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Cyclooxygenase-2 Expression in Cultured Cortical Thick Ascending Limb of Henle Increases in Response to Decreased Extracellular Ionic Content by Both Transcriptional and Post-transcriptional Mechanisms

2002; Elsevier BV; Volume: 277; Issue: 47 Linguagem: Inglês

10.1074/jbc.m206040200

1083-351X

Huifang Cheng, Raymond C. Harris,

Inflammatory mediators and NSAID effects

We showed previously that decreased extracellular salt or chloride up-regulates the cortical thick ascending limb of Henle (cTALH) COX-2 expression via a p38-dependent pathway. The present studies determined that low salt medium increased COX-2 mRNA expression 3.9-fold control by 6 h in cultured cTALH, which was blocked by actinomycin D pretreatment, suggesting transcriptional regulation. Luciferase activity (normalized to β-galactosidase activity) of the full-length (−3400) COX-2 promoter in cTALH increased from 1.8 ± 0.3 in control media to 5.8 ± 0.7 in low salt (n = 9;p < 0.01). Low chloride medium had similar effects as low salt has on COX-2 promoter activity. Deletion constructs −815, −512, and −410 were similarly stimulated, but −385 could not be stimulated significantly by low salt (1.8 ± 0.3versus 2.4 ± 0.5, n = 10). This suggested involvement of an NF-κB cis-element located in this region, which was confirmed by utilizing a construct with a point mutation of this NF-κB-binding site that was not stimulated by low salt medium. Co-incubation of the specific p38 inhibitor, SB203580 or PD169316, inhibited a low salt-induced increase in luciferase activity of the intact COX-2 promoter (5.8 ± 0.7versus 1.1 ± 0.2, n = 8 and 1.4 ± 0.4, n = 4 respectively, p < 0.01). Mobility shift assays indicated that the low salt medium stimulated NF-κB binding activity, and this stimulation was inhibited by p38 inhibitors. To test whether p38 also increased COX-2 expression by increasing mRNA stability, cTALH were incubated in low salt for 2 h, and actinomycin was then added with or without SB203580. p38 inhibition led to a decreased half-life of COX-2 mRNA (from 68 to 18 min, n = 4–7, p < 0.05). Therefore, these studies indicate that p38 stimulates COX-2 expression in cTALH and macula densa by transcriptional regulation predominantly via a NF-κB-dependent pathway and by post-transcriptional increases in mRNA stability. We showed previously that decreased extracellular salt or chloride up-regulates the cortical thick ascending limb of Henle (cTALH) COX-2 expression via a p38-dependent pathway. The present studies determined that low salt medium increased COX-2 mRNA expression 3.9-fold control by 6 h in cultured cTALH, which was blocked by actinomycin D pretreatment, suggesting transcriptional regulation. Luciferase activity (normalized to β-galactosidase activity) of the full-length (−3400) COX-2 promoter in cTALH increased from 1.8 ± 0.3 in control media to 5.8 ± 0.7 in low salt (n = 9;p < 0.01). Low chloride medium had similar effects as low salt has on COX-2 promoter activity. Deletion constructs −815, −512, and −410 were similarly stimulated, but −385 could not be stimulated significantly by low salt (1.8 ± 0.3versus 2.4 ± 0.5, n = 10). This suggested involvement of an NF-κB cis-element located in this region, which was confirmed by utilizing a construct with a point mutation of this NF-κB-binding site that was not stimulated by low salt medium. Co-incubation of the specific p38 inhibitor, SB203580 or PD169316, inhibited a low salt-induced increase in luciferase activity of the intact COX-2 promoter (5.8 ± 0.7versus 1.1 ± 0.2, n = 8 and 1.4 ± 0.4, n = 4 respectively, p < 0.01). Mobility shift assays indicated that the low salt medium stimulated NF-κB binding activity, and this stimulation was inhibited by p38 inhibitors. To test whether p38 also increased COX-2 expression by increasing mRNA stability, cTALH were incubated in low salt for 2 h, and actinomycin was then added with or without SB203580. p38 inhibition led to a decreased half-life of COX-2 mRNA (from 68 to 18 min, n = 4–7, p < 0.05). Therefore, these studies indicate that p38 stimulates COX-2 expression in cTALH and macula densa by transcriptional regulation predominantly via a NF-κB-dependent pathway and by post-transcriptional increases in mRNA stability. Prostaglandins are metabolized from arachidonic acid through cyclooxygenase (COX)-dependent 1The abbreviations used are: COX, cyclooxygenase; cTALH, cortical thick ascending limb of Henle; EMSA, electrophoretic mobility shift assay; CREB, cAMP-response element-binding protein.pathways (1Needleman P. Turk J. Jakschik B.A. Morrison A.R. Lefkowith J.B. Annu. Rev. Biochem. 1986; 55: 69-102Google Scholar) and modulate vascular tone, expression and secretion of renin, and salt and water homeostasis in the mammalian kidney. Two isoforms of COX have been found: COX-1, which is constitutive, and COX-2, which is inflammation-mediated and glucocorticoid-sensitive (2Masferrer J.L. Zweifel B.S. Seibert K. Needleman P. J. Clin. Invest. 1990; 86: 1375-1379Google Scholar, 3Kujubu D.A. Fletcher B.S. Varnum B.C. Lim R.W. Herschman H.R. J. Biol. Chem. 1991; 266: 12866-12872Google Scholar, 4O'Banion M.K. Winn V.D. Young D.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4888-4892Google Scholar). We originally reported that salt restriction increases renal cortical COX-2 expression selectively in tubular epithelial cells of macula densa and the surrounding cortical thick ascending limb of Henle (cTALH) (5Harris R.C. McKanna J.A. Akai Y. Jacobson H.R. Dubois R.N. Breyer M.D. J. Clin. Invest. 1994; 94: 2504-2510Google Scholar), which was confirmed by subsequent studies (6Jensen B.L. Kurtz A. Kidney Int. 1997; 52: 1242-1249Google Scholar). The macula densa is recognized to be important for regulation of renal renin expression and regulation of afferent renal arteriolar tone through the process of tubuloglomerular feedback (7Schnermann J. Am. J. Physiol. 1998; 274: R263-R279Google Scholar). Up-regulation of COX-2 in these cells was also found in other high renin states, such as angiotensin-converting enzyme inhibitor (8Cheng H.F. Wang J.L. Zhang M.Z. Miyazaki Y. Ichikawa I. McKanna J.A. Harris R.C. J. Clin. Invest. 1999; 103: 953-961Google Scholar, 9Wolf K. Castrop H. Hartner A. Goppelt-Strube M. Hilgers K.F. Kurtz A. Hypertension. 1999; 34: 503-507Google Scholar), diuretic administration (10Mann B. Hartner A. Jensen B.L. Kammerl M. Kramer B.K. Kurtz A. Kidney Int. 2001; 59: 62-68Google Scholar), and experimental renovascular hypertension (11Wang J.L. Cheng H.F. Harris R.C. Hypertension. 1999; 34: 96-101Google Scholar). We previously demonstrated that COX-2 is an important mediator for renin release from macula densa (8Cheng H.F. Wang J.L. Zhang M.Z. Miyazaki Y. Ichikawa I. McKanna J.A. Harris R.C. J. Clin. Invest. 1999; 103: 953-961Google Scholar, 12Cheng H.F. Wang J.L. Zhang M.Z. Wang S.W. McKanna J.A. Harris R.C. Am. J. Physiol. 2001; 280: F449-F456Google Scholar). It has been suggested that decreased intraluminal chloride concentration is the signal for macula densa stimulation of renin secretion (13Lorenz J.N. Weihprecht H. Schnermann J. Skott O. Briggs J.P. Am. J. Physiol. 1990; 259: F186-F193Google Scholar, 14Persson A.E. Salomonsson M. Westerlund P. Greger R. Schlatter E. Gonzalez E. Kidney Int. 1991; 32 (suppl.): 39-44Google Scholar). Ion substitution experiments in perfusion of isolated cortical thick ascending limbs with associated juxtaglomerular apparati indicated that only when chloride was replaced by alternative anions was renin secretion increased; no increase in renin expression or secretion could be seen in response to sodium substitution by other cations (15Lorenz J.N. Weihprecht H. Schnermann J. Skott O. Briggs J.P. Am. J. Physiol. 1991; 260: F486-F493Google Scholar). Our previous studies indicated that decreased extracellular NaCl or selective decreases in extracellular chloride up-regulated cTALH COX-2 expression, and this up-regulation of COX-2 in cTALH occurred via a p38-dependent pathway (16Cheng H.F. Wang J.L. Zhang M.Z. McKanna J.A. Harris R.C. J. Clin. Invest. 2000; 106: 681-688Google Scholar). The current study investigated the transcriptional and post-transcriptional regulation of COX-2 by alteration of extracellular salt or chloride. Goat anti-human uromucoid antibody was from ICN (Costa Mesa, CA). Anti-goat IgG (H+L) was from Vector Laboratory (Burlingame, CA). Anti-p65, -p50, -p52, and -c-Rel antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Rat COX-2 cDNA was from Oxford Biomedical Research (Oxford, MI). The luciferase assay system and gel shift assay systems were from Promega (Madison, WI). SB203580, PD169316, and Bay 11–7082 were from Calbiochem Bioscience Inc. (La Jolla, CA). [32P]CTP (3,000 Ci/mmol) and [γ-32P]ATP (3,000 Ci/mmol at 10 mCi/ml) were from PerkinElmer Life Sciences. LipofectAMINE was from Invitrogen. Other reagents were purchased from Sigma. cTALH cells were isolated from homogenates of rabbit renal cortex by immunodissection with anti-Tamm Horsfall antibody, as previously described (8Cheng H.F. Wang J.L. Zhang M.Z. Miyazaki Y. Ichikawa I. McKanna J.A. Harris R.C. J. Clin. Invest. 1999; 103: 953-961Google Scholar, 17Allen M.L. Nakao A. Sonnenburg W.K. Burnatowska-Hledin M. Spielman W.S. Smith W.L. Am. J. Physiol. 1988; 255: F704-F710Google Scholar, 18Cheng H.F. Wang J.L. Zhang M.Z. McKanna J.A. Harris R.C. Am. J. Physiol. 2000; 279: F122-F129Google Scholar). Briefly, the renal cortex was dissected, minced, and digested with 0.1% collagenase. After blocking with 10% bovine serum albumin, the sieved homogenates were incubated with goat anti-human Tamm Horsfall antiserum (50 mg/ml) for 30 min on ice, followed by washing and addition to plastic Petri dishes coated with anti-goat IgG (8 mg/ml). Attached cells resistant to washing were dislodged and grown to confluence in Dulbecco's modified Eagle' medium/Ham's F-12 medium with 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in 95% air, 5% CO2. The proximal 3.2 kb of the mouse COX-2 promoter was cloned by reverse transcription-PCR from mouse kidney RNA with the 5′ primer (CAT GAA TTC TGT TCT GCC CTC ATG TGT ATG) and the 3′ primer (TAA GGT ACC GGT GGA GCT GGC AGG ATG CAG TGC A) and then subcloned into a luciferase reporter vector (Promega). The −815 and −512 deletion luciferase reporter constructs were a generous gift from Dr. Yamamoto (19Yamamoto K. Arakawa T. Ueda N. Yamamoto S. J. Biol. Chem. 1995; 270: 31315-31320Google Scholar). The mouse COX-2 promoter was cut withEcoRI and PpuMI, blunted, and self-ligated to make the −385 deletion construct. The XhoI cutting site was introduced by point mutation to produce the −410 deletion construct. NF-κB and CREB point mutants were also constructed (Stratagene, La Jolla, CA). Briefly, the luciferase reporter plasmid containing the mutation for NF-κB or CREB was developed. After PCR amplification, the product was treated with endonuclease DpnI and then transformed to XL 1-Blue supercompetent cells. All of the mutants and other plasmid constructs were verified by sequencing. Primary cultured cTALH cells at 50–60% confluence were transiently transfected with LipofectAMINE reagent (Invitrogen). When the cells grew to confluence (48 h later), they were made quiescent with serum-free medium for 16 h and then changed to the indicated condition (e.g.low salt or low chloride medium) for 6 h. The content of low salt or low chloride medium was as previously described (16Cheng H.F. Wang J.L. Zhang M.Z. McKanna J.A. Harris R.C. J. Clin. Invest. 2000; 106: 681-688Google Scholar). The cells were extracted with lysis buffer (luciferase assay system; Promega), and their luciferase activities were measured with a LumiCount Microplate Luminometer (model AL10000; Packard Bioscience Co., Meridian, CT). The results were normalized to β-galactosidase activity as previously described (20Arakawa T. Nakamura M. Yoshimoto T. Yamamoto S. FEBS Lett. 1995; 363: 105-110Google Scholar). Primary cultured cTALH cell RNA was extracted by the acid guanidium thiocyanate-phenol chloroform method (21Wang J.L. Cheng H.F. Shappell S. Harris R.C. Kidney Int. 2000; 57: 2334-2342Google Scholar). RNA samples were electrophoresed in denatured agarose gel, transferred to nitrocellulose membranes, and hybridized with a 32P-labeled cDNA of rat COX-2. The membranes were then stripped and rehybridized with glyceraldehyde-3-phosphate dehydrogenase. Nuclear protein was extracted as previously described (22Zhang X. Huang C.J. Nazarian R. Ritchie T. de Vellis J.S. Noble E.P. BioTechniques. 1997; 22: 848-850Google Scholar,23Sakai T. Ichiyama T. Whitten C.W. Giesecke A.H. Lipton J.M. Can. J. Anaesth. 2000; 47: 1019-1024Google Scholar). Briefly, the cells were homogenized with a Dounce homogenizer in buffer containing 10 mm HEPES, pH 7.9, 10 mmKCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mmdithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, and 0.1% leupeptin. 10% Nonidet P-40 was added to make a final concentration of 0.5%, incubated on ice for 30 min, and centrifuged. After washing, the pellet was resuspended in 20 mm HEPES, pH 7.9, 400 mm NaCl, 1 mm EDTA, 1 mm EGTA, I mm dithiothreitol, 1 mmphenylmethylsulfonyl fluoride, 10% glycerol, and 0.1% leupeptin with 1/10 volume of 4 m KCl. EMSA was performed as described (24Cheng G. Hagen T.P. Dawson M.L. Barnes K.V. Menick D.R. J. Biol. Chem. 1999; 274: 12819-12826Google Scholar). Double-stranded NF-κB and CREB oligonucleotides were end-labeled with [32P]ATP by T4 polynucleotide kinase. 5 μg of nuclear protein was added to the reaction mixture, incubated for 30 min at 25 °C, and resolved on 6% nondenatured polyacrylamide gels. Antibody supershift EMSA was performed by incubating nuclear extracts with anti-p65, -p50, -p52, or -c-Rel antibody or irrelevant control antibody for 10 min at room temperature before adding the labeled probe. Renal cortices were homogenized with RIPA buffer and centrifuged, heated to 100 °C for 5 min with sample buffer, separated on SDS gels under reducing conditions, and transferred to Immobilon-P transfer membranes (Millipore, Bedford, MA). The blots were blocked overnight with 100 mm Tris-HCl, pH 7.4, containing 5% nonfat dry milk, 3% albumin, and 0.5% Tween 20, followed by incubation for 16 h with monoclonal IκB or pIκB antibodies. The second biotinylated antibody reagent was detected using avidin and biotinylated horseradish peroxidase (Pierce) and exposed on film using ECL (Amersham Biosciences). All of the values are presented as the means ± S.E. Analysis of variance and Bonferroni ttests were used for statistical analysis, and the differences were considered significant when p < 0.05. In previous studies we demonstrated that immunoreactive COX-2 protein increased in primary cultured cTALH in response to exposure to low salt or low chloride medium. Substitution of other cations for sodium did not affect COX-2 expression, whereas substitution of other anions for chloride led to increased COX-2 expression (16Cheng H.F. Wang J.L. Zhang M.Z. McKanna J.A. Harris R.C. J. Clin. Invest. 2000; 106: 681-688Google Scholar). In the present studies, we determined that COX-2 mRNA expression increased in low salt medium, with an apparent peaking within 6 h (3.9 ± 0.4-fold control) and increased expression observed for up to 16 h (3.4 ± 0.5-fold control) (Fig.1 A). When cells were preincubated with 5 μg/ml actinomycin D (25Han S.W. Lei Z.M. Rao C.V. Mol. Cell. Endocrinol. 1999; 147: 7-16Google Scholar) for 20 min prior to exposure to low salt medium, increases in COX-2 mRNA expression were blocked (Fig. 1 B). The time course of COX-2 mRNA increase and inhibition by actinomycin D were similar in low chloride medium (not shown). Informative deletions of the murine COX-2 promoter were constructed (Fig. 2 A) and transfected into cTALH cells. After quiescence, the cells were exposed to normal or low salt medium for 6 h, and luciferase activity was measured, normalized by β-galactosidase activity, and expressed as fold control (Fig. 2 B). Luciferase activity of the vector transfected cells was identical if grown in normal medium or low salt medium (1.2 ± 0.1-fold control, n = 14–18, NS). There were no significant differences of luciferase activity among the six groups when grown in normal medium. Low salt medium elevated the luciferase activity of full-length COX-2 promoter-transfected cells (5.2 ± 0.4-fold control, n = 10,p < 0.01). Low salt medium induced similar increases in promoter activity with the −815 construct (5.4 ± 0.6-fold control, n = 17–18, p < 0.01) and the −512 deletion construct (5.6 ± 0.5-fold control in low salt medium, n = 17–18, p < 0.01). However, there was no significant increase in luciferase activity by low salt medium in the −385 deletion construct (1.8 ± 0.2 in normal medium versus 2.4 ± 0.5-fold control in low salt medium, n = 10, NS). Of note, the murine COX-2 promoter contains putative CREB and NF-κB sites between −512 and −385. An additional deletion at −410 removed the putative CREB site but did not interrupt the NF-κB site. Fig. 2 A indicated that the −410 deletion still responded to low salt stimulation (1.6 ± 0.2 in normal medium versus 5.5 ± 0.8-fold control in low salt medium, n = 8,p < 0.01). The NF-κB inhibitor, BAY 11–7082 (10 μm) (26Pierce J.W. Schoenleber R. Jesmok G. Best J. Moore S.A. Collins T. Gerritsen M.E. J. Biol. Chem. 1997; 272: 21096-21103Google Scholar) also inhibited the low salt medium-induced increase in luciferase activity in the −815 construct (to 2.7 ± 0.6-fold control, n = 8, p < 0.01) (Fig. 3).Figure 3NF-κB and p38 inhibitors block low salt-induced COX-2 promoter activity. cTALH cells were transfected with the −815 COX-2 promoter construct in the absence or presence of the NF-κB inhibitor BAY11–7082 (10 μm) or the p38 inhibitors SB203580 (10 μm) or PD169316 (1 μm) (n = 8). **,p < 0.01. LS, low salt.View Large Image Figure ViewerDownload (PPT) To confirm a role for the putative NF-κB site in low salt medium-induced increases in luciferase activity, inactive mutations of the indicated NF-κB and CREB sites were constructed (19Yamamoto K. Arakawa T. Ueda N. Yamamoto S. J. Biol. Chem. 1995; 270: 31315-31320Google Scholar) (Fig.4 A). The CREB mutant construct had a significant increase in luciferase activity in response to the low salt medium, although the stimulation was somewhat less than what was observed with the wild type construct. In contrast, the NF-κB mutation completely blocked low salt medium-induced activation (Fig.4 B). Similar responses were seen in the low chloride medium (−815 deletion, from 1.7 ± 0.2- to 4.6 ± 1.0-fold control,n = 4, p < 0.01; NF-κB mutation, from 1.2 ± 0.1- to 1.3 ± 0.3-fold control,n = 4, NS). (Fig. 4 C). In EMSA of cTALH nuclear extracts incubated with a consensus NF-κB oligonucleotide, two specific bands were detected (Fig.5 A). Low salt medium incubation stimulated the NF-κB binding activity (Fig.5 B). CREB binding by nuclear extracts from cTALH cells exposed to low salt medium was also slightly decreased compared with control (Fig. 5 C). Supershift assay indicated that the two bands seen in the NF-κB EMSAs were p65 and p50 (Fig.5 D). Because activation of cytoplasmic NF-κB and translocation to the nucleus is regulated by phosphorylation of IκB, we determined IκB and pIκB expression in cTALH cells. Low salt medium decreased IκB expression (0.4 ± 0.1-fold control, n = 7,p < 0.01) (Fig.6 A) and increased immunoreactive pIκB expression (2.2 ± 0.2-fold control,n = 8, p < 0.05) (Fig.6 B). When a p38-specific inhibitor, PD169316 (1 μm) (16Cheng H.F. Wang J.L. Zhang M.Z. McKanna J.A. Harris R.C. J. Clin. Invest. 2000; 106: 681-688Google Scholar) or SB 203580 (10 μm) (27Leonard M. Ryan M.P. Watson A.J. Schramek H. Healy E. Kidney Int. 1999; 56: 1366-1377Google Scholar), was added to low salt or low chloride medium, stimulation of luciferase activity in the −815 construct was decreased (PD169316, from 5.4 ± 0.3- to 1.4 ± 0.4-fold control, n = 6, p < 0.01; SB203580, 1.1 ± 0.2-fold control, n = 8,p < 0.05) (Fig. 3). EMSA further confirmed that SB203580 reduced the increases in NF-κB binding ability (Fig.5 B). SB203580 also inhibited increases in IκB phosphorylation stimulated by low salt (1.4 ± 0.1 of control medium, n = 8, NS) and prevented decreases in IκB levels (0.9 ± 0.1, n = 7, NS) (Fig. 6). In other cell systems, alterations in COX-2 mRNA expression have been attributed to both transcriptional and post-transcriptional regulation. To test the possibility that there was a component of post-transcriptional stabilization of COX-2 mRNA in response to low salt or low chloride medium, cTALH cells were stimulated by low salt medium for 2 h, and then actinomycin D was added with or without the specific p38 inhibitor, SB203580. p38 inhibition led to an increased decay rate of COX-2 mRNA (from 68 to 18 min,n = 4–7, p < 0.05) (Fig.7), suggesting that p38 activity may also regulate COX-2 mRNA stability with low salt stimulation. In vivo, dietary salt restriction increases COX-2 expression in the macula densa and the surrounding cTALH cells (5Harris R.C. McKanna J.A. Akai Y. Jacobson H.R. Dubois R.N. Breyer M.D. J. Clin. Invest. 1994; 94: 2504-2510Google Scholar, 6Jensen B.L. Kurtz A. Kidney Int. 1997; 52: 1242-1249Google Scholar). Macula densa/cTALH regulate renal renin expression and renal hemodynamics in response to alterations in tubular luminal chloride concentration (15Lorenz J.N. Weihprecht H. Schnermann J. Skott O. Briggs J.P. Am. J. Physiol. 1991; 260: F486-F493Google Scholar). Macula densa sensing of luminal chloride is dependent on net apical transport, mediated by the luminal Na+/K+/2Cl− co-transporter, BSC-1 (28Schlatter E. Salomonsson M. Persson A.E. Greger R. Pfluegers Arch. Eur. J. Physiol. 1989; 414: 286-290Google Scholar, 29Salomonsson M. Gonzalez E. Westerlund P. Persson A.E. Kidney Int. 1991; 32 (suppl.): 51-54Google Scholar, 30Nielsen S. Maunsbach A.B. Ecelbarger C.A. Knepper M.A. Am. J. Physiol. 1998; 275: F885-F893Google Scholar). Na+/K+/2Cl−co-transporter has a high affinity for Na+ and K+ but a lower affinity for Cl− (in the 30–50 mmol/l range), resulting in sensitivity to physiologic changes in luminal chloride (31Greger R. Nephrol. Dial. Transplant. 1997; 12: 2215-2217Google Scholar). The potential role of prostaglandins in mediation of macula densa function has prompted further studies to investigate the signals mediating this increased COX-2 expression. In our previous studies utilizing primary cultured cTALH cells, we determined that immunoreactive COX-2 expression increased significantly when medium NaCl was decreased without alterations in extracellular osmolality or following administration of the Na+/K+/2Cl− co-transport inhibitor, bumetanide. Selective substitution of chloride led to increased COX-2 expression, whereas selective substitution of sodium had no effect (16Cheng H.F. Wang J.L. Zhang M.Z. McKanna J.A. Harris R.C. J. Clin. Invest. 2000; 106: 681-688Google Scholar). Similarly, it has been reported recently that low chloride stimulates prostaglandin E2 release and COX-2 expression in MMDD1 cells, which are derived from mouse macula densa (32Yang T. Park J.M. Arend L. Huang Y. Topaloglu R. Pasumarthy A. Praetorius H. Spring K. Briggs J.P. Schnermann J.B. J. Biol. Chem. 2000; 275: 37922-37929Google Scholar). These studies suggested that decreased extracellular [Cl−] initiates the overexpression of COX-2 observedin vivo in response to salt restriction. The present studies indicate that a lessened extracellular chloride-mediated increase in p38 activity stimulates COX-2 expression in cTALH and macula densa by transcriptional regulation via an NF-κB-dependent pathway and by post-transcriptional increases in mRNA stability. In other systems, different members of the mitogen-activated protein kinase superfamily have been shown to increase COX-2 expression, including extracellular signal-regulated kinase- (33LaPointe M.C. Isenovic E. 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Furthermore, the inhibition of NF-κB binding activity by the p38 inhibitor SB203580 indicated that the p38 pathway mediates NF-κB activation. CREB promotes recruitment of the transcriptional co-activator CBP and p300. It has been shown to function as a classic intracellular second messenger in glucose homeostasis, growth factor-dependent cell survival, and involvement in learning and memory (54Mayr B. Montminy M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 599-609Google Scholar). Although NF-κB activation appears to be absolutely necessary for low salt-mediated increases in COX-2 transcription, our luciferase activity and EMSA data also suggest the possibility of an additional role for CREB. In this regard, in activated macrophages both CREB and NF-κB, as well as C/EBP β and δ, have been identified as key factors in coordinately orchestrating COX-2 transcription (55Caivano M. Gorgoni B. Cohen P. Poli V. J. Biol. Chem. 2001; 276: 48693-48701Google Scholar). 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In addition to the transcriptional induction of COX-2, stabilization of the COX-2 mRNA at the post-transcriptional level is important for maximal expression (66Jang B.C. Sanchez T. Schaefers H.J. Trifan O.C. Liu C.H. Creminon C. Huang C.K. Hla T. J. Biol. Chem. 2000; 275: 39507-39515Google Scholar, 67Ristimaki A. Narko K. Hla T. Biochem. J. 1996; 318: 325-331Google Scholar, 68Lasa M. Mahtani K.R. Finch A. Brewer G. Saklatvala J. Clark A.R. Mol. Cell. Biol. 2000; 20: 4265-4274Google Scholar). A crucial role for AU-rich sequence elements in the COX-2 3′-untranslated region to stabilize COX-2 mRNA expression has been shown in human lung fibroblast cells (66Jang B.C. Sanchez T. Schaefers H.J. Trifan O.C. Liu C.H. Creminon C. Huang C.K. Hla T. J. Biol. Chem. 2000; 275: 39507-39515Google Scholar), HeLa-To cells (68Lasa M. Mahtani K.R. Finch A. Brewer G. Saklatvala J. Clark A.R. Mol. Cell. Biol. 2000; 20: 4265-4274Google Scholar), and murine macrophage-like cells (69Dean J.L. Wait R. Mahtani K.R. Sully G. Clark A.R. Saklatvala J. Mol. Cell. Biol. 2001; 21: 721-730Google Scholar). Further studies will be required to determine whether p38 activation by decreased extracellular chloride increases COX-2 mRNA stability by a similar mechanism. In summary, these studies indicate that p38 stimulates COX-2 expression in cultured cTALH by transcriptional regulation predominantly via a NF-κB-dependent pathway and by post-transcriptional increases in mRNA stability.