Control of the Calcitonin Gene-related Peptide Enhancer by Upstream Stimulatory Factor in Trigeminal Ganglion Neurons
2008; Elsevier BV; Volume: 283; Issue: 9 Linguagem: Inglês
10.1074/jbc.m708662200
ISSN1083-351X
AutoresKi-Youb Park, Andrew F. Russo,
Tópico(s)Neuroendocrine regulation and behavior
ResumoThe neuropeptide calcitonin gene-related peptide (CGRP) is a key player in migraine. However, the transcription factors controlling CGRP expression in the migraine-relevant trigeminal ganglion neurons are unknown. Previous in vitro studies demonstrated that upstream stimulatory factor (USF) 1 and USF2 bind to the CGRP neuroendocrine-specific 18-bp enhancer, yet discrepant overexpression results in cell lines, and the ubiquitous nature of the USF cast doubts about its role. To test the functional role of USF, we first demonstrated that small interfering RNAs directed against USF1 and USF2 reduced endogenous CGRP RNA and preferentially targeted the USF binding site at the 18-bp enhancer in the neuronal-like CA77 cell line. In cultured rat trigeminal ganglion neurons, knockdown of either USF1 or USF2 reduced CGRP promoter activity. Conversely, overexpression of USF1 or USF2 increased promoter activity. The activation was even greater upon cotransfection with an upstream activator of mitogen-activated protein kinases and was synergistic in a heterologous cell line. To begin to address the paradox of how ubiquitous USF proteins might direct neuronal-specific activity, we examined USF expression and used a series of adenoviral reporters in the cultured ganglia. Unexpectedly, there was more intense USF immunostaining in neurons than nonneuronal cells. Importantly, the 18-bp USF enhancer driving a minimal promoter was sufficient for neuronal specificity, although it was not the only site that directed neuronal expression. These results demonstrate that USF1 and USF2 are important contributors to neuronal-specific and mitogen-activated protein kinase regulation of the CGRP gene in trigeminal ganglion neurons. The neuropeptide calcitonin gene-related peptide (CGRP) is a key player in migraine. However, the transcription factors controlling CGRP expression in the migraine-relevant trigeminal ganglion neurons are unknown. Previous in vitro studies demonstrated that upstream stimulatory factor (USF) 1 and USF2 bind to the CGRP neuroendocrine-specific 18-bp enhancer, yet discrepant overexpression results in cell lines, and the ubiquitous nature of the USF cast doubts about its role. To test the functional role of USF, we first demonstrated that small interfering RNAs directed against USF1 and USF2 reduced endogenous CGRP RNA and preferentially targeted the USF binding site at the 18-bp enhancer in the neuronal-like CA77 cell line. In cultured rat trigeminal ganglion neurons, knockdown of either USF1 or USF2 reduced CGRP promoter activity. Conversely, overexpression of USF1 or USF2 increased promoter activity. The activation was even greater upon cotransfection with an upstream activator of mitogen-activated protein kinases and was synergistic in a heterologous cell line. To begin to address the paradox of how ubiquitous USF proteins might direct neuronal-specific activity, we examined USF expression and used a series of adenoviral reporters in the cultured ganglia. Unexpectedly, there was more intense USF immunostaining in neurons than nonneuronal cells. Importantly, the 18-bp USF enhancer driving a minimal promoter was sufficient for neuronal specificity, although it was not the only site that directed neuronal expression. These results demonstrate that USF1 and USF2 are important contributors to neuronal-specific and mitogen-activated protein kinase regulation of the CGRP gene in trigeminal ganglion neurons. Calcitonin gene-related peptide (CGRP) 2The abbreviations used are:CGRPcalcitonin gene-related peptidehCGRPhuman CGRPUSFupstream stimulatory factorERKextracellular signal-regulated kinaseJNKc-Jun NH2-terminal kinaseMAPmitogen-activated proteinMEKmitogen-activated extracellular signal-regulated kinase kinaseMEKKMEKK kinaseRTreverse transcriptionqPCRquantitative PCRkbkilobase pair(s)GFPgreen fluorescent proteinPBSphosphate-buffered salinesiRNAsmall interfering RNATKthymidine kinaseCMVcytomegalovirusGAPDHglyceraldehyde-3-phosphate dehydrogenase.2The abbreviations used are:CGRPcalcitonin gene-related peptidehCGRPhuman CGRPUSFupstream stimulatory factorERKextracellular signal-regulated kinaseJNKc-Jun NH2-terminal kinaseMAPmitogen-activated proteinMEKmitogen-activated extracellular signal-regulated kinase kinaseMEKKMEKK kinaseRTreverse transcriptionqPCRquantitative PCRkbkilobase pair(s)GFPgreen fluorescent proteinPBSphosphate-buffered salinesiRNAsmall interfering RNATKthymidine kinaseCMVcytomegalovirusGAPDHglyceraldehyde-3-phosphate dehydrogenase. is a potent vasodilatory neuropeptide (1Brain S.D. Grant A.D. Physiol. Rev. 2004; 84: 903-934Crossref PubMed Scopus (616) Google Scholar) that has been implicated in the pathology of migraine (2Goadsby P.J. Expert Opin. Emerg. Drugs. 2006; 11: 419-427Crossref PubMed Scopus (21) Google Scholar, 3Waeber C. Moskowitz M.A. Neurology. 2005; 64: 9-15Crossref PubMed Google Scholar, 4Durham P.L. Headache. 2006; 46: 3-8Crossref PubMed Scopus (201) Google Scholar). Although the mechanisms underlying migraine remain controversial, there is a growing acceptance of the involvement of the trigeminal ganglion neurons, which express CGRP and relay nociceptive signals from the vasculature and dura to the brainstem (5Goadsby P.J. Lipton R.B. Ferrari M.D. N. Engl. J. Med. 2002; 346: 257-270Crossref PubMed Scopus (1558) Google Scholar, 6Pietrobon D. Striessnig J. Nat. Rev. Neurosci. 2003; 4: 386-398Crossref PubMed Scopus (476) Google Scholar). Most notably, systemic administration of CGRP induces migraine-like symptoms among migraineurs (7Lassen L.H. Haderslev P.A. Jacobsen V.B. Iversen H.K. Sperling B. Olesen J. Cephalalgia. 2002; 22: 54-61Crossref PubMed Scopus (726) Google Scholar), and a CGRP receptor antagonist can attenuate migraine (8Olesen J. Diener H.C. Husstedt I.W. Goadsby P.J. Hall D. Meier U. Pollentier S. Lesko L.M. N. Engl. J. Med. 2004; 350: 1104-1110Crossref PubMed Scopus (1024) Google Scholar). The possibility that CGRP synthesis is elevated during migraine is suggested by elevation of serum CGRP levels during spontaneous migraine (9Goadsby P.J. Edvinsson L. Ekman R. Ann. Neurol. 1990; 28: 183-187Crossref PubMed Scopus (1208) Google Scholar, 10Juhasz G. Zsombok T. Jakab B. Nemeth J. Szolcsanyi J. Bagdy G. Cephalalgia. 2005; 25: 179-183Crossref PubMed Scopus (153) Google Scholar). Given the generally long duration of migraine, it seems reasonable that these elevated CGRP levels might be sustained by increased transcription. Hence, an understanding of CGRP regulation in trigeminal neurons may provide clues regarding the pathophysiology of migraine. calcitonin gene-related peptide human CGRP upstream stimulatory factor extracellular signal-regulated kinase c-Jun NH2-terminal kinase mitogen-activated protein mitogen-activated extracellular signal-regulated kinase kinase MEKK kinase reverse transcription quantitative PCR kilobase pair(s) green fluorescent protein phosphate-buffered saline small interfering RNA thymidine kinase cytomegalovirus glyceraldehyde-3-phosphate dehydrogenase. calcitonin gene-related peptide human CGRP upstream stimulatory factor extracellular signal-regulated kinase c-Jun NH2-terminal kinase mitogen-activated protein mitogen-activated extracellular signal-regulated kinase kinase MEKK kinase reverse transcription quantitative PCR kilobase pair(s) green fluorescent protein phosphate-buffered saline small interfering RNA thymidine kinase cytomegalovirus glyceraldehyde-3-phosphate dehydrogenase. We have previously reported that a heterodimer of the transcription factor USF1 and USF2 binds to the 18-bp enhancer of the CGRP gene in vitro (11Lanigan T.M. Russo A.F. J. Biol. Chem. 1997; 272: 18316-18324Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). In addition to the binding data, USF overexpression increased CGRP promoter activity in a lung carcinoma cell line (12Viney T.J. Schmidt T.W. Gierasch W. Sattar A.W. Yaggie R.E. Kuburas A. Quinn J.P. Coulson J.M. Russo A.F. J. Biol. Chem. 2004; 279: 49948-49955Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). However, the activation by USF was only observed in this non-neuronal cell line that does not express the endogenous CGRP gene. In contrast, in another non-neuronal cell line (COS7) and in the neuronal-like CA77 thyroid C cell line, USF overexpression failed to stimulate promoter activity. 3T. J. Viney and A. F. Russo, unpublished observations.3T. J. Viney and A. F. Russo, unpublished observations. Furthermore, the 18-bp enhancer is active only in neuroendocrine thyroid C cell lines (11Lanigan T.M. Russo A.F. J. Biol. Chem. 1997; 272: 18316-18324Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), yet USF is ubiquitous (13Sirito M. Lin Q. Maity T. Sawadogo M. Nucleic Acids Res. 1994; 22: 427-433Crossref PubMed Scopus (291) Google Scholar). These discrepancies raised the need to demonstrate whether USF is indeed a regulator of the CGRP 18-bp enhancer in neurons. USF was initially identified as a cellular transcription factor for the adenovirus-2 major late gene (14Hough P.V. Mastrangelo I.A. Wall J.S. Hainfeld J.F. Sawadogo M. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4826-4830Crossref PubMed Scopus (12) Google Scholar, 15Moncollin V. Miyamoto N.G. Zheng X.M. Egly J.M. EMBO J. 1986; 5: 2577-2584Crossref PubMed Scopus (68) Google Scholar). Because of this initial finding, USF has been identified as a transcription factor for many genes involved in a range of cellular processes, including proliferation (16Luo X. Sawadogo M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1308-1313Crossref PubMed Scopus (111) Google Scholar), stress responses (17Galibert M.D. Carreira S. Goding C.R. EMBO J. 2001; 20: 5022-5031Crossref PubMed Scopus (178) Google Scholar), and metabolism (18Read M.L. Clark A.R. Docherty K. Biochem. J. 1993; 295: 233-237Crossref PubMed Scopus (58) Google Scholar). The two USF proteins, USF1 and USF2, share 44% identity overall and 70% identity within the C-terminal region, which includes basic-helix-loop-helix and leucine zipper domains (13Sirito M. Lin Q. Maity T. Sawadogo M. Nucleic Acids Res. 1994; 22: 427-433Crossref PubMed Scopus (291) Google Scholar). The two proteins can form homodimers, although the heterodimer is usually the most abundant form (13Sirito M. Lin Q. Maity T. Sawadogo M. Nucleic Acids Res. 1994; 22: 427-433Crossref PubMed Scopus (291) Google Scholar, 19Viollet B. Lefrancois-Martinez A.M. Henrion A. Kahn A. Raymondjean M. Martinez A. J. Biol. Chem. 1996; 271: 1405-1415Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 20Sirito M. Lin Q. Deng J.M. Behringer R.R. Sawadogo M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3758-3763Crossref PubMed Scopus (131) Google Scholar). USF1 and USF2 are ubiquitously expressed, including in the nervous system (13Sirito M. Lin Q. Maity T. Sawadogo M. Nucleic Acids Res. 1994; 22: 427-433Crossref PubMed Scopus (291) Google Scholar). A paradox is that several helix-loop-helix proteins that are ubiquitously expressed, including USF, can also be involved in cell-specific expression (21Corre S. Galibert M.D. Pigm. Cell Res. 2005; 18: 337-348Crossref PubMed Scopus (197) Google Scholar, 22Massari M.E. Murre C. Mol. Cell. Biol. 2000; 20: 429-440Crossref PubMed Scopus (1372) Google Scholar). The USF proteins can be regulated by phosphorylation. In vitro kinase assays have shown that p38 MAP kinase, but not Jun N-terminal kinase (JNK), phosphorylates threonine 153 of USF1 (17Galibert M.D. Carreira S. Goding C.R. EMBO J. 2001; 20: 5022-5031Crossref PubMed Scopus (178) Google Scholar). Phosphorylation of USF1 by p38 MAP kinase is necessary for transcriptional activation of the tyrosinase promoter (17Galibert M.D. Carreira S. Goding C.R. EMBO J. 2001; 20: 5022-5031Crossref PubMed Scopus (178) Google Scholar). A physical interaction between phosphorylated extracellular signal-regulated kinase (ERK) and USF1 has been suggested (23Kutz S.M. Higgins C.E. Samarakoon R. Higgins S.P. Allen R.R. Qi L. Higgins P.J. Exp. Cell Res. 2006; 312: 1093-1105Crossref PubMed Scopus (57) Google Scholar). Additionally, ERK MAP kinase appears to act through USF to stimulate the Cox-2 promoter (24Juttner S. Cramer T. Wessler S. Walduck A. Gao F. Schmitz F. Wunder C. Weber M. Fischer S.M. Schmidt W.E. Wiedenmann B. Meyer T.F. Naumann M. Hocker M. Cell. Microbiol. 2003; 5: 821-834Crossref PubMed Scopus (77) Google Scholar). USF1 and USF2 are also phosphorylated in response to phorbol ester and forskolin stimulation (25Sayasith K. Lussier J.G. Sirois J. J. Biol. Chem. 2005; 280: 28885-28893Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 26Chen J. Malcolm T. Estable M.C. Roeder R.G. Sadowski I. J. Virol. 2005; 79: 4396-4406Crossref PubMed Scopus (36) Google Scholar). Because the CGRP 18-bp enhancer is stimulated by the ERK MAP kinase pathway (27Durham P.L. Russo A.F. J. Neurosci. 2003; 23: 807-815Crossref PubMed Google Scholar) and probably other MAP kinases (28Bowen E.J. Schmidt T.W. Firm C.S. Russo A.F. Durham P.L. J. Neurochem. 2006; 96: 65-77Crossref PubMed Scopus (95) Google Scholar), it is possible that MAP kinases may also activate the CGRP promoter via USF proteins in trigeminal neurons. In this report we demonstrate that USF proteins are activators of the CGRP promoter in cultured neurons derived from rat trigeminal ganglia. USF knockdown and overexpression resulted in a decrease and increase, respectively, of CGRP promoter activity. Moreover, overexpression of the MAP kinase activators, mitogen-activated/ERK kinase (MEK) kinase (MEKK) or MEK1, with USF1 or USF2 further increased CGRP promoter activation, whereas USF knockdown reduced MEKK activation of the CGRP promoter. Finally, immunocytochemistry showed that the 18-bp enhancer containing the USF site is sufficient for neuronal-specific CGRP promoter activity. Transfection of Cell Lines—Culture conditions for CA77 and NCI-H460 cells have been described (12Viney T.J. Schmidt T.W. Gierasch W. Sattar A.W. Yaggie R.E. Kuburas A. Quinn J.P. Coulson J.M. Russo A.F. J. Biol. Chem. 2004; 279: 49948-49955Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). CA77 cells were transfected when they reached ∼80% confluence in a 12-well plate (Falcon), as described (12Viney T.J. Schmidt T.W. Gierasch W. Sattar A.W. Yaggie R.E. Kuburas A. Quinn J.P. Coulson J.M. Russo A.F. J. Biol. Chem. 2004; 279: 49948-49955Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar), with some modifications. CA77 cells (1 ml) were cotransfected with 20 nm nonspecific control, USF1, or USF2 siRNA duplexes and 0.25 μg of reporter plasmid. The plasmids 18-bp-TK-luc, TK-luc, 1.25-rCGRP-luc, and 1.25-rCGRP Bam mut have been described (12Viney T.J. Schmidt T.W. Gierasch W. Sattar A.W. Yaggie R.E. Kuburas A. Quinn J.P. Coulson J.M. Russo A.F. J. Biol. Chem. 2004; 279: 49948-49955Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 29Tverberg L.A. Russo A.F. J. Biol. Chem. 1993; 268: 15965-15973Abstract Full Text PDF PubMed Google Scholar). At 48–96 h after cotransfection, cell lysates were assayed for luciferase activity using reagents from Promega. NCI-H460 cells were transfected by electroporation as described (12Viney T.J. Schmidt T.W. Gierasch W. Sattar A.W. Yaggie R.E. Kuburas A. Quinn J.P. Coulson J.M. Russo A.F. J. Biol. Chem. 2004; 279: 49948-49955Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Luciferase activity was normalized to protein using Bradford reagent (Bio-Rad) and to β-galactosidase activity measured using Tropix Galacto-Light Plus Assay System (Tropix). For all transfections (CA77, NCI-H460, and primary cultures) plasmid concentrations were held constant with pSV40-β-galactosidase (30Wood J.L. Russo A.F. J. Biol. Chem. 2001; 276: 21262-21271Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), pCMV5 (27Durham P.L. Russo A.F. J. Neurosci. 2003; 23: 807-815Crossref PubMed Google Scholar), or pMyc-His (Invitrogen) as indicated. siRNA Duplexes—USF1 and USF2 siRNA duplexes were purchased from Invitrogen. Three different siRNA duplexes were initially transfected into CA77 cells and tested for their effects on CGRP promoter activity. Two USF1 siRNA duplexes decreased promoter activity, whereas the other duplex did not affect activity. The most potent duplex was used for later studies. Only one USF2 siRNA duplex decreased promoter activity. Rat USF1 siRNA is 5′-CCCAACGUCAAGUACGUCUUCCGAA-3′; rat USF2 siRNA is 5′-GCAUCCUGUCCAAGGCUUGCGAUUA-3′. Stealth™ RNAi Negative Control Medium GC Duplex (Invitrogen) was used as the nonspecific control siRNA duplex. Reverse Transcription (RT) and Quantitative PCR (qPCR)—Transfection of siRNA duplexes was performed as described above. A plasmid encoding cytomegalovirus (CMV) promoter-driven green fluorescent protein (GFP) was cotransfected with either 5 nm nonspecific control siRNA or USF siRNA (2.5 nm USF1 siRNA and 2.5 nm USF2 siRNA) duplexes. After 72 h, GFP-positive cells were collected by flow cytometry, and RNA was extracted using a QIAshredder column and RNeasy Mini kit (Qiagen). For each sample, about 500 ng of DNase I-treated RNA was applied per RT reaction using a random primer as recommended (Applied Biosystems). One-tenth of the cDNA was subjected to real-time qPCR using SYBR Green as described (31Zhang Z. Winborn C.S. Marquez de Prado B. Russo A.F. J. Neurosci. 2007; 27: 2693-2703Crossref PubMed Scopus (192) Google Scholar) with 50 nm CGRP primers or 333 nm 18 S rRNA primers. For each sample qPCR was performed in triplicate. The PCR protocol was 50 °C for 2 min, 95 °C for 10 min, 40 cycles of denaturing at 95 °C for 15 s, annealing at 60.7 °C for 30 s, and extension at 72 °C for 1 min. PCR primers were: rat CGRP (GenBank M11597) sense, 5′-AACCTTAGAAAGCAGCCCAGGCATG-3′, and antisense, 5′-GTGGGCACAAAGTTGTCCTTCACCA-3′; rat 18 S rRNA (GenBank V0127), sense, 5′-ATGGCCGTTCTTAGTTGGTG-3′, and antisense, 5′-AACGCCACTTGTCCCTCTAA-3′. Relative quantification of CGRP mRNA level was determined using the ΔΔCt method (32Livak K.J. Schmittgen T.D. Methods. 2001; 25: 402-408Crossref PubMed Scopus (120953) Google Scholar). Isolation and Culture of Neurons from Rat Trigeminal Ganglia—Ganglia were removed from Sprague-Dawley rat pups (2–4 days old) and cultured as previously described with some modifications (31Zhang Z. Winborn C.S. Marquez de Prado B. Russo A.F. J. Neurosci. 2007; 27: 2693-2703Crossref PubMed Scopus (192) Google Scholar). Four ganglia were used per sample. Cells were resuspended in complete medium (10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 10 ng/ml mouse 2.5 S nerve growth factor (Alomone Labs), L-15 medium (Leibovitz)) and plated onto laminin (Roche Applied Science)-coated coverslips placed in a 6-well dish. The laminin-coated coverslips were prepared by loading 4 μg of laminin dissolved in 1 ml of phosphate-buffered saline (PBS) onto each 4-cm2 coverslip and subsequent overnight incubation at 4 °C. Transfection of Rat Trigeminal Ganglia Cultures—The 2.24-kb-human CGRP promoter-luciferase plasmid (hCGRP-luc) was generated by homologous recombination of pDestination C-Luc and pENTR-hCGRP. pDestination C-Luc was generated by subcloning the β-globin/IgG chimeric intron from pCI (Promega) into BamHI and PstI sites of pGEM®-4Z (Promega) to make pStec1 and firefly luciferase from pGL3-Basic (Promega) into XhoI and XbaI sites of pStec1. The resultant plasmid (pStec1-luc) was linearized by digestion with PstI, treated with mung bean nuclease, then ligated with Gateway® Reading Frame Cassette C.1 (Invitrogen). pENTR-hCGRP was generated by subcloning a PCR fragment of the 2.24-kb hCGRP promoter into pGEM-T Easy (Promega) then into the EcoRI site of Gateway® pENTR™11 vector (Invitrogen). Human USF1 and mouse USF2 expression vectors have been described (12Viney T.J. Schmidt T.W. Gierasch W. Sattar A.W. Yaggie R.E. Kuburas A. Quinn J.P. Coulson J.M. Russo A.F. J. Biol. Chem. 2004; 279: 49948-49955Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). The T153A and T153E mutant USF1 vectors were generated from the USF1 vector using the QuikChange® site-directed mutagenesis kit (Stratagene). The MEKK (amino acids 380–672) plasmid from Stratagene has been described (30Wood J.L. Russo A.F. J. Biol. Chem. 2001; 276: 21262-21271Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Within 24 h of culturing the dissociated cells in each well of a 6-well dish (Falcon) were transfected using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. Plasmids were mixed with Lipofectamine 2000 (ratio, 1 μg to 1 or 2 μl) in warm L-15 medium. After incubation with transfecting solutions, cultured cells were scraped with 1 ml of PBS and transferred to Eppendorf tubes. Cells were collected by centrifugation at 14,000 rpm for 3 min. Cell lysates were prepared with 50 μl of 1× reporter lysis buffer (Promega) and subjected to freeze-thawing to aid lysis. For luciferase activity assays, 20 μl of lysate was mixed with reagents from Promega. Transfections of siRNA duplexes involved procedures similar to the plasmid transfections. After 48–72 h, the cells were lysed and assayed for luciferase activity and Western blotting. Adenoviral Infections of Trigeminal Ganglia Cultures—AdrCGRP-luc, an adenovirus containing the 1.25-rCGRP fused with firefly luciferase in pGL3 has been described (28Bowen E.J. Schmidt T.W. Firm C.S. Russo A.F. Durham P.L. J. Neurochem. 2006; 96: 65-77Crossref PubMed Scopus (95) Google Scholar). The AdrCGRP-Bam-luc adenoviral vector has a BamHI linker inserted into the 1.25-rCGRP. The 1.25-kb rCGRP-Bam mutant promoter fragment was obtained by digestion of the 1.25-kb rCGRP-Bam-luc plasmid (12Viney T.J. Schmidt T.W. Gierasch W. Sattar A.W. Yaggie R.E. Kuburas A. Quinn J.P. Coulson J.M. Russo A.F. J. Biol. Chem. 2004; 279: 49948-49955Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar) with XbaI and SacI, then subcloned into the Nhe and SacI sites of the pGL3 luciferase vector (Promega) and transferred as an XbaI-KpnI fragment into pacAd5K-NpA adenoviral shuttle vector. Adenoviruses were generated and purified by the University of Iowa Gene Transfer Core Facility. An adenoviral vector containing a minimal TK promoter with three copies of the 18-bp enhancer and the β-galactosidase reporter gene (Ad18-bp-TK-lacZ) was generated from a lacZ shuttle plasmid and the previously described 18-bp-TK-luciferase plasmid (29Tverberg L.A. Russo A.F. J. Biol. Chem. 1993; 268: 15965-15973Abstract Full Text PDF PubMed Google Scholar). The AdCMV-β-galactosidase (AdCMV-lacZ) adenoviral vector has been described (33Durham P.L. Dong P.X. Belasco K.T. Kasperski J. Gierasch W.W. Edvinsson L. Heistad D.D. Faraci F.M. Russo A.F. Brain Res. 2004; 997: 103-110Crossref PubMed Scopus (25) Google Scholar). Trigeminal ganglia cultures were infected with adenovirus 24 h after plating. Cultures were incubated with 200 μl of L-15 media containing 1.1 × 108 plaque-forming units of AdrCGRP-luc or AdrCGRP-Bam-luc per sample for 4-h at 37 °C and ambient CO2. Then 2 ml of the complete medium described above was added. After 24 h of incubation, cultures were subjected to immunocytochemistry. For infections of Ad18-bp-TK-lacZ and AdCMV-lacZ, 2 × 109 or 2.4 × 109 plaque-forming units, respectively, were used. After 36 h of incubation immunocytochemistry was performed. Immunocytochemistry—Cultures were rinsed in PBS and fixed in cold methanol for 10 min at –20 °C. After washing with PBS, the cells were incubated with 10% bovine serum albumin in PBS for 30 min. This was followed by 1 h of incubation with primary antibodies, a monoclonal mouse anti-β-tubulin III antibody (1:800 dilution, Sigma), and a polyclonal rabbit anti-β-galactosidase antibody (1:100 dilution, Santa Cruz Biotechnology). The primary antibodies were diluted in 1.5% bovine serum albumin containing PBS. After washing with PBS, the cells were incubated in 10% bovine serum albumin containing PBS for 30 min. Then rhodamine anti-rabbit IgG and fluorescein isothiocyanate-anti-mouse IgG (1:200 dilution, Jackson ImmunoResearch Laboratories) were added to the cells. After washing with PBS, the cells were incubated with ToPro3 (1:1000 diluted in dimethyl sulfoxide, Molecular Probes) for 5 min. For USF1 and USF2 immunocytochemistry, a similar process was followed. Primary antibodies were rabbit IgG anti-USF1 (sc-229) and anti-USF2 (sc-861) used at a dilution of 1:50. For immunocytochemistry of NCI-H460 cells, cells were transfected with 20 μg of pCMV-GFP and 20 μg of USF expression vector. After 3 days of incubation, a similar process was performed using a mouse monoclonal anti-GFP antibody (1:800 dilution, G 6539, Sigma) and rabbit IgG anti-USF antibody (1:50–1:500 dilution). For luciferase immunocytochemistry, infected rat trigeminal ganglia cultures were fixed with 4% paraformaldehyde for 10 min at room temperature. After washing with PBS, cells were incubated with 1:1 (v/v) acetone:water for 3 min at 4 °C followed by acetone for 5 min at 4 °C. Then cells were incubated with 1:1 (v/v) acetone:water for 3 min at room temperature. After rinsing with PBS for 3 min, samples were blocked with 1% fetal bovine serum (diluted in PBS) for 15 min. Samples were incubated with a goat anti-luciferase antibody (1:50 dilution, Promega) and a mouse anti-β-tubulin III antibody (1:800 dilution) in 0.1% fetal bovine serum for 1 h. After washing 3 × 5 min with PBS, samples were incubated with fluorescein isothiocyanate-anti-goat IgG and rhodamine-anti-mouse IgG (1:200 dilution, Jackson ImmunoResearch Laboratories) for 30 min. After 3 × 5 min washes with PBS, ToPro3 was added for 5 min. Images were taken by using confocal microscope (Zeiss). For analysis of nuclear versus cytoplasmic staining, USF1 and USF2 images were analyzed at different focal planes with z-stack program then compiled to generate one image for the figure. USF staining results were confirmed by blind analyses done by a second individual. Western Blotting—Cell lysates were analyzed as described (12Viney T.J. Schmidt T.W. Gierasch W. Sattar A.W. Yaggie R.E. Kuburas A. Quinn J.P. Coulson J.M. Russo A.F. J. Biol. Chem. 2004; 279: 49948-49955Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar), except that transfers were done at 45 V for 2 h at 4 °C. Primary antibodies were used at 1:1000 dilutions overnight at 4 °C, and secondary antibodies were diluted 1:5,000–10,000 for 0.5–1-h incubations. The membrane was stripped and reprobed with new primary antibody after blocking. The rabbit IgG anti-USF1 (sc-229) and anti-USF2 (sc-861), goat anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, sc-20357), and donkey anti-goat horseradish peroxidase secondary antibodies (used to detect the anti-GAPDH antibodies) were all from Santa Cruz Biotechnology. To detect the anti-USF antibodies, donkey anti-rabbit horseradish peroxidase secondary antibodies (GE Healthcare) were used. The mean value from the histogram analysis performed using the NIH ImageJ software was used for the quantification of protein band intensity. USF Knockdown Decreases CGRP mRNA Levels in CA77 Cells—Our first test was to determine whether USF proteins regulate the endogenous CGRP gene. This was particularly important because our previous evidence for USF activation of the CGRP promoter (12Viney T.J. Schmidt T.W. Gierasch W. Sattar A.W. Yaggie R.E. Kuburas A. Quinn J.P. Coulson J.M. Russo A.F. J. Biol. Chem. 2004; 279: 49948-49955Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar) was not observed in the neuronal-like CA77 thyroid C cell line, which expresses CGRP. To resolve this issue, we used siRNA-mediated knockdown of USF proteins followed by RT-qPCR measurement of CGRP mRNA. The use of the CA77 cells was necessary because initial attempts to reduce endogenous CGRP RNA by USF1 and USF2 siRNAs in trigeminal ganglia cultures were not successful (data not shown). This may have been due to a more stable pool of CGRP mRNA in neurons than reported in a cell line (half-life about 17 h) (34Cote G.J. Gagel R.F. J. Biol. Chem. 1986; 261: 15524-15528Abstract Full Text PDF PubMed Google Scholar), which has been observed for the mRNAs of heavy and mid-sized neurofilament subunits (35Schwartz M.L. Shneidman P.S. Bruce J. Schlaepfer W.W. J. Biol. Chem. 1992; 267: 24596-24600Abstract Full Text PDF PubMed Google Scholar). Another possible reason could have been the cellular heterogeneity of the cultures if siRNA duplexes were taken up by non-neuronal cells more easily than by neurons. To resolve these technical problems, we turned to the homogenous CA77 cell line to examine the endogenous CGRP gene. A combination of siRNAs targeting USF1 and USF2 was transfected into CA77 cells. The pCMV-GFP reporter plasmid was included to allow for the selection of transfected cells by flow cytometry before RNA extraction. CGRP mRNA levels in the samples were measured by RT-qPCR and were normalized to 18 S ribosomal RNA levels in the same samples. The data were then compared with the signal obtained after treatment of the cells with nonspecific control siRNA duplexes. We found that the combined transfection of siRNAs targeting USF1 and USF2 decreased the level of the endogenous CGRP mRNA to about 60% that in controls (Fig. 1A). Likewise, the protein levels of USF1 and USF2 were reduced to 42 and 81%, respectively, that in samples transfected with the control siRNA (Fig. 1B). As a loading and specificity control, GAPDH levels were not affected (96–104% of the levels after transfection with control siRNA) (Fig. 1B). Overall, these results indicate that expression of the endogenous CGRP gene in CA77 cells requires USF proteins. Specificity of USF siRNA Duplexes—Given that the USF proteins regulate many genes, the siRNA-mediated knockdown of USF could potentially ind
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