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作者：Hui-Fang Cheng, Ming-Zhi Zhang, and Raymond C. Harris作者单位：George M. O‘Brien Kidney and Urologic Diseases Center and Division of Nephrology, Vanderbilt University School of Medicine and Nashville Veterans Affairs Hospital, Nashville, Tennessee
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【摘要】
0 }! Q+ m) u8 k: i1 @ To examine the interaction of nitric oxide (NO) and cyclooxygenase (COX-2) and the signaling pathway involved, primary cultured rabbit cortical thick ascending limb (cTAL) were used. In these cells, immunoreactive COX-2 and vasodilatory prostaglandins were increased by a NO donor, S -nitros- N -acetylpenicillamine (SNAP; 2.5 ± 0.3-fold control, n = 6, P < 0.01). SNAP increased expression of phosphorylated p38 (pp38; 2.4 ± 0.3-fold control; n = 5; P < 0.01), which was inhibited by the p38 inhibitor SB-203580 (1.3 ± 0.1-fold control, n = 5, P < 0.01). SB-203580 inhibited SNAP-induced COX-2 expression [1.4 ± 0.2-fold control, n = 6, not significant (NS) vs. control] and levels of PGE 2 significantly. In cTAL cells transfected with a luciferase reporter driven by the wild-type mouse COX-2 promoter, SNAP stimulated luciferase activity, which was reversed by SB-203580 (control vs. SNAP vs. SNAP SB-203580: 1.4 ± 0.2-, 8.3 ± 1.4-, and 0.4 ± 0.1-fold control, respectively, n = 4, P < 0.01). Electrophoretic mobility shift assay indicated that SNAP stimulated nuclear factor (NF)- B binding activity in cTAL that was also inhibited by the p38 inhibitor. SNAP was not able to stimulate a mutant COX-2 promoter construct that is not activated by NF- B (0.9 ± 0.1, 1.2 ± 0.1, and 1.0 ± 0.2 respectively, n = 4, NS). Low chloride increased COX-2 expression (2.7 ± 0.4-fold control, n = 6, P < 0.01) and pp38 expression (2.8 ± 0.3-fold; n = 5, P < 0.01), which were reversed by the specific NO synthase (NOS) inhibitor 7-nitroindazole. Administration of a low-salt diet increased immunoreactive COX-2 and neuronal NOS (nNOS) in the macula densa and surrounding cTAL of kidneys of wild-type mice but did not significantly elevate COX-2 expression in nNOS -/- mice. In summary, these studies indicate that, in cTAL, NO can increase COX-2 expression in cTAL and macula densa through p38-dependent signaling pathways via activation of NF- B.
' Z" m: E0 r3 o' x3 \ 【关键词】 nitric oxide cylooxygenase nuclear factor B p transcriptional regulation$ ~3 L; g* M6 i7 {) i' e" l/ ~
NITRIC OXIDE (NO) exhibits a broad spectrum of biological effects, including modulation of vascular tone, neurotransmission, hormone release, inflammation, and cell growth. In the kidney, NO is an important regulator of renal plasma flow, glomerular filtration rate, the renin-angiotensin system, and sodium excretion ( 4, 19 ). The three isoforms of nitric oxide synthase (NOS) have distinct localization patterns in the mammalian kidney. Type I NOS, the brain or neural isoform (nNOS), is highly expressed in the macula densa ( 3 ) and is thought to contribute to regulation of tubuloglomerular feedback and activation of renin production and release by the juxtaglomerular cells ( 41, 45 ). These effects may involve interactions between NO and other vasoconstrictors, such as ANG II and thromboxane ( 16, 37 ) and oxygen radicals and cyclooxygenase (COX)-2 ( 21 )./ |2 ^+ T' q P6 x) s
) @" e8 o1 @- m/ ^& U% {, ZIn the kidney cortex, COX-2 is also localized to macula densa and adjacent cortical thick ascending limb (cTAL) ( 12, 20 ). Previous studies have shown that in vivo COX-2 expression in cTAL and macula densa increased in response to low salt ( 7, 20, 38, 48 ), and, in cultured cTAL and macula densa cells, COX-2 expression increased in response to decreased extracellular chloride ( 9, 47 ). Previous studies by our group indicated that administration of NO donors or cell-permeable cGMP analogs also stimulated COX-2 expression in cultured cTAL cells, and nNOS inhibitors decreased COX-2 expression in rats on a low-salt diet ( 11 ). The present studies examined whether common signal pathways are involved and whether these stimuli are necessarily integrated in regulating cortical COX-2 expression.
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! `( ?" j. y1 u" q5 y r' gMATERIALS AND METHODS3 Q' G5 a5 v/ P5 {4 V: _
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Materials. The selective COX-2 inhibitor SC-58236, which exhibits a COX-2/COX-1 selectivity of 1,780-fold ( 32 ), was a gift from Searle Monsanto (St. Louis, MO). Rabbit anti-COX-2 antibody and PGE 2, 6-keto-PGF 1, and thromboxane B 2 (TxB 2 ) EIA kits were from Cayman Chemical, rabbit anti-nNOS antibody was from Zymed Laboratories (San Francisco, CA), and monoclonal anti-pp38 and goat anti- -actin antibody were from Santa Cruz Biotechnology (Santa Cruz, CA). The enhanced chemiluminescence (ECL) kit and ECL Hyperfilm were from Amersham (Arlington Heights, IL). The BCA protein assay reagent kit, immunopure ABC peroxidase staining kit, and biotin-labeled mouse anti-rabbit or rabbit anti-goat IgG (H L) antibodies were from Pierce (Rockford, IL). 7-Nitroindazole (7-Ni) was from Calbiochem (La Jolla, CA). Other reagents were purchased from Sigma Chemical (St. Louis, MO).
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' O% N. k- t6 U$ d% mPrimary culture of rabbit cTAL cells. cTAL cells were isolated from homogenates of rabbit renal cortex by immunodissection with anti-Tamm Horsfall antibody, as previously described ( 1, 11, 13 ). Briefly, the renal cortex was dissected, minced, and digested with 0.1% collagenase. After being blocked with 10% BSA, 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 DMEM-F-12 with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in 95% air-5% CO 2.
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' E# @0 q. c9 Y- M; S$ n' }Transfection and luciferase reporter assay. Primary cultured cTALH cells at 50-60% confluence were transiently transfected with lipofectamine reagent (GIBCO-BRL) according to instructions. Briefly, after changing to serum-free culture medium, a DNA-lipofectamine mixture (3 µg DNA and 10 µl lipofectamine reagent in 150 µl DMEM-F-12) was added to the culture medium and allowed to react with cells at 37°C, 5% CO 2. A pCMV- -gal plasmid (1 µg) encoding -galactosidase regulated by the CMV promoter was cotransfected with the pGAL constructs as a control for transfection efficiency. After transfection with lipofectamine for 6-8 h, DMEM-F-12 containing 20% FBS was added back to the cell culture. The mouse COX-2 -815 luciferase reporter construct was a generous gift from Dr. K. Kamamoto ( 46 ), and we have previously reported that it mediates similar activity as the full-length promoter ( 9 ). As previously described ( 9 ), the nuclear factor (NF)- B mutant represents a point mutation from GA to CC at the NF- B binding site at -410 in the COX-2 -815 luciferase reporter construct. When 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 for 6 h. The cells were extracted with lysis buffer (Luciferase Assay System; Promega), and their luciferase activity was measured with a LumiCount Microplate Luminometer (model: AL10000; Packard Bioscience, Meridian, CT). The results were normalized to -galactosidase activity as previously described ( 2 ).6 t. w6 o5 N* T, a6 m! F# m; f6 c
+ O. t; y7 n7 t$ N5 }& FAnimal model and genotype. Mice with genetic deletion of the COX-2 gene maintained on a mixed B6/129 background were originally generated by Dinchuk et al. ( 17 ), and heterozygous breeding pairs were obtained from Jackson Laboratories (Bar Harbor, ME). Mice were genotyped by PCR and confirmed by Southern hybridization with a specific internal probe, as described previously ( 14 ). nNOS knockout mice on B6/129 background were purchased from Jackson Laboratories.
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Age-matched male adult (8 wk) COX-2 -/-, nNOS -/-, and wild-type mice were maintained on either control or sodium-deficient (0.02-0.03% Na ) diet (ICN, Irvine, CA) for 21 days before death.+ n% q* H: n/ s6 _+ |
; Z# G7 H( ^& ^) P. m& dImmunoblotting. For COX-2, renal cortexes were homogenized in 30 mM Tris·HCl, pH 8.0, and 100 µM phenylmethylsulfonyl fluoride (PMSF; 1:9 wt/vol). After a 10-min centrifugation at 10,000 g, the supernatant was centrifuged for 60 min at 110,000 g to prepare microsomes, as described previously ( 10 ). Proteins were resuspended in SDS-sample buffer. For detection of other proteins, renal cortexes 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 polyclonal rabbit COX-2 antiserum or other indicated antibodies. The second biotinylated antibody reagent was detected using avidin and biotinylated horseradish peroxidase (Pierce) and exposed on film using ECL (Amersham). The membrane was rehybridized with goat anti- -actin antibody (Santa Cruz) to normalize protein loading.- b# d: V5 h. ?8 w
3 V _+ ^9 ?9 u4 t: L2 a& }, s' hPGE 2, 6-keto-PGF 1, and TxB 2 measurement. cTAL cells were cultured under conditions indicated in the text; 10 -5 M arachidonic acid was added to the medium and incubated at 37°C for 1 h before the supernatant was removed. PGE 2, 6-keto-PGF 1, and TxB 2 were measured by EIA according to the manufacturer's instructions.( i* O4 R8 W4 |8 G6 @) v
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NO generation. After quiescence, the cTAL cells were incubated in low or normal chloride medium for 16 h. NO production was measured by the concentration of nitrite, a stable NO metabolite, using Griess reagent (Nitrate/Nitrite Colorimetric Assay Kit; Cayman Chemical), as previously described( 25 ).! v# b C; N a* N) d. v
/ l7 X. D; r# @. gNuclear extracts and electrophoretic mobility shift assay. Nuclear protein was extracted as previously described ( 49 ). Briefly, cells were homogenized with a Dounce homogenizer in buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT), 0.5 mM PMSF, and 0.1% leupeptin. Nonidet P-40 (10%) was added to make a final concentration of 0.5%, incubated on ice for 30 min, and centrifuged. After being washed, the pellet was resuspended in 20 mM HEPES (pH 7.9), 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 10% glycerol, and 0.1% leupeptin with 8 X/ W8 k- g3 l9 q; f
) |, |' s0 F8 M1 p( V# F+ x& gImmunohistochemistry. Under deep anesthesia with Nembutal (70 mg/kg ip), mice were exsanguinated with 50 ml/100 g heparinized saline (0.9% NaCl, 2 U/ml heparin, 0.02% sodium nitrite) through a transcardial aortic cannula and fixed with glutaraldehyde-periodate acid saline (GPAS) for COX-2 staining as previously described. GPAS contains final concentrations of 2.5% glutaraldehyde, 0.011 M sodium metaperiodate, 0.04 M sodium phosphate, 1% acetic acid, and 0.1 M NaCl and provides excellent preservation of tissue structure and antigenicity. Antigen was retrieved in 0.01 M citrate buffer, pH 6.0, by microwave for 2 min.1 E* F9 L( ~: t* }3 ]
8 ^$ i) ~+ c* q; O; Y3 PCOX-2 immunoreactivity was localized with COX-2 antiserum diluted to 2.5 ng/ml; nNOS immunoreactivity was with anti-nNOS antibody (5 µg/ml). The first antibody was localized using Vectastain ABC-Elite (Vector, Burlingame, CA) with diaminobenzidine as the chromogen, followed by a light counterstain with toluidine blue. The fixed kidneys were dehydrated through a graded series of ethanols, embedded in paraffin, sectioned at 4 µm thickness, and mounted on glass slides./ K% s# i+ S/ |9 r+ I
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Statistical analysis. All values are presented as means ± SE. ANOVA and Bonferroni t -tests were used for statistical analysis, and differences were considered significant when P ; C5 i: }) P3 S. n5 n; F9 {
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The NO donor, S-nitros-N-acetylpenicillamine, stimulated COX-2 expression and prostaglandin production in cTAL cells. Cultured cTAL cells that were incubated with S -nitros- N -acetylpenicillamine (SNAP, 10 -4 M) for 16 h increased expression of immunoreactive COX-2 (2.5 ± 0.3-fold control, n = 6, P & U$ X7 q+ i j6 j# u0 @" a7 }
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Fig. 1. A : the nitric oxide (NO) donor S -nitros- N -acetylpenicillamine (SNAP) increased immunoreactive (ir)-cyclooxygenase (COX)-2 expression in cortical thick ascending limb (cTAL) cells that was inhibited by a p38 inhibitor, SB-203580. After quiescence, primary cultured cTAL cells were incubated with normal medium (as control) or 10 -4 M SNAP or coincubated with 10 -4 M SNAP and 10 -5 M SB-203580 for 16 h. ir-COX-2 was detected by immunoblotting and expressed as a degree of control. -Actin was used as a protein-loading control ( n = 6 experiments). ** P
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Fig. 2. SNAP increased PGE 2 and 6-keto-PGF 1 production but did not change thromboxane B 2 (TxB 2 ) in cTAL cells. Cultured cTAL cells were treated as in Fig. 1 and then preincubated with 10 -5 M arachidonic acid at 37°C for 1 h before the supernatant was removed. PGE2 ( A ), 6-keto-PGF 1 ( B ), and TxB 2 ( C ) were measured by EIA ( n = 4 experiments). * P
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5 Q& e( O( ?/ W! |2 h5 sThe p38 mitogen-activated protein kinase pathway regulated NO-mediated increases in COX-2 expression. To explore the effect of p38 in signaling of COX-2 by NO, a specific p38 inhibitor, SB-203580 (10 -5 M), was added to the medium of cTAL cells during coincubation with SNAP. Increases in COX-2 expression were significantly blunted (from 2.5 ± 0.3- to 1.4 ± 0.2-fold control, n = 6, NS vs. control; Fig. 1 ). The increase in PGE 2 production was also prevented by SB-203580 (to 37.4 ± 13.5 ng/mg protein, n = 4, NS vs. control; Fig. 2, A and B ). Expression of the active form of p38, phosphorylated p38 (pp38), was increased in cTAL cells by SNAP (2.4 ± 0.3-fold control; n = 5; P 5 d7 G" s2 m9 i& A
- P4 H, R' N* @% s% }6 ]+ O9 N& DFig. 3. SNAP stimulated COX-2 transcriptional regulation mediated by p38 via nuclear factor (NF)- B. A : NF- B mutation prevented the stimulation of SNAP on COX-2 promoter. Cultured cTAL cells were transiently transfected with vector (as control), a luciferase reporter construct driven by the wild-type COX-2 promoter, or the correspondent NF- B mutant COX-2 promoter construct. Transfection, SNAP stimulation, and luciferase activity measurement were described in MATERIALS AND METHODS ( n = 4 experiments). ** P
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NO-induced transcriptional regulation of COX-2 was mediated by p38-dependent NF- B activation. Our previous studies indicated that decreased extracellular chloride induced COX-2 upregulation in cTAL at the transcriptional level via NF- B ( 9 ). To investigate if the regulation of COX-2 by NO was also mediated by NF- B, cultured cTAL cells were transiently transfected with a luciferase reporter construct driven by the proximal 815 bp of wild-type murine COX-2 promoter, which we had previously determined to mediate similar activity as the full-length promoter ( 9 ). Compared with vector-transfected cells, basal COX-2 promoter-mediated luciferase activity was not elevated significantly; in contrast, SNAP significantly stimulated luciferase activity, which was reversed by SB-203580 (1.4 ± 0.2-, 8.3 ± 1.4-, and 0.4 ± 0.1-fold control, respectively, n = 4, P + U L7 M' g. {( m* m7 w j3 ?
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Fig. 4. The neuronal nitric oxide synthase (nNOS) inhibitor 7-nitroindazole (7-Ni) inhibited upregulation of COX-2 ( A ) and pp38 ( B ) expression in cultured cTAL by decreased extracellular chloride concentration ( n = 6 experiments). * P 9 l6 ^0 t& }7 }- `) C) c
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Fig. 5. COX-2 upregulation induced by low salt was attenuated in nNOS -/-- mice ( A and B ) but nNOS expression increased in both wild-type and COX-2 -/- mice with salt depletion ( C and D ). Wild-type, nNOS -/-, and COX-2 -/- mice were fed a low-salt diet for 3 wk, and renal cortex-COX-2 and nNOS expression was detected with immunohistochemistry ( A and C ) and immunoblotting ( B and D ). Increased ir-COX-2 expression in macula densa/cTAL from wild-type mice on a low-salt diet is indicated by arrows ( A ) but was not seen in nNOS -/- kidney ( A and B; n = 6 experiments). KO, knockout. * P $ N1 [, z. ]+ [2 _7 C% P
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nNOS inhibition diminished the upregulation of COX-2 by low extracellular chloride in cTAL cells. Previously, we have demonstrated that low-chloride-induced increases in COX-2 expression from cultured cTAL cells ( 12 ). In cultured cTAL cells, basal NO was found to be 3.2 ± 0.5 µM, whereas in cells exposed to the low-chloride medium for 16 h, there was a significant stimulation of NO production (7.8 ± 0.2 µM, n = 4, P 1 d6 O$ `9 N6 N( x( `0 M1 j
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Increases in renal COX-2 by low salt were attenuated in nNOS -/- mice. We further investigated the role of nNOS in macula densa/cTAL COX-2 expression in vivo. Administration of a low-salt diet for 3 wk increased immunoreactive COX-2 in the kidneys of wild-type mice (3.3 ± 0.5-fold control, n = 6, P
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DISCUSSION
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* Z$ S% v! a; K* a$ }, B- ~" DOur results indicated that the NO donor SNAP stimulates COX-2 expression and activity in primary cultured cTAL cells through a p38 MAP kinase-dependent pathway, mediated by NF- B-dependent transcriptional regulation. In addition, the activation of p38 and upregulation of COX-2 induced by decreased extracellular chloride ( 9 ) were also significantly inhibited by NOS inhibition. In vivo studies demonstrated that increased renal cortical COX-2 seen in wild-type mice was blunted in nNOS null mice, further supporting a role for NO in COX-2 regulation in macula densa and cTAL.! F) F. a! o1 d, @7 \+ u5 c
1 h/ a/ ^1 h5 V. ^3 J. Y) W# i6 [Because of the colocalization of nNOS and COX-2 in renal cortex and the parallel increase of renin, COX-2, and nNOS gene expression in response to salt restriction, a number of previous studies have investigated their potential interactions ( 27, 31, 34, 39, 42 ). In cultured cTAL cells and macula densa cells, the same stimulus for macula densa mediation of increased renin secretion, namely low extracellular chloride, also stimulated COX-2 expression ( 12, 47 ). The induced nNOS was predominantly shown in macula densa by immunostain ( Fig. 5 ), although the immunoreactive nNOS was detectable ( 11 ) and could be stimulated by low-chloride medium in cultured cTAL cells. Recently, Paliege et al. ( 31 ) reported that absence of either COX-2 or nNOS is associated with suppressed renin secretion, and COX-2-derived PGE 2 inhibited nNOS expression.
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Consistent with our previous studies ( 11 ), the current studies indicated that, in cultured cTAL, the NO donor SNAP stimulated COX-2 expression and production of vasodilatory PGs, and the nNOS specific inhibitor 7-Ni blocked increased COX-2 expression by low extracellular chloride, suggesting that NO may modulate COX-2 expression in these cells. This result was further confirmed by in vivo studies with nNOS knockout mice. However, previous immunohistochemical studies in nNOS knockout mice by Theilig et al. ( 40 ) did not confirm a requirement for active nNOS to detect stimulation of COX-2 expression by short- and long-term unilateral renal artery stenosis, low salt, or furosemide. Although we do not know the underlying reasons for the discrepancy, we note that mice were subjected to a longer period of salt restriction in our studies than in the previously reported studies of Theilig et al., although 1 or 2 wk of salt restriction have shown the effect to induce COX-2 expression. Furthermore, immunohistochemical studies are very helpful for the localization of antigen expression but may have certain limitations for quantitation, especially when the number of immunoreactive-positive cells is the only indicator without additional information concerning the relative level of expression (density) within a positive cell. In our studies, immunoreactive COX-2 was measured by quantitative immunoblot analysis in addition to immunohistochemical analysis. With these complementary approaches, we failed to detect significant upregulation of cortical COX-2 expression by salt restriction in nNOS null mice.
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There are multiple potential mechanisms by which NO may mediate activity and expression of COX-2-derived metabolites. It has been suggested that peroxynitrite produced by the reaction of NO with superoxide can initiate lipid peroxidation, thereby releasing arachidonic acid from the cell membrane and potentially increasing COX activity ( 15, 24 ). Additionally, NO may directly react with, and thereby remove, free radicals that can inactivate COX-2 ( 18, 36 ) or bind to the heme-Fe 2 group of COX-2, thus directly activating the enzyme ( 35, 43 ).
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$ u( h" s" o: I# G I2 UPrevious studies have indicated parallel regulation of COX-2 and nNOS proteins in response to administration of diets of varying salt content ( 29, 42 ). Our previous studies demonstrated that decreased extracellular salt/chloride upregulated COX-2 expression in cTAL cells through a p38 MAP kinase pathway via NF- B activation, which was correlated with increased inhibitory factor B phosphorylation ( 9 ). The current results suggested that, in cultured cTAL, NO stimulates COX-2 expression by a similar signaling mechanism. NO has previously been shown in other cell types to activate p38 ( 26, 33 ), and, in the present studies, we determined that the NO donor SNAP could activate p38.% C6 e% G( }; Z% x5 g9 [9 {" S
) E" W) Q2 s- A- H% BThe mechanisms by which NO or low intracellular chloride activates p38 were not elucidated in the current studies. Because cell-permeable cGMP analogs stimulate COX-2 expression in cultured cTAL ( 11 ), one possible mechanism by which NO might increase COX-2 expression is by decreasing intracellular chloride concentrations by cGMP-dependent inhibition of the Na -K -Cl - cotransporter ( 29, 30 ). NO may activate p38 MAP kinase ( 6 ) and increase NF- B (p50/p65) DNA binding activity ( 22 ) in a cGMP-dependent fashion. Alternatively, low chloride could directly increase NO production, which then directly activates p38. There was a partial but significant inhibition of low-chloride-induced p38 activation and COX-2 expression in response to nNOS-specific NOS inhibitors. Further studies will be required to determine the mechanisms that underlie this effect.3 v8 H& M( \" ^4 L' f% x2 ]4 s* I
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Both NO and PGs are modulators of vascular tone, cell growth, inflammation, hormone release, renal hemodynamics, renin production, and tubuloglomerular feedback, and previous studies have suggested that prostanoids derived from COX-2 may serve as mediators or modulators of at least some of the biological actions of nNOS ( 5, 23, 28, 44 ). Our in vivo and in vitro studies indicating that inhibition of nNOS significantly inhibits increases in cTAL/macula densa COX-2 expression in response to low salt implicate macula densa-derived NO as a modulator of COX-2 expression in these cells.
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GRANTS
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-3961, U01 DK-61018 (Animal Models of Diabetic Complications Consortium), U24 DK-59637, and DK-62794, by funds from the Veterans Administration, and a grant from the Satellite Health Care System.3 m$ ?; `! u) |
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