J. Künstle G. Obata T. Sha M. Noguchi M. Mol. Cell. 2000; 6: 395-407Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar), and immunoblotted with anti-phospho antibody (#9601, Cell Signaling). To detect non-phosphorylated (or phosphorylated) Akt and Nur77 (Figs. 4 and 5), PC12 cells were harvested at indicated time points after NGF stimulation (50 ng/ml, murine NGF (7S), 13290-010, Invitrogen), lysed with Brij97 lysis buffer (4Laine J. Künstle G. Obata T. Sha M. Noguchi M. Mol. Cell. 2000; 6: 395-407Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar), resolved onto SDS-PAGE, and immunoblotted by TCL1 polyclonal antibody (533, generated by recombinant human TCL1), anti-Akt Abs, or anti-phospho-Ser-473 Akt Abs using ECL (Amersham Biosciences). To detect phosphorylated Nur77, cells were harvested, lysed, immunoprecipitated with anti-Nur77 antibody (M-210, sc5569, Santa Cruz Biotechnology), and immunoblotted by anti-phospho antibody (9601, Cell Signaling). Structure of the TCL1 Promoter Region—To clarify the molecular regulation of TCL1, we cloned 1123 bp of the TCL1 5′-proximal promoter (from –931 to +192) from human 293 cells (ATCC). Sequence analysis of the 5′-TCL1 promoter region revealed a TATA box with the following transcriptional regulatory elements of FKHRL: Nur77, MyoD, IRF, NFκB, or Sp1 (Fig. 1A). Recently, it was reported that Akt activation directly phosphorylates and regulates Nur77 (NGFI-B or TR3) and fork head transcription factor (FKHRL) (27Matsuyama N. Onishi K. Mori Y. Ueno T. Takayama Y. Gotoh Y. J. Biol. Chem. 2001; 274: 32799-32805Abstract Full Text Full Text PDF Scopus (137) Google Scholar, 28Pekarsky Y. Hallas C. Palamarchuk A. Koval A. Bullrich F. Hirata Y. Bichi R. Letofsky J. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3690-3694Crossref PubMed Scopus (153) Google Scholar, 31Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5454) Google Scholar). In this regard it was striking that a putative FKHRL consensus binding site (CAAAATAA, located from –857 to 850) as well as a Nur77/NGFI-B binding site (NBRE, CCAAGGTCA, located from –340 to –332) were present within 1000 of the 5′-TCL1 proximal promoter region (Fig. 1B). Nur77 is an orphan receptor superfamily transcription factor that is implicated in neuronal development and apoptosis (20Milbrandt J. Neuron. 1988; 1: 183-188Abstract Full Text PDF PubMed Scopus (531) Google Scholar). It is noteworthy that none of the molecular targets of Nur77/NGFI-B in cell death and survival machinery have been identified in the literature (21Winoto A. Littman D.R. Cell. 2002; 109: S57-S66Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). Recognition sequences of NGFI-B orphan re

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