Critical Role of cAMP Response Element Binding Protein Expression in Hypoxia-elicited Induction of Epithelial Tumor Necrosis Factor-α
1999; Elsevier BV; Volume: 274; Issue: 27 Linguagem: Inglês
10.1074/jbc.274.27.19447
ISSN1083-351X
AutoresCormac T. Taylor, Nana Fueki, Azin Agah, Robert M. Hershberg, Sean P. Colgan,
Tópico(s)Immune Response and Inflammation
ResumoTissue hypoxia is intimately associated with a number of chronic inflammatory conditions of the intestine. In this study, we investigated the impact of hypoxia on the expression of a panel of inflammatory mediators by intestinal epithelia. Initial experiments revealed that epithelial (T84 cell) exposure to ambient hypoxia evoked a time-dependent induction of the proinflammatory markers tumor necrosis factor-α (TNF-α), interleukin-8 (IL-8), and major histocompatibility complex (MHC) class II (37 ± 6.1-, 7 ± 0.8-, and 9 ± 0.9-fold increase over normoxia, respectively, each p < 0.01). Since the gene regulatory elements for each of these molecules contains an NF-κB binding domain, we investigated the influence of hypoxia on NF-κB activation. Cellular hypoxia induced a time-dependent increase in nuclear p65, suggesting a dominant role for NF-κB in hypoxia-elicited induction of proinflammatory gene products. Further work, however, revealed that hypoxia does not influence epithelial intercellular adhesion molecule 1 (ICAM-1) or MHC class I, the promoters of which also contain NF-κB binding domains, suggesting differential responses to hypoxia. Importantly, the genes for TNF-α, IL-8, and MHC class II, but not ICAM-1 or MHC class I, contain cyclic AMP response element (CRE) consensus motifs. Thus, we examined the role of cAMP in the hypoxia-elicited phenotype. Hypoxia diminished CRE binding protein (CREB) expression. In parallel, T84 cell cAMP was diminished by hypoxia (83 ± 13.2% decrease, p < 0.001), and pharmacologic inhibition of protein kinase A induced TNF-α and protein release (9 ± 3.9-fold increase). Addback of cAMP resulted in reversal of hypoxia-elicited TNF-α release (86 ± 3.2% inhibition with 3 mm 8-bromo-cAMP). Furthermore, overexpression of CREB but not mutated CREB by retroviral-mediated gene transfer reversed hypoxia-elicited induction of TNF-α defining a causal relationship between hypoxia-elicited CREB reduction and TNF-α induction. Such data indicate a prominent role for CREB in the hypoxia-elicited epithelial phenotype and implicate intracellular cAMP as an important second messenger in differential induction of proinflammatory mediators. Tissue hypoxia is intimately associated with a number of chronic inflammatory conditions of the intestine. In this study, we investigated the impact of hypoxia on the expression of a panel of inflammatory mediators by intestinal epithelia. Initial experiments revealed that epithelial (T84 cell) exposure to ambient hypoxia evoked a time-dependent induction of the proinflammatory markers tumor necrosis factor-α (TNF-α), interleukin-8 (IL-8), and major histocompatibility complex (MHC) class II (37 ± 6.1-, 7 ± 0.8-, and 9 ± 0.9-fold increase over normoxia, respectively, each p < 0.01). Since the gene regulatory elements for each of these molecules contains an NF-κB binding domain, we investigated the influence of hypoxia on NF-κB activation. Cellular hypoxia induced a time-dependent increase in nuclear p65, suggesting a dominant role for NF-κB in hypoxia-elicited induction of proinflammatory gene products. Further work, however, revealed that hypoxia does not influence epithelial intercellular adhesion molecule 1 (ICAM-1) or MHC class I, the promoters of which also contain NF-κB binding domains, suggesting differential responses to hypoxia. Importantly, the genes for TNF-α, IL-8, and MHC class II, but not ICAM-1 or MHC class I, contain cyclic AMP response element (CRE) consensus motifs. Thus, we examined the role of cAMP in the hypoxia-elicited phenotype. Hypoxia diminished CRE binding protein (CREB) expression. In parallel, T84 cell cAMP was diminished by hypoxia (83 ± 13.2% decrease, p < 0.001), and pharmacologic inhibition of protein kinase A induced TNF-α and protein release (9 ± 3.9-fold increase). Addback of cAMP resulted in reversal of hypoxia-elicited TNF-α release (86 ± 3.2% inhibition with 3 mm 8-bromo-cAMP). Furthermore, overexpression of CREB but not mutated CREB by retroviral-mediated gene transfer reversed hypoxia-elicited induction of TNF-α defining a causal relationship between hypoxia-elicited CREB reduction and TNF-α induction. Such data indicate a prominent role for CREB in the hypoxia-elicited epithelial phenotype and implicate intracellular cAMP as an important second messenger in differential induction of proinflammatory mediators. interferon-γ tumor necrosis factor-α Hanks' balanced salt solution cyclic AMP response element cyclic AMP response element binding protein electrophoretic mobility shift assay major histocompatibility complex intercellular adhesion molecule 1 protein kinase A interleukin enzyme-linked immunosorbent assays polymerase chain reaction phorbol 12-myristate 13-acetate polyacrylamide gel electrophoresis protein kinase A inhibitor The human intestine is lined with a single layer of protective epithelial cells that possess properties such as barrier and ion (and subsequent fluid) transport functions (1Powell D. Am. J. Physiol. 1981; 241: G275-G288PubMed Google Scholar, 2Powell D.W. Johnson L.R. Physiology of the Gastrointestinal Tract. 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Invest. 1994; 93: 2056-2065Crossref PubMed Scopus (200) Google Scholar) on intestinal epithelial cells. Based on this previous work, we have proposed that epithelial cells have the unique ability to “phenotype-switch,” whereby epithelia lose classic qualities of epithelia (i.e. barrier function, ion transport properties, etc.) and assume features resembling immune-type cells (i.e.surface expression of MHC class I and II, antigen presentation properties, regulated polymorphonuclear leukocyte trafficking, etc.) (5Colgan S.P. Parkos C.A. Matthews J.B. D'Andrea L. Awtrey C.S. Lichtman A. Delp C. Madara J.L. Am. J. Physiol. 1994; 267: C402-C410Crossref PubMed Google Scholar). Tissue ischemia/hypoxia often occurs concomitantly with other inflammatory processes, and thus, appropriate models to study mechanisms of inflammation should also account for conditions of cellular hypoxia. Surprisingly little is known about the mechanisms of hypoxic signal transduction. Several reports indicate that hypoxia down-regulates activity of cellular cAMP-generating machinery and that such diminished cAMP signaling can influence gene expression and end point cellular functions (14Taylor C.T. Lisco S.J. Awtrey C.S. Colgan S.P. J. Pharmacol. Exp. Ther. 1998; 284: 568-575PubMed Google Scholar, 15Ogawa S. Koga S. Kuwabara K. Brett J. Morrow B. Morris S.A. Bilezikian J.P. Silverstein S.C. Stern D. Am. J. Physiol. 1992; 262: C546-C554Crossref PubMed Google Scholar, 16Ogawa S. Shreeniwas R. Butura C. Brett J. Stern D. Adv. Exp. Med. Biol. 1990; 281: 303-312Crossref PubMed Google Scholar, 17Stevens T. Rodman D.M. Endothelium. 1995; 3: 1-11Crossref Scopus (12) Google Scholar, 18Zünd G. Nelson D.P. Neufeld E.J. Dzus A.L. Bischoff J. Mayer J.E. Colgan S.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7075-7080Crossref PubMed Scopus (51) Google Scholar). At the transcriptional level, a primary target for cAMP-mediated signaling is cAMP response element binding proteins, a family of 43-kDa leucine zipper transcription factors that share certain structural motifs, bind DNA as dimers, and regulate transcription of target genes (19Meyer T.E. Habener J.F. Endocr. Rev. 1993; 14: 269-290PubMed Google Scholar). At present, it is not known whether hypoxia-mediated gene expression is directly related to CREB activity or CREB expression. Recently, we have studied the role of epithelia in intestinal ischemia using a model epithelium, T84 cells. In response to conditions of ambient hypoxia, epithelia express a more classical immune-like phenotype, including release of proinflammatory cytokines and chemokines (20Colgan S.P. Dzus A.L. Parkos C.A. J. Exp. Med. 1996; 184: 1003-1015Crossref PubMed Scopus (78) Google Scholar, 21Taylor C.T. Dzus A.L. Colgan S.P. Gastroenterology. 1998; 114: 657-668Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar), regulated neutrophil transmigration (20Colgan S.P. Dzus A.L. Parkos C.A. J. Exp. Med. 1996; 184: 1003-1015Crossref PubMed Scopus (78) Google Scholar), diminished ion transport (14Taylor C.T. Lisco S.J. Awtrey C.S. Colgan S.P. J. Pharmacol. Exp. Ther. 1998; 284: 568-575PubMed Google Scholar), and induction of major histocompatibility complex class II expression (21Taylor C.T. Dzus A.L. Colgan S.P. Gastroenterology. 1998; 114: 657-668Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). The basis for these changes in epithelial function are not well understood, and thus, in the studies defined here, we hypothesized a basic mechanism of hypoxia-induced phenotype switch. These studies revealed that hypoxia specifically induces a panel of epithelial proteins that bear cyclic AMP response elements (CRE) within the region important for regulation of gene expression. Further mechanistic insight was defined with one gene product, epithelial-derived TNF-α, and revealed a critical role for CRE and CRE-binding proteins (CREB) in induction of TNF-α. Such results define a primary role for CRE in the hypoxia-elicited epithelial phenotype and implicate CREB down-regulation as a mechanism of differential induction of proinflammatory mediators by cellular hypoxia. The T84 cell line is a human colonic carcinoma cell line (22Dharmsathaphorn K. Mandel K.G. McRoberts J. Tisdale L.D. Masui H. Am. J. Physiol. 1984; 246: G204-G208Crossref PubMed Google Scholar) which, when plated on permeable membrane supports, forms polarized monolayers of columnar intestine-like epithelial cells. T84 cells are functionally well differentiated with regard to electrogenic Cl− secretion and ion transport and serve as excellent models of crypt columnar epithelial cells (22Dharmsathaphorn K. Mandel K.G. McRoberts J. Tisdale L.D. Masui H. Am. J. Physiol. 1984; 246: G204-G208Crossref PubMed Google Scholar, 23Dharmsathaphorn K. Madara J.L. Methods Enzymol. 1990; 192: 354-389Crossref PubMed Scopus (153) Google Scholar). T84 cells were grown as monolayers in a 1:1 mixture of Dulbecco/Vogt modified Eagle's medium and Ham's F-12 medium, supplemented with 15 mmHEPES buffer (pH 7.5), 14 mm NaHCO3, 40 mg/ml penicillin, 8 mg/ml ampicillin, 90 mg/ml streptomycin, and 5% newborn calf serum. Monolayers were subcultured from flasks every 7–14 days by brief trypsin treatment (0.1% trypsin and 0.9 mm EDTA in Ca2+- and Mg2+-free phosphate-buffered saline). Epithelial exposure to hypoxia was performed as described previously (20Colgan S.P. Dzus A.L. Parkos C.A. J. Exp. Med. 1996; 184: 1003-1015Crossref PubMed Scopus (78) Google Scholar). T84 cell growth media (1:1 Ham's F-12/Dulbecco's modified Eagle's medium) were replaced with fresh, pre-equilibrated hypoxic media, and cells were placed in a humidified environment within the hypoxia chamber (Coy Laboratory Products, Ann Arbor, MI) and maintained at 37 °C. Standard hypoxic conditions, based on previous work (20Colgan S.P. Dzus A.L. Parkos C.A. J. Exp. Med. 1996; 184: 1003-1015Crossref PubMed Scopus (78) Google Scholar), were pO2 20 torr, pCO2 35 torr, with the balance made up of nitrogen and water vapor. Normoxic controls were cells exposed to the same experimental protocols under conditions of atmospheric oxygen concentrations (pO2 147 torr and pCO2 35 torr within a tissue culture incubator). Epithelial expression of indicated surface proteins was quantified using a cell-surface ELISA, as described before (5Colgan S.P. Parkos C.A. Matthews J.B. D'Andrea L. Awtrey C.S. Lichtman A. Delp C. Madara J.L. Am. J. Physiol. 1994; 267: C402-C410Crossref PubMed Google Scholar). Epithelial cells were grown and assayed for antibody binding following exposure to normoxia or hypoxia for the indicated periods. Cells were washed with ice-cold HBSS (Sigma), blocked with media for 30 min at 4 °C. Anti-ICAM-1 monoclonal antibody (clone P2A4 (24Dittel B.N. Wayner E.A. McCarthy J.B. LeBien T.W. Blood. 1993; 81: 2272-2282Crossref PubMed Google Scholar) obtained from the Developmental Studies Hybridoma Bank, Iowa City, IA, used as undiluted cell culture supernatant), anti-MHC class I (25Barnstable C.J. Bodmer W.F. Brown G. Galfre G. Milstein C. Williams A.F. Ziegler A. Cell. 1978; 14: 9-20Abstract Full Text PDF PubMed Scopus (1600) Google Scholar) (clone W6/32 obtained from the American Type Culture Collection, used as 1:100 diluted ascitic fluid), or anti-MHC class II (clone L243(5) obtained from the American Type Culture Collection, used as undiluted cell culture supernatant) were used to examine protein surface expression by ELISA. After washing with HBSS, a peroxidase-conjugated sheep anti-mouse secondary antibody (Cappel, West Chester, PA) was added. Secondary antibody (10 μg/ml) was diluted in media containing 10% fetal bovine serum. After washing, plates were developed by addition of peroxidase substrate (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), 1 mm final concentration, Sigma) and read on a microtiter plate spectrophotometer at 405 nm (Molecular Devices). Controls consisted of media only and secondary antibody only. Data are presented as the mean ± S.E. optical density at 405 nm (secondary antibody background subtracted). IFN-γ (1000 units/ml, 48 h) served as a positive control for induction of these molecules, as described before (5Colgan S.P. Parkos C.A. Matthews J.B. D'Andrea L. Awtrey C.S. Lichtman A. Delp C. Madara J.L. Am. J. Physiol. 1994; 267: C402-C410Crossref PubMed Google Scholar). Cytokine (TNF-α) and chemokine (IL-8) levels were quantified by capture ELISA as described previously (20Colgan S.P. Dzus A.L. Parkos C.A. J. Exp. Med. 1996; 184: 1003-1015Crossref PubMed Scopus (78) Google Scholar, 21Taylor C.T. Dzus A.L. Colgan S.P. Gastroenterology. 1998; 114: 657-668Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Phorbol myristate pre-exposure (10 ng/ml, 12 h) served as a positive control for induction of IL-8 (20Colgan S.P. Dzus A.L. Parkos C.A. J. Exp. Med. 1996; 184: 1003-1015Crossref PubMed Scopus (78) Google Scholar) and TNF-α (21Taylor C.T. Dzus A.L. Colgan S.P. Gastroenterology. 1998; 114: 657-668Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Following experimental treatment of epithelial cells, whole cell extracts (for examination of CREB and phospho-CREB) were prepared as described previously (26Whitley M.Z. Thanos D. Read M.A. Maniatis T. Collins T. Mol. Cell. Biol. 1994; 14: 6464-6475Crossref PubMed Scopus (177) Google Scholar). For analysis of nuclear extracts (NF-κB activation), confluent monolayers of T84 cells on 100-mm Petri dishes were washed in ice-cold phosphate-buffered saline and lysed by incubation in 500 μl of buffer A (10 mm HEPES (pH 8.0), 1.5 mm MgCl2, 10 mm KCl, 0.5 mm dithiothreitol, 200 mm sucrose, 0.5 mm phenylmethanesulfonyl fluoride, 1 μg of both leupeptin and aprotinin per ml, 0.5% Nonidet P-40) for 5 min at 4 °C. The crude nuclei released by lysis were collected by microcentrifugation (15 s). Nuclei were rinsed once in buffer A and resuspended in 100 μl of buffer C (20 mmHEPES (pH 7.9), 1.5 mm MgCl2, 420 mm NaCl, 0.2 mm EDTA, 0.5 mmphenylmethanesulfonyl fluoride, 1.0 mm dithiothreitol, 1 μg/ml of both leupeptin and aprotinin). Nuclei were incubated on a rocking platform at 4 °C for 30 min and clarified by microcentrifugation for 5 min. Proteins were measured (DC protein assay, Bio-Rad). Samples (25 μg/lane, as indicated) of T84 cell lysates were separated by non-reducing SDS-PAGE, transferred to nitrocellulose, and blocked overnight in blocking buffer (250 mm NaCl, 0.02% Tween 20, 5% goat serum, and 3% bovine serum albumin). For Western blotting, anti-NF-κB (rabbit polyclonal antibody specific for p65 subunit of NF-κB, Biomol, Plymouth Meeting, PA), anti-CREB (Upstate Biotechnology, Inc., Lake Placid, NY), or anti-phospho-CREB (Upstate Biotechnology, Inc., Lake Placid, NY) were added for 3 h; blots were washed, and species-matched peroxidase-conjugated secondary antibody was added, exactly as described previously (27Zünd G. Uezono S. Stahl G.L. Dzus A.L. McGowan F.X. Hickey P.R. Colgan S.P. Am. J. Physiol. 1997; 273: C1571-C1580Crossref PubMed Google Scholar). Labeled bands from washed blots were detected by ECL. Resulting bands were quantified from scanned images using NIH Image software (Bethesda). Nuclear extracts of cells exposed to indicated experimental conditions were obtained as described above. The following synthetic oligonucleotide probes were synthesized (Genosys Biotechnologies, Inc.; The Woodlands, TX) and used as probes in EMSAs; the CRE-like motif (bold) lies at −115/−93 relative to the transcription start site in the TNF-α promoter, 5′-GTCGACCTCCAGATGACGTCATGGGTTGTC-3′. Oligonucleotide probes for EMSA were digoxigenin-labeled according to manufacturer's instructions (Gel Shift kit, Roche Molecular Biochemicals). Labeled oligonucleotides were incubated with nuclear lysates for 10 min at 37 °C and separated by electrophoresis on a 6% nondenaturing polyacrylamide gel. DNA-protein complexes were transblotted to nylon membrane, probed with anti-digoxigenin-peroxidase, and developed by ECL. Controls consisted of free probe alone and excess unlabeled probe. For supershift analysis, protein-DNA complexes were incubated with anti-phospho-CREB antibodies (Upstate Biotechnology, Inc., Lake Placid, NY; 1:1000 dilution) for 1 h at 4 °C prior to electrophoresis. Confluent T84 monolayers on 6-well plates were exposed to indicated experimental conditions, and cAMP was quantified exactly as before (14Taylor C.T. Lisco S.J. Awtrey C.S. Colgan S.P. J. Pharmacol. Exp. Ther. 1998; 284: 568-575PubMed Google Scholar). Briefly, cells were cooled to 4 °C; cAMP was extracted from washed monolayers with extraction buffer (66% EtOH, 33% HBSS containing the phosphodiesterase inhibitor isobutylmethylxanthine, 5 mm, Sigma), and lysates were cleared by spinning at 10,000 × g for 5 min and dried under vacuum to remove EtOH. Samples were reconstituted in assay buffer, and cAMP was quantified using displacement ELISAs (Amersham Pharmacia Biotech) according to manufacturer's instructions. Data were converted to concentration using a daily standard curve, and concentrations were expressed as cAMP per μg of total protein. Epithelial cells grown on permeable supports were exposed basolaterally to Rp-cAMPs (0–50 μm Sigma), protein kinase A inhibitor amide (0–30 μm; Calbiochem), or 8-bromo-cAMP (0–3 mm; Sigma) for 48 h prior to harvesting of cells for CREB determination or basolateral supernatants for TNF-α assay as described above. Total RNA was purified according to the manufacturer's procedure from normoxic and hypoxic (6, 12 or 24 h) T84 cells using TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH). 10–50 μg of each RNA sample was treated with DNase I (GenHunter Corp., Nashville, TN), and protein contaminants were then removed through a phenol/chloroform extraction step. RNA samples were ethanol-precipitated and resuspended in diethyl pyrocaronate-treated water. The reaction was set up according to Promega's Reverse Transcription System protocol (Promega Corp., Madison, WI). Briefly, 1 μg of RNA was added to the reaction mixture consisting of 4 μl of 25 mmol/liter MgCl2, 2 μl of 10× Reverse Transcriptase Buffer, 2 μl of 10 mmol/liter of each dNTP, 0.5 μl of rRNase in ribonuclease inhibitor (20 units total), 15 units of avian myeloblastosis virus reverse transcriptase, and 0.5 μg of oligo(dT)15 primer in a total volume of 20 μl. The single-stranded cDNA was synthesized (MJ Research, Inc., Thermocycler Model PTC-200) using one cycle at 25 °C for 10 min, one cycle at 42 °C for 45 min, and one cycle at 95 °C for 5 min followed by a final cycle at 4 °C for 5 min. The PlatinumTM Taq DNA Polymerase High Fidelity PCR System from Life Technologies, Inc., was used in the amplification step. The PCR reaction for human TNF-α contained 1 μmeach of the sense primer (5′-CGGGACGTGGAGCTGGCCGAGGAG-3′) and the antisense primer (5′-CACCAGCTGGTTATCTCTCAGCTC-3′), 5 μl of Reverse Transcriptase reaction, 5 μl of 10× PlatinumTM Taq high fidelity PCR buffer, 2 μl of 50 mmMgSO4, 0.2 mmol of dNTP, and 2.5 units of PlatinumTM Taq High Fidelity enzyme mix in a total volume of 50 μl. The amplification reaction included a 5-min denaturation at 94 °C and a 5-min annealing at 60 °C, followed by 30 cycles at 72 °C of 1.5 min, 94 °C for 45 s, and 60 °C for 45 s, with a final extension at 72 °C for 10 min. The PCR reactions were then visualized on a 1% agarose gel containing 5 μg/ml ethidium bromide. A 355-base pair fragment corresponding to TNF-α was observed. In order to ensure that an equal amount of template was used in each amplification reaction, 5 μl of Reverse Transcriptase reaction was used as template with 1 μmeach of human β-actin sense primer (5′-TGACGGGGTCACCCACACTGTGCCCATCTA-3′) and antisense primer (5′-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3′) in identical reactions; a 661-base pair amplified fragment with equal intensity was observed in all samples. Retroviral-mediated gene transfer of T84 cells with CREB and mutant CREB (serine to alanine mutated at site 133, the PKA phosphorylation site) containing vectors was performed using a previously described technique (28Hershberg R.M. Framson P.E. Cho D.H. Lee L.Y. Kovats S. Beitz J. Blum J.S. Nepom G.T. J. Clin. Invest. 1997; 100: 204-215Crossref PubMed Scopus (190) Google Scholar). Briefly, CREB or mutant CREB cDNA (a kind gift from Dr. Marc Montminy, Harvard Medical School) was expressed under the control of the CMV-IE promoter, and the cDNA for the dominant selectable marker (neomycin resistance) was expressed under control of the viral long terminal repeat. 106 epithelial cells were plated 24 h prior to infection. Cells were washed once, and then 3–4 ml of fresh, 0.45-μm filtered, viral supernatant supplemented with 4 μg/ml Polybrene were added to the adherent cells. After 8 h, 5 ml of fresh complete medium was added, and the cells were cultured for 48 h before drug selection. Cytokine, nucleotide, and cell-surface ELISA data were evaluated by analysis of variance and by Student'st test with p < 0.05 considered significant. All values are given as mean ± S.E. for nexperiments. Recently, we demonstrated that hypoxia enhances epithelial responses to the cytokine IFN-γ, and such responses were subsequently attributed to induction of epithelial TNF-α release from the basolateral surface (21Taylor C.T. Dzus A.L. Colgan S.P. Gastroenterology. 1998; 114: 657-668Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). These studies, however, did not reveal significant insight into mechanisms that evoke induction of TNF-α, and therefore, we sought to understand such findings at the mechanistic level. As an initial screen, a panel of epithelial proinflammatory markers were examined to determine whether such signaling by hypoxia was specific for TNF-α or is generalized to other molecules. As shown in Fig. 1, responses to hypoxia were specific. Indeed, epithelial exposure to hypoxia induced TNF-α release (maximal 37.0 ± 6.1-fold increase, p < 0.01), IL-8 release (maximal 7.0 ± 0.8-fold increase,p < 0.01), and induced surface expression of MHC class II (maximal 9.0 ± 0.9-fold increase, p < 0.01). Hypoxia did not, however, influence epithelial ICAM-1 expression (p = not significant compared with normoxia) or MHC class I (p = not significant compared with normoxia) indicating a degree of specificity for signaling by hypoxia. In an attempt to gain insight into such specificity, we began by examining induction pathways of these pro-inflammatory genes. The regulatory regions of each of these genes contain a binding site for NF-κB, a transcription factor important in induction of a number of proinflammatory genes (29Collins T. Read M.A. Neish A.S. Whitley M.Z. Thanos D. Maniatis T. FASEB J. 1995; 9: 899-909Crossref PubMed Scopus (1578) Google Scholar). As shown in Fig. 2, Western blot analysis of nuclear extracts derived from epithelia exposed to hypoxia (measured pO2 20 torr for 0–48 h) revealed cytoplasmic-to-nuclear localization of the p65 subunit NF-κB, a reliable readout of NF-κB activation (27Zünd G. Uezono S. Stahl G.L. Dzus A.L. McGowan F.X. Hickey P.R. Colgan S.P. Am. J. Physiol. 1997; 273: C1571-C1580Crossref PubMed Google Scholar). Indeed, periods of hypoxia as short as 6 h revealed a significant cytoplasmic-to-nuclear localization of the p65 subunit of NF-κB. Such responses to hypoxia were maximal by 24 h (densitometric measurement of 360.5 versus normoxic control value of 82.2 relative units) and were similar to our positive control PMA (497.9 relative units). We next concentrated on elucidating pathways of epithelial gene induction by hypoxia, specifically using TNF-α to define these principles. TNF-α gene induction and protein release can be regulated in a number of ways including transcriptional and post-translational pathways. Thus we examined whether hypoxia induces transcription of the epithelial TNF-α gene. As shown in Fig. 3, reverse transcriptase-PCR analysis revealed a time-dependent induction of TNF-α. Such induction was detectable at 12 h, maximal at 24 h, and exceeded that of our positive control (PMA, 10 ng/ml for 12 h). These data reveal that hypoxia activates transcriptional pathways. In addition to a binding site for the transcription factor NF-κB, the TNF-α, IL-8, and MHC class II genes bear a cAMP responsive consensus binding site, termed a cAMP response element (CRE) (26Whitley M.Z. Thanos D. Read M.A. Maniatis T. Collins T. Mol. Cell. Biol. 1994; 14: 6464-6475Crossref PubMed Scopus (177) Google Scholar, 27Zünd G. Uezono S. Stahl G.L. Dzus A.L. McGowan F.X. Hickey P.R. Colgan S.P. Am. J. Physiol. 1997; 273: C1571-C1580Crossref PubMed Google Scholar). Importantly, the regulatory regions of those molecules not induced by hypoxia (MHC class I and ICAM-1, see Fig. 1) do not contain a CRE. These CRE DNA consensus motifs serve to regulate transcription of genes through protein kinase A and calcium-dependent pathways. In general, increases in intracellular cAMP are associated with decreased TNF-α release (30Tannenbaum C.S. Hamilton T.A. J. Immunol. 1989; 142: 1274-1279PubMed Google Scholar). Phosphorylation of nuclear proteins, termed cAMP response element binding proteins (CREB), positively or negatively regulate the activation of CRE-containing genes (e.g. cAMP can increase or decrease transcription) depending on the gene (31Montminy M. Annu. Rev. Biochem. 1997; 66: 807-822Crossref PubMed Scopus (860) Google Scholar). Since a number of different consensus motifs serve as functional CREs, we examined the contribution of the CRE found within the TNF-α promoter. To do this, we developed an EMSA using oligonucleotides flanking the TNF-α CRE (32Newell C.L. Deisseroth A.B. Lopez-Berestein G. J. Leukocyte Biol. 1994; 56: 27-35Crossref PubMed Scopus (117) Google Scholar). As shown in Fig.4, proteins from nuclear extracts of T84 cells bind to the CRE region of the TNF-α promoter. Furthermore, addition of antibody to t
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