- Email: [email protected]
iNOS signaling interacts with COX-2 pathway in colonic fibroblasts
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Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/yexcr
Research Article
iNOS signaling interacts with COX-2 pathway in colonic fibroblasts Yingting Zhua,b,n,1, Min Zhua,1, Peter Lancea a
Arizona Cancer Center, the University of Arizona, Tucson, AZ 85724, USA Tissue Tech Inc, Miami, FL 33173, USA
b
article information
abstract
Article Chronology:
COX-2 and iNOS are two major inflammatory mediators implicated in colorectal inflammation
Received 11 April 2012
and cancer. Previously, the role of colorectal fibroblasts involved in regulation of COX-2 and
Received in revised form
iNOS expression was largely ignored. In addition, the combined interaction of COX-2 and iNOS
25 May 2012
signalings and their significance in the progression of colorectal inflammation and cancer within
Accepted 28 May 2012
the fibroblasts have received little investigation. To address those issues, we investigated the
Available online 5 June 2012
role of colonic fibroblasts in the regulation of COX-2 and iNOS gene expression, and explored
Keywords:
possible mechanisms of interaction between COX-2 and iNOS signalings using a colonic CCD-
Cyclooxygenase-2
18Co fibroblast line and LPS, a potential stimulator of COX-2 and iNOS. Our results clearly
Prostaglandin E2
demonstrated that LPS activated COX-2 gene expression and enhanced PGE2 production,
Inducible nitric oxide synthase
stimulated iNOS gene expression and promoted NO production in the fibroblasts. Interestingly,
Nitric oxide
activation of COX-2 signaling by LPS was not involved in activation of iNOS signaling, while
Lypopolysaccharide
activation of iNOS signaling by LPS contributed in part to activation of COX-2 signaling. Further
Protein kianse C
analysis indicated that PKC plays a major role in the activation and interaction of COX-2 and iNOS signalings induced by LPS in the fibroblasts. & 2012 Elsevier Inc. All rights reserved.
Introduction Prostaglandins are small lipid messengers and have a wide variety of biological activities. They participate in pain and inflammation responses, and may be partially responsible for the occurrence of colorectal cancer [1]. The main form of prostaglandins is prostaglandin E2 (PGE2). A rate limiting step in the synthesis of prostaglandins is the enzyme cyclooxygenase
(COX). Two COX isoforms, COX-1 and COX-2, have been identified. COX-1 is constitutively expressed in all tissues and is involved in many physiological functions [2], while COX-2 is a rate-limiting enzyme for generation of prostaglandins from arachidonic acid and is associated with inflammation and tumorigenesis [3–7]. Increased levels of COX-2 mRNA and protein have been found in the great majority of colorectal cancer compared with levels in adjacent histologically normal mucosa [8]. COX-2
Abbreviations: LPS, Lypopolysaccharide; COX, Cyclooxygenase; PGE2, Prostaglandin E2; iNOS, Inducible nitric oxide; NO, Nitric oxide; PKC, Protein kinase C; NS 398, N-[2-(Cyclohexyloxy)-4-nitrophenyl]methanesulfonamide; 1400 W, N-[3-(Aminomethyl)benzyl]acetamidine; STA, Staurosporine; BIM, Bisindoylmalemide I n
Corresponding author at: University of Arizona, Tissue Tech Inc., 7000 SW 97th Avenue Suite 212, Miami, Florida 33173, United States. Fax: þ305 274 1297. E-mail address: [email protected] (Y. Zhu). 1 The first two authors contributed equally to this work. 0014-4827/$ - see front matter & 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yexcr.2012.05.027
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over-expression with carcinogenic potential has been reported at the cellular level [9,10]. However, the origin of COX-2 and its product, PGE2, is still not well defined. Traditionally, the causation and pathogenesis of colorectal cancers are attributed largely to genetic alterations within the epithelium. However, some evidences indicate that factors originated from the non-epithelial compartment also play a critical role in tumorigenesis [11–14]. Underlying the epithelium, the stroma is the compartment that serves as the connective tissue framework, and includes large numbers of fibroblasts. A study revealed that fibroblasts have a more profound influence on the development and progression of carcinomas than was appreciated [15]. Previously, We have shown that inflammatory factors such as IL1-b, TNF-a and deoxycholic acid stimulate a sustained COX-2 expression and PGE2 production in CCD-18Co and primary colonic fibroblasts [16–18]. Since lipopolysaccharide (LPS) is a major component of the outer membrane of Gramnegative bacteria and a prototypical endotoxin that exists in the colon environment, we wondered whether inflammatory LPS could also upregulate the expression of COX-2 at mRNA and protein levels and promote the production of PGE2 in the colonic fibroblasts. We were also curious whether LPS might affect the production of COX-2 and PGE2 related factors such as inducible nitric oxide synthase (iNOS), its product nitric oxide (NO), and whether COX-2 pathway would interact with iNOS signaling. NO is a small signal molecule that directly regulates the activity of intracellular proteins promoting inflammation, tumor angiogenesis and invasiveness in human colorectal cancer cells [19,20]. NO is produced by the deamination of arginine, catalyzed by NO synthases. Two NO synthases, constitutive NOS (cNOS) and iNOS, have been reported. Production of NO from cNOS, such as neural and endothelial NOS, is a key Ca2þdependent regulator of homeostasis, whereas the generation of NO by iNOS plays an important role in Ca2þ-independent hostdefense response [21]. Although COX-2 is a potential target for NO because it contains an iron-heme center at their active site [22–25], little attention was paid to a potential interaction between the two signaling pathways in the fibroblasts. The aim of this study is to investigate (1) whether LPS activates COX-2 and iNOS signalings, (2) whether COX-2 and iNOS signalings interact, and (3) what the possible mechanisms of interaction are in the colonic CCD-18Co fibroblasts.
Materials and methods Materials CCD-18Co, a colonic fibroblast strain derived from normal human fetal colon, was purchased from the American Type Culture Collection (Manassas, VA, USA). Other fibroblast cultures were initiated from colonoscopic biopsy, obtained with approval of the Institutional Review Boards of the Buffalo Veterans Administration Medical Center and the Albany Medical College [16–18]. Tissue culture reagents were purchased from Sigma (St. Louis, MO, USA). Salmonella minnesota LPS, N-[2-(Cyclohexyloxy)-4nitrophenyl]methanesulfonamide (NS 398) and N-(3-(Aminomethyl)benzyl)acetamidine (1400 W) were purchased from Sigma. Bisindoylmalemide I (BIM) was obtained from Calbiochem (La Jolla, CA, USA). RNeasy Mini Kit was purchased from
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Qiagen (Valencia, CA, USA). COX-2, iNOS siRNAs, control scRNA and HiPerFects siRNA transfection reagents were obtained from Qiagen (Valencia, CA, USA).
Fibroblast culture CCD-18Co or normal human colonic fibroblasts were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% FBS, nonessential amino acids (0.1 mM) and sodium pyruvate (1 mM). To determine the effects of LPS, selected cultures were incubated in medium supplemented with LPS.
siRNA transfection For the short pulse siRNA knockdown, parallel fibroblast cultures were subjected to 100 nM scRNA or siRNA transfection following our protocol [26]. The tranfected cultures were incubated at 37 1C for 24 h before termination for further analysis.
Real-time PCR analysis of iNOS and COX-2 mRNAs Total RNAs were extracted using RNeasy Mini Kit (Qiagen) and were reverse-transcribed using High Capacity Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). cDNA of each cell sample was amplified by real-time RT-PCR using specific primer-probe mixtures and DNA polymerase in 7000 Real-time PCR System (Applied Biosystems). Real-time RT-PCR profile consisted of 10 min of initial activation at 95 1C, followed by 40 cycles of 15 s denaturation at 95 1C, and 1 min annealing and extension at 60 1C. The genuine identity of each PCR product was confirmed by the size determination using 2% agarose gels followed by ethidium bromide staining together with PCR marker in EC3 Imaging System (BioImaging System, Upland, CA, USA). Each experiment was repeated 4 times.
Western blotting analysis of iNOS and COX-2 proteins iNOS and COX-2 protein levels were determined as described [16] with modifications. Confluent cultured fibroblasts were treated with test compounds. Lysates were electrophoresed on polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS-PAGE) and electroblotted to an Immobilon-P membrane (Millipore Corporation, Bedford, MA, USA). The membrane was incubated for 4 h in PBS containing 10% non-fat milk and 0.1% Tween 20 at pH 7.4. This was followed by a 2 h incubation at room temperature with rabbit anti-human iNOS or COX-2 antibody (Cayman, Ann Arbor, MI, USA) diluted 1:1000 in PBS containing 10% non-fat milk and 0.1% Tween 20 at pH 7.4. The membrane was washed three times in PBS containing 0.1% Tween 20 at pH 7.4, then incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma, St. Louis, MO, USA) for detection of iNOS or COX-2 protein. The membrane was washed four times in PBS containing 0.1% Tween 20 at pH 7.4. Bound antibodies were detected by chemiluminescence using reagents purchased from KPL (Gaithersburg, MD, USA). The resulting bands were analyzed densitometrically with J imaging and the loading difference was corrected by analyzing density of b-actin.
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PGE2 Assay Fibroblasts were seeded in 24-well plastic culture plates at a density of 5 105 cells/cm2 and grown to confluence in DMEM supplemented with 10% FBS. It is well recognized that serum is a potent inducer of COX-2 and, thus, PGE2 [16]. Therefore, 24 h before administering the treatments to fibroblast cultures, medium was replaced with DMEM supplemented with 1% FBS. To determine the effects of LPS, selected cultures were incubated in medium supplemented with LPS. For the final 30 min before harvesting of cultures, medium was replaced with PBS supplemented with 1% gelatin. Test compounds were present during this final incubation period. PGE2 levels in harvested PBS-gelatin were determined using Radioimmunoassay Kit (Amersham, Arlington Heights, IL, USA) according to the manufacturer’s instructions. Protein content in the harvest was determined by the method of Bradford.
Nitric oxide assay Supernatants were harvested after the period of testing from LPS control and cultures supplemented with LPS, (1 mg/ml). Nitrite concentrations ([NO]) in cell-free supernatants were
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measured spectrophotometrically using Nitric Oxide Assay Kit, Colorimetric (Calbiochem, La Jolla, CA, USA).
iNOS and COX-2 inhibitor assays 1400 W (10 mM), a selective inhibitor of iNOS [27] and NS 398 (10 mM), a selective inhibitor of COX-2 activity [28], were combined with LPS in selected fibroblast cultures.
PKC and PKC inhibitor assay Involvement of protein kinase C (PKC) signaling was addressed by measurement of PKC activity. CCD-18Co cells were seeded in 60 mm dishes at a density of 5 105/well and cultured until 70% confluence. Selected cultures were then treated with LPS for periods of 1 to 10 min. Treated and control cultures were washed with PBS and homogenized in buffer containing 50 mM Tris/HCl at pH 7.5, 0.3% b-mercaptoethanol, 50 mg/ml phenylmethylsulphonyl fluoride, and 10 mM benzamidine. The homogenate was centrifuged at 100,000 g. The pellet was resuspended in the same buffer supplemented with 0.2% Triton X-100 and mixed on ice for 60 min before re-centrifugation at 100,000 g. Solubilized particulate fractions were assayed for PKC activity using the
Fig. 1 – LPS Stimulated PGE2 Synthesis in Normal Human Colonic and CCD-18Co Fibroblasts. Cells were cultured without (control) or in the presence of LPS (1 lg/ml) for the time indicated. PGE2 levels in harvested medium were determined by radioimmunoassay (RIA). Bars represent the mean of results from 4 separated cultures7SE. po0.05, po0.01when compared to the control.
Fig. 2 – LPS Elevated NO Synthesis in Normal Human Colonic and CCD-18Co Fibroblasts. Cells were cultured without (control) or in the presence of LPS (1 lg/ml) for the time indicated. NO levels in harvested medium were determined by Nitric Oxide Assay Kit. Bars represent the mean of results from 4 separated cultures7SE. po0.05, po0.001when compared to the control.
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Fig. 3 – LPS-stimulated PGEs Synthesis was through Inducing COX-2 gene expression in the fibroblasts. (A) LPS Induced COX-2 mRNA expression. Cells were incubated for the times indicated with LPS (1 lg/ml). Total RNA isolated was reverse transcribed and analyzed by real-time PCR with corresponding primers and probes. (B) LPS Upregulated COX-2 protein synthesis. Lysates from the cells incubated from times indicated with LPS (1 lg/ml) were electrophoresed and transferred to membranes, which were then incubated with the polyclonal antibody for COX-2. Bound antibody was detected by chemiluminescence. The results were normalized by b-actin. Bars represent the mean of results from 4 separated cultures7SE. po0.05, po0.01when compared to the control.
Fig. 4 – LPS-stimulated NO Synthesis was through promoting iNOS Gene Expression in the Fibroblasts. A. LPS induced iNOS mRNA Expression. Cells were incubated for the time indicated with LPS (1 lg/ml). Total RNA isolated was reverse transcribed and analyzed by realtime PCR with corresponding primers and probes. B. LPS Promoted iNOS Protein Synthesis. Lysates from the cells incubated from times indicated with LPS (1 lg/ml) were electrophoresed and transferred to membranes, which were then incubated with polyclonal antibodies to iNOS. Bound antibodies were detected by chemiluminescence. The results were normalized by b-actin. Bars represent the mean of results from 4 separated cultures7SE. po0.05, po0.01 when compared to the control.
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BiotrakTM PKC Assay System (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The system is based upon PKC-catalyzed transfer of [g-32P]ATP to a PKC-specific peptide and activity is expressed as pmol/min/mg protein (n ¼4). In further experiments, a nonselective protein kinase inhibitor, staurosporine (STA, 20 mM, Sigma), or a selective PKC inhibitor, bisindoylmalemide I (BIM, 10 mM, Calbiochem), was combined with LPS. Their inhibitory effects on PGE2 and NO synthesis were examined as described above.
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Statistics All summary data were reported as means7S.E. calculated for each group and compared using Analysis of Variance test and Student’s unpaired t-test by MicroSoft ExcelTM (MicroSoft, Redmont, WA, USA). Test results were reported as two-tailed p values, where po0.05 was considered statistically significant.
Results Cell viability Cell viability was determined by trypan blue exclusion [18]. Cells were seeded at the same time from a single parent culture. Cultures were incubated without (control) or with LPS (1 mg/ml). Treated and control cultures were harvested at the same time, after careful washing to remove cells that had detached during incubation. Harvested cells were incubated with trypan blue and counted using a hemacytometer. From each culture, four fields of triplicate preparations were counted for the percentage of cells that excluded the dye. Cell viability was 495% after all treatment periods (data not shown).
LPS upregulated PGE2 production in both normal human colonic and CCD18Co fibroblasts The effective dosages of LPS to induce PGE2 and NO expression were reported in a wide range, from 10 ng to10 mg/ml. [29–31] The optimized dosage of LPS for PGE2 and NO induction in normal human colonic and CCD18Co cells was determined at 1 mg/ml. We measured PGE2 levels in both normal human colonic and CCD18Co fibroblasts after exposure to LPS. The mean basal PGE2 level in normal human colonic fibroblasts from 4 separate experiments was 3 ng/mg protein. An increase in PGE2 level was first evident 12 h after exposure to LPS and rose 8-fold to a
Fig. 5 – COX-2 inhibitor NS398 or COX-2 siRNA attenuated LPS-stimulated PGE2 synthesis, but not NO Production in the fibroblasts. (A) NS398 completely blocked LPS-stimulated PGE2 synthesis in the fibroblasts. CCD-18Co cells were incubated with LPS (1 lg/ml) and NS398 (10 lM) for 24 h. PGE2 levels in harvested medium were determined by RIA. Bars represent the mean results from 4 experiments7SE. (B) COX-2 siRNA blocked LPS-stimulated PGE2 production. Lyophilized duplex siRNAs corresponding to the COX-2 sequence and control (scrambled) RNA were used for transfection. After the fibroblast cultures were incubated in the transfection medium mixture for 24 h, fresh medium (control) or fresh medium supplemented with LPS (1 lg/ ml) was added to each well for a further 24 h. PGE2 analysis was performed as we described. Bars represent the mean results from 4 experiments7SE. (C) NS398 did not affect LPS-stimulated NO synthesis. CCD-18Co cells were incubated with LPS (1 lg/ml) and NS398 (10 lM) for 72 h. NO concentrations in harvested medium were determined by nitric oxide assay kit. Bars represent the mean results from 4 experiments7SE. D. COX-2 siRNA did not Alter LPS-stimulated NO production. Lyophilized duplex siRNAs corresponding to the COX-2 sequence and control (scrambled) RNA were used for transfection. After the fibroblast cultures were incubated in the transfection medium mixture for 24 h, fresh medium (control) or fresh medium supplemented with LPS (1 lg/ml) was added to each well for a further 24 h. NO analysis was performed as described above. Bars represent the mean of results from 4 separated cultures7SE. po0.01, po0.001 when compared to the control.
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maximal level of 24 ng/mg protein at 24 h and remained high through 48 h (Fig. 1, npo0.05, nnnpo0.001 when compared to the control). In contrast, the mean basal PGE2 level in CCD-18CO fibroblasts from 4 separate experiments was also 3 ng/mg protein. An increase in PGE2 level was also evident 12 h after exposure to LPS and rose 7-fold at 24 h (22 ng/mg protein) and remained elevated through 48 h (Fig. 1, npo0.05, nnpo0.01 when compared to the control). The results indicated that LPS is a stimulant for the production of PGE2 with similar intensity in both normal human colonic and CCD-18Co fibroblasts, indicating that CCD-18Co fibroblasts is a good cell model for evaluating PGE2 and its related signaling.
LPS induced NO synthesis in both normal human colonic and CCD18Co fibroblasts In the present study, the effect of LPS on NO synthesis was conducted using both normal human colonic and CCD-18Co fibroblasts. Our results indicate that LPS increased NO production in both normal human colonic and CCD18Co fibroblasts. The NO production in normal human colonic fibroblasts increased to 63, 167, 643 nmoles/107 cells after 6, 12, 24 h of LPS stimulation, respectively and remained high after 48 h (Fig. 2, npo0.05, nnn po0.001 when compared to the control). In contrast, the NO production in CCD18Co fibroblasts increased to 37, 161, 596 nmoles/107 cells after 6, 12, 24 h of LPS stimulation
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respectively and remained high after 48 h (Fig. 2, npo0.05, po0.001 when compared to the control), indicating that LPS stimulated similar levels of NO production in the normal human colonic and CCD-18Co fibroblasts. Thus we used CCD-18Co fibroblasts to conduct further mechanistic studies since it was difficult to grow primary human colonic fibroblasts in large numbers ex vivo. nnn
LPS induced PGE2 production was through upregulating COX-2 mRNA and protein expression in CCD-18Co fibroblasts Real-time PCR and Western analysis were performed to analyze basal expression and upregulation of COX-2 mRNA and protein in response to LPS. Expression of COX-2 mRNA, barely detectable in cellular RNA from CCD-18Co fibroblasts, increased 3-fold by 6 h and 5-fold by 12 h and 7-fold by 24 h (Fig. 3A, npo0.05, npo0.01 when compared to the LPS control). In contrast, COX-1 mRNA level was unaffected by LPS treatment (data not shown). Correspondingly, COX-2 protein was low in CCD-18Co cells and upregulated 2-fold by 6 h, 3-fold by 12 h and 6-fold by 24 h respectively after LPS treatment (Fig. 3B, npo0.05, nnnpo0.01 when compared to the control). COX-1 protein levels were also unaffected by LPS treatment (data not shown). These data demonstrated that LPS-triggered PGE2 production was largely through induction of COX-2 mRNA and protein expression.
Fig. 6 – Inhibition of PGE2 production by COX-2 inhibitor NS398 or COX-2 siRNA was through down regulating COX-2 mRNA and protein expression in the fibroblasts. (A) NS398 blocked LPS-stimulated COX-2 mRNA expression. CCD-18Co cells were incubated with LPS (1 lg/ml) and NS398 (10 lM) for 24 h. COX-2 mRNA levels were determined by RT-realtime PCR. Bars represent the mean results from 4 experiments7SE. (B) NS398 blocked LPS-stimulated COX-2 protein expression. CCD-18Co cells were incubated with LPS (1 lg/ml) and NS398 (10 lM) for 24 h. COX-2 protein levels were determined by Western blotting. Bars represent the mean results from 4 experiments7SE. (C) COX-2 siRNA Attenuated LPS-stimulated COX-2 mRNA expression. CCD18Co cells were incubated with LPS (1 lg/ml) and COX-2 siRNA (100 nM) for 24 h. COX-2 mRNA levels were determined by RTrealtime PCR. Bars represent the mean results from 4 experiments7SE. (D) COX-2 siRNA attenuated LPS-stimulated COX-2 protein expression. CCD-18Co cells were incubated with LPS (1 lg/ml) and COX-2 siRNA (100 nM) for 24 h. COX-2 protein levels were determined by Western blotting. The results were normalized by b-actin. Bars represent the mean of results from 4 separated cultures7SE. po0.01 when compared to the control.
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LPS-stimulated NO production was through promoting iNOS mRNA and protein expression in CCD18Co fibroblasts Real-time PCR and Western blotting analysis were used to characterize basal iNOS mRNA and protein levels and their up-regulation in response to LPS. Expression of iNOS mRNA, barely detectable in cellular RNA from CCD-18Co fibroblasts, increased 3-, 4-, and 6fold at 6, 12 and 24 h and remained high at 48 h (Fig. 4A, npo0.05, nn po0.01 when compared to the LPS control). Western blotting analysis demonstrated that iNOS protein expression was almost undetectable in untreated CCD-18Co cells, but increased 2-, 3-, and 5-fold after 6, 12 and 24 h of LPS treatment but only 2-fold at 48 h (Fig. 4B, npo0.05, nnpo0.01 when compared to the control). The results clearly suggest that LPS-stimulated NO production was through induction of iNOS gene expression.
COX-2 inhibitor NS-398 or COX-2 siRNA attenuated PGE2, but not NO production induced by LPS NS-398 is a selective COX-2 inhibitor. Treatment with NS-398 (10 mM for 24 h) caused complete attenuation of the PGE2
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response to LPS in CCD-18Co fibroblasts (Fig. 5A, p40.05 when compared to the LPS control). The above result was confirmed by knockdown of COX-2 by COX-2 siRNA (Fig. 5B, p40.05 when compared to the LPS control). However, NS398 or COX-2 siRNA had no effect on NO synthesis (Fig. 5C and D, p40.05 when compared to the LPSþ control). These data demonstrated that LPS-stimulated PGE2 production is COX-2 specific and LPS enhanced NO formation is not COX-2 dependent.
Inhibition of PGE2 production by NS398 or COX-2 siRNA was through Downregulating COX-2 mRNA and protein expression in the fibroblasts NS398 or COX-2 siRNA caused complete attenuation of COX-2 mRNA (Fig. 6A and C, p40.05 when compared to the LPS control) and COX-2 protein expression (Fig. 6B and D, p40.05 when compared to the LPS control), indicating an inhibition of PGE2 production by NS398 or COX-2 siRNA was through inhibition of COX-2 mRNA and Protein Expression in the CCD18Co fibroblasts.
Fig. 7 – iNOS inhibitor 1400 W or iNOS siRNA reduced LPS-stimulated PGE2 synthesis, but blocked NO production in the fibroblasts. (A) 1400 W reduced LPS-stimulated PGE2 synthesis in the fibroblasts. CCD-18Co cells were incubated with LPS (1 lg/ ml) and 1400 W (10 lM) for 24 h. PGE2 levels in harvested medium were measured by RIA. Bars represent the mean results from 4 experiments7SE. (B) 1400 W completely blocked LPS-stimulated NO synthesis in the fibroblasts. CCD-18Co cells were coincubated with LPS (1 lg/ml) and 1400 W (10 lM) for 24 h. NO concentrations in harvested medium were determined by nitric oxide assay kit. Bars represent the mean results from 4 experiments7SE. (C) iNOS siRNA reduced LPS-stimulated PGE2 production in the fibroblasts. Lyophilized duplex siRNAs corresponding to the iNOS sequence and control (scrambled) RNA were used for transfection. The PGE2 experimental procedures were described above. Bars represent the mean results from 4 experiments7SE. (D) iNOS siRNA blocked LPS-stimulated NO production in the fibroblasts. Lyophilized duplex siRNAs corresponding to the iNOS sequence and control (scrambled) RNA were used for transfection. The NO experimental procedures were described above. Bars represent the mean of results from 4 separated cultures7SE. po0.05, po0.01 when compared to the control. #po0.05 when compared to LPSþ control.
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iNOS inhibitor 1400 W or iNOS siRNA not only attenuated NO production, but also significantly decreased PGE2 production stimulated by LPS Treatment with iNOS inhibitor 1400 W or iNOS siRNA caused complete attenuation of the iNOS and NO response to LPS in CCD-18Co fibroblasts (Fig. 7B and D, p40.05 when compared to that of LPS control), but reduced PGE2 production by approximately 55% and 50%, respectively (Fig. 7A and C, #po0.05 when compared to the LPSþ control). The results suggest that LPSstimulated NO production is fully iNOS signaling-dependent and LPS-stimulated PGE2 production is partially iNOS signalingdependent.
The partial inhibition of PGE2 production by 1400 W or iNOS siRNA was through inhibition of COX-2 mRNA and protein expression in CCD18Co fibroblasts 1400 W or iNOS siRNA caused partial attenuation of COX-2 mRNA (Fig. 8A and C, #po0.05 when compared to the LPSþ control) and COX-2 protein expression (Fig. 8B and D, #po0.05 when compared to the LPSþ control), indicating that an inhibition of PGE2 production by 1400 W or iNOS siRNA was partially through inhibition of COX-2 mRNA and Protein Expression in the CCD18Co fibroblasts.
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The inhibition of NO production by 1400 W or iNOS siRNA was through inhibition of iNOS mRNA and protein expression in CCD18Co fibroblasts 1400 W or iNOS siRNA caused complete attenuation of iNOS mRNA (Fig. 9A and C, p40.05 when compared to the LPS control) and iNOS protein expression (Fig. 9B and D, p40.05 when compared to the LPS control), indicating an inhibition of NO production by 1400 W or iNOS siRNA was through inhibition of iNOS mRNA and Protein Expression in the CCD18Co fibroblasts.
LPS-stimulated PGE2 and NO synthesis was mediated by protein kinase C (PKC), not Ca2þ signaling The PKC superfamily participates in a wide array of cellular responses. Activation of PKC by LPS peaked at 3 min (Fig. 10, n po0.05, nnpo0.01 when compared to the control) and appeared Ca2þ-independent since no intracellular calcium activation was recorded in CCD18-Co cells treated with LPS (data not shown). Immediate addition of protein kinase inhibitor, STA or PKC inhibitor BIM (20 mM and 10 mM respectively) to the cultures treated with LPS (1 mg/ml) for 24 h completely blocked LPSstimulated synthesis of PGE2 and NO (Fig. 11, p40.05 when
Fig. 8 – Decrease of PGE2 production by 1400 W or iNOS siRNA was partially Through downregulating COX-2 Gene Expression in the Fibroblasts. (A) 1400 W reduced LPS-stimulated COX-2 mRNA expression. CCD-18Co cells were incubated with LPS (1 lg/ml) and 1400 W (10 lM) for 24 h. iNOS mRNA levels were determined by RT-realtime PCR. Bars represent the mean results from 4 experiments7SE. (B) 1400 W downregulated LPS-stimulated COX-2 protein expression. CCD-18Co cells were incubated with LPS (1 lg/ml) and 1400 W (10 lM) for 24 h. COX-2 protein levels were determined by Western blotting. Bars represent the mean results from 4 experiments7SE. (C) iNOS siRNA decreased LPS-stimulated COX-2 mRNA expression. CCD-18Co cells were incubated with LPS (1 lg/ml) and iNOS siRNA (100 nM) for 24 h. COX-2 mRNA levels were determined by RT-realtime PCR. Bars represent the mean results from 4 experiments7SE. (D) iNOS siRNA downregulated LPS-stimulated COX-2 mRNA expression. CCD-18Co cells were incubated with LPS (1 lg/ml) and iNOS siRNA (100 nM) for 24 h. COX-2 protein levels were determined by Western blotting. The results were normalized by b-actin. Bars represent the mean of results from 4 separated cultures7SE. n po0.05, nnpo0.01 when compared to the control. #po0.05 when compared to the LPSþ control.
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Fig. 9 – Inhibition of NO production by 1400 W or iNOS siRNA was through downregulating iNOS gene expression in the fibroblasts. (A) 1400 W blocked LPS-stimulated iNOS mRNA expression. CCD-18Co cells were incubated with LPS (1 lg/ml) and 1400 W (10 lM) for 24 h. iNOS mRNA levels were determined by RT-realtime PCR. Bars represent the mean results from 4 experiments7SE. (B) 1400 W blocked LPS-stimulated iNOS protein expression. CCD-18Co cells were incubated with LPS (1 lg/ml) and 1400 W (10 lM) for 24 h. COX-2 protein levels were determined by Western blotting. Bars represent the mean results from 4 experiments7SE. (C) iNOS siRNA attenuated LPS-stimulated iNOS mRNA expression. CCD-18Co cells were incubated with LPS (1 lg/ml) and iNOS siRNA (100 nM) for 24 h. iNOS mRNA levels were determined by RT-realtime PCR. Bars represent the mean results from 4 experiments7SE. (D) iNOS siRNA Attenuated LPS-stimulated iNOS protein expression. CCD-18Co cells were incubated with LPS (1 lg/ml) and iNOS siRNA (100 nM) for 24 h. iNOS protein levels were determined by Western blotting. The results were normalized by b-actin. Bars represent the mean of results from 4 separated cultures7SE. po0.01 when compared to the LPS control.
Discussion
Fig. 10 – LPS activated PKC activity in the fibroblasts. CCD18Co cells were exposed to LPS (1 lg/ml) for periods of 1–10 min. Cell homogenates were pelleted, and particulate fractions were resolubilized. PKC activity of these samples was determined from transfer of [32]ATP to a PKC-specific peptide and were expressed as pmol/min/mg protein. Bars represent the mean of results from 4 separated cultures7SE. po0.05, po0.01 when compared to the control.
compared to the LPS control). The results suggest that LPSstimulated COX-2 and iNOS gene expression is PKC-dependent and Ca2þ-independent.
In this article, we first tested whether CCD-18CO cells resemble primary human colonic fibroblasts producing PGE2 and NO. To our surprise, CCD18Co cells produced similar levels of PGE2 and NO when compared to those of primary human colonic fibroblasts during the entire experimental period (Figs. 1 and 2). LPS stimulated PGE2 and NO production peaked at 24 h and remained high up to 48 h in both primary human colonic and CCD18Co fibroblasts (Figs. 1 and 2), indicating that CCD18Co fibroblasts are a good model for our experimental purpose. To test whether this PGE2 induction induced by LPS is caused by increased gene expression of COX-2, we performed RT-realtime PCR and Western blotting. Indeed LPS induced timedependent COX-2 mRNA and corresponding protein expression in the cells (Fig. 3), similar to our previous reports that IL1b, TNFa and DCA induced COX-2 expression [16–18]. The results clearly indicated that LPS stimulated PGE2 production was through induction of COX-2 mRNA and protein expression. NO produced by iNOS was reported to enhance the activity of COX-2 [32]. These effects may involve ‘‘cross-talk’’ between the two enzyme systems and be responsible for enhanced growth in some tumors [33]. However, it is still largely unknown whether colonic fibroblasts are main sources of COX-2 and iNOS, and if so, whether those fibroblasts are involved in the cross-talk between COX-2 and iNOS pathways, and whether the cross-talk plays a
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Fig. 11 – Protein kinase inhibitors blocked activation of PKC and synthesis of PGE2 and NO. CCD-18Co cells were coincubated with LPS (1 lg/ml) and staurosporine (STA, 20 lM) or bisindoylmalemide (BIM, 10 lM) for 24 h. PGE2 levels in harvested medium were determined by RIA and NO levels in harvested medium were determined by nitric oxide assay kit. Bars represent the mean of results from 4 separated cultures7SE. po0.01, po0.001 when compared to the LPS control.
significant role in inflammation and progression of colonic cancer. As the results indicated, LPS was found not only to induce a time-dependent NO production (peaked at 24 h, Fig. 2), but also a time-dependent iNOS mRNA and protein expression in the fibroblasts (peaked at 24 h, Fig. 4) in CCD18Co fibroblasts. We suggest that the fibroblasts play a significant role in regulating the production of nitric oxide. Cross-talk of COX-2 and NO signalings between tumor spheroids and normal cells has recently been investigated [34]. However, whether cross-talk between COX-2 and NO signalings within the fibroblasts occurs is not known. To investigate whether the cross-talk between COX-2 and iNOS pathways in CCD18Co cells occurs after LPS treatment, we used specific inhibitors of COX-2 and iNOS, NS 398 and 1400 W, and knockdown of COX-2 and iNOS by COX-2 siRNA and iNOS siRNA respectively. Our results demonstrated that NS398 or COX-2 siRNA completely blocked PGE2 induction, but had no effect on NO production induced by LPS, indicating that the products of COX-2 pathway or COX-2 itself did not affect iNOS signaling since a specific COX-2 inhibitor, NS398 or knockdown of COX-2 by COX-2 siRNA did not inhibit NO production (Fig. 5). Further real-time PCR and Western analysis indicated that an inhibition of PGE2 production by NS398 or COX-2 siRNA was through
attenuating COX-2 mRNA and protein expression (Fig. 6). In contrast, activation of NO pathway was contributed to increased COX-2 gene expression and PGE2 production, since specific iNOS inhibitors, 1400 W or knockdown of iNOS by iNOS siRNA reduced PGE2 induction by 55% and 50%, respectively (Fig. 7). The inhibition was due to inhibition of COX-2 gene expression (Fig. 8). In addition, 1400 W or iNOS siRNA completely abolished the effect on NO production induced by LPS (Fig. 7). Such an inhibition is due to inhibition of iNOS gene expression (Fig. 9). We speculate that products generated by activation of iNOS signaling by LPS or activation of iNOS itself were involved in regulation of production of proinflammatory PGE2, which might contribute to inflammatory response and progression of colorectal cancer [19,35]. Up to now, how NO signaling regulates COX-2 expression is not well understood. Two major mechanisms proposed were that NO influences its intracellular targets are stimulation of guanylyl cyclase by direct binding of NO to iron in heme at the active site of guanylyl cyclase [36], or S-nitrosylation of protein targets on appropriate cysteines [37,38]. Because COX-2 has heme at its active site [39] and contains 13 cysteines [40], it was proposed that iNOS binds S-nitrosylates and activates COX-2 [25]. Whether this is the case in our cell model system requires further
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investigation. Interestingly, the interaction of COX-2 and iNOS signalings did not affect the cell viability during the entire study, probably because the dosage of LPS to the cells was well within physiological tolerance. Furthermore, we suggest that protein kinase C, not Ca2þ, was involved in LPS-induced activation of COX-2 and iNOS signalings in CCD-18Co fibroblasts. LPS treatment to the fibroblasts increased cellular PKC activity within 3 min after administration (Fig. 10). The selective PKC inhibitors, STA and BIM, attenuated the PKC activation and induction of PGE2 and NO in the CCD18Co cells (Fig. 11). We speculate that protein Kinase C, not Caþ, plays a major role in mediating COX-2 and iNOS activation in response to LPS, similar to the action mechanisms elicited by deoxycholic acid we previously reported [16–18]. To conclude, colonic fibroblasts are major sources of PGE2 and iNOS. Products from iNOS signaling is involved in cross-talk with COX-2 pathway in human colonic fibroblasts. The schematic diagram showing components and inhibitors has been summarized as follows:
We speculate that drugs targeting COX-2 and iNOS signalings might be useful for the control of colonic inflammation and colorectal cancer.
Acknowledgments This work was supported in part by Arizona Cancer Center (NIH Institutional Research Grant # 7400128) and a small research grant from the University of Arizona. No potential conflict of interest was noted.
references
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Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/yexcr
Research Article
iNOS signaling interacts with COX-2 pathway in colonic fibroblasts Yingting Zhua,b,n,1, Min Zhua,1, Peter Lancea a
Arizona Cancer Center, the University of Arizona, Tucson, AZ 85724, USA Tissue Tech Inc, Miami, FL 33173, USA
b
article information
abstract
Article Chronology:
COX-2 and iNOS are two major inflammatory mediators implicated in colorectal inflammation
Received 11 April 2012
and cancer. Previously, the role of colorectal fibroblasts involved in regulation of COX-2 and
Received in revised form
iNOS expression was largely ignored. In addition, the combined interaction of COX-2 and iNOS
25 May 2012
signalings and their significance in the progression of colorectal inflammation and cancer within
Accepted 28 May 2012
the fibroblasts have received little investigation. To address those issues, we investigated the
Available online 5 June 2012
role of colonic fibroblasts in the regulation of COX-2 and iNOS gene expression, and explored
Keywords:
possible mechanisms of interaction between COX-2 and iNOS signalings using a colonic CCD-
Cyclooxygenase-2
18Co fibroblast line and LPS, a potential stimulator of COX-2 and iNOS. Our results clearly
Prostaglandin E2
demonstrated that LPS activated COX-2 gene expression and enhanced PGE2 production,
Inducible nitric oxide synthase
stimulated iNOS gene expression and promoted NO production in the fibroblasts. Interestingly,
Nitric oxide
activation of COX-2 signaling by LPS was not involved in activation of iNOS signaling, while
Lypopolysaccharide
activation of iNOS signaling by LPS contributed in part to activation of COX-2 signaling. Further
Protein kianse C
analysis indicated that PKC plays a major role in the activation and interaction of COX-2 and iNOS signalings induced by LPS in the fibroblasts. & 2012 Elsevier Inc. All rights reserved.
Introduction Prostaglandins are small lipid messengers and have a wide variety of biological activities. They participate in pain and inflammation responses, and may be partially responsible for the occurrence of colorectal cancer [1]. The main form of prostaglandins is prostaglandin E2 (PGE2). A rate limiting step in the synthesis of prostaglandins is the enzyme cyclooxygenase
(COX). Two COX isoforms, COX-1 and COX-2, have been identified. COX-1 is constitutively expressed in all tissues and is involved in many physiological functions [2], while COX-2 is a rate-limiting enzyme for generation of prostaglandins from arachidonic acid and is associated with inflammation and tumorigenesis [3–7]. Increased levels of COX-2 mRNA and protein have been found in the great majority of colorectal cancer compared with levels in adjacent histologically normal mucosa [8]. COX-2
Abbreviations: LPS, Lypopolysaccharide; COX, Cyclooxygenase; PGE2, Prostaglandin E2; iNOS, Inducible nitric oxide; NO, Nitric oxide; PKC, Protein kinase C; NS 398, N-[2-(Cyclohexyloxy)-4-nitrophenyl]methanesulfonamide; 1400 W, N-[3-(Aminomethyl)benzyl]acetamidine; STA, Staurosporine; BIM, Bisindoylmalemide I n
Corresponding author at: University of Arizona, Tissue Tech Inc., 7000 SW 97th Avenue Suite 212, Miami, Florida 33173, United States. Fax: þ305 274 1297. E-mail address: [email protected] (Y. Zhu). 1 The first two authors contributed equally to this work. 0014-4827/$ - see front matter & 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yexcr.2012.05.027
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over-expression with carcinogenic potential has been reported at the cellular level [9,10]. However, the origin of COX-2 and its product, PGE2, is still not well defined. Traditionally, the causation and pathogenesis of colorectal cancers are attributed largely to genetic alterations within the epithelium. However, some evidences indicate that factors originated from the non-epithelial compartment also play a critical role in tumorigenesis [11–14]. Underlying the epithelium, the stroma is the compartment that serves as the connective tissue framework, and includes large numbers of fibroblasts. A study revealed that fibroblasts have a more profound influence on the development and progression of carcinomas than was appreciated [15]. Previously, We have shown that inflammatory factors such as IL1-b, TNF-a and deoxycholic acid stimulate a sustained COX-2 expression and PGE2 production in CCD-18Co and primary colonic fibroblasts [16–18]. Since lipopolysaccharide (LPS) is a major component of the outer membrane of Gramnegative bacteria and a prototypical endotoxin that exists in the colon environment, we wondered whether inflammatory LPS could also upregulate the expression of COX-2 at mRNA and protein levels and promote the production of PGE2 in the colonic fibroblasts. We were also curious whether LPS might affect the production of COX-2 and PGE2 related factors such as inducible nitric oxide synthase (iNOS), its product nitric oxide (NO), and whether COX-2 pathway would interact with iNOS signaling. NO is a small signal molecule that directly regulates the activity of intracellular proteins promoting inflammation, tumor angiogenesis and invasiveness in human colorectal cancer cells [19,20]. NO is produced by the deamination of arginine, catalyzed by NO synthases. Two NO synthases, constitutive NOS (cNOS) and iNOS, have been reported. Production of NO from cNOS, such as neural and endothelial NOS, is a key Ca2þdependent regulator of homeostasis, whereas the generation of NO by iNOS plays an important role in Ca2þ-independent hostdefense response [21]. Although COX-2 is a potential target for NO because it contains an iron-heme center at their active site [22–25], little attention was paid to a potential interaction between the two signaling pathways in the fibroblasts. The aim of this study is to investigate (1) whether LPS activates COX-2 and iNOS signalings, (2) whether COX-2 and iNOS signalings interact, and (3) what the possible mechanisms of interaction are in the colonic CCD-18Co fibroblasts.
Materials and methods Materials CCD-18Co, a colonic fibroblast strain derived from normal human fetal colon, was purchased from the American Type Culture Collection (Manassas, VA, USA). Other fibroblast cultures were initiated from colonoscopic biopsy, obtained with approval of the Institutional Review Boards of the Buffalo Veterans Administration Medical Center and the Albany Medical College [16–18]. Tissue culture reagents were purchased from Sigma (St. Louis, MO, USA). Salmonella minnesota LPS, N-[2-(Cyclohexyloxy)-4nitrophenyl]methanesulfonamide (NS 398) and N-(3-(Aminomethyl)benzyl)acetamidine (1400 W) were purchased from Sigma. Bisindoylmalemide I (BIM) was obtained from Calbiochem (La Jolla, CA, USA). RNeasy Mini Kit was purchased from
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Qiagen (Valencia, CA, USA). COX-2, iNOS siRNAs, control scRNA and HiPerFects siRNA transfection reagents were obtained from Qiagen (Valencia, CA, USA).
Fibroblast culture CCD-18Co or normal human colonic fibroblasts were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% FBS, nonessential amino acids (0.1 mM) and sodium pyruvate (1 mM). To determine the effects of LPS, selected cultures were incubated in medium supplemented with LPS.
siRNA transfection For the short pulse siRNA knockdown, parallel fibroblast cultures were subjected to 100 nM scRNA or siRNA transfection following our protocol [26]. The tranfected cultures were incubated at 37 1C for 24 h before termination for further analysis.
Real-time PCR analysis of iNOS and COX-2 mRNAs Total RNAs were extracted using RNeasy Mini Kit (Qiagen) and were reverse-transcribed using High Capacity Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). cDNA of each cell sample was amplified by real-time RT-PCR using specific primer-probe mixtures and DNA polymerase in 7000 Real-time PCR System (Applied Biosystems). Real-time RT-PCR profile consisted of 10 min of initial activation at 95 1C, followed by 40 cycles of 15 s denaturation at 95 1C, and 1 min annealing and extension at 60 1C. The genuine identity of each PCR product was confirmed by the size determination using 2% agarose gels followed by ethidium bromide staining together with PCR marker in EC3 Imaging System (BioImaging System, Upland, CA, USA). Each experiment was repeated 4 times.
Western blotting analysis of iNOS and COX-2 proteins iNOS and COX-2 protein levels were determined as described [16] with modifications. Confluent cultured fibroblasts were treated with test compounds. Lysates were electrophoresed on polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS-PAGE) and electroblotted to an Immobilon-P membrane (Millipore Corporation, Bedford, MA, USA). The membrane was incubated for 4 h in PBS containing 10% non-fat milk and 0.1% Tween 20 at pH 7.4. This was followed by a 2 h incubation at room temperature with rabbit anti-human iNOS or COX-2 antibody (Cayman, Ann Arbor, MI, USA) diluted 1:1000 in PBS containing 10% non-fat milk and 0.1% Tween 20 at pH 7.4. The membrane was washed three times in PBS containing 0.1% Tween 20 at pH 7.4, then incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma, St. Louis, MO, USA) for detection of iNOS or COX-2 protein. The membrane was washed four times in PBS containing 0.1% Tween 20 at pH 7.4. Bound antibodies were detected by chemiluminescence using reagents purchased from KPL (Gaithersburg, MD, USA). The resulting bands were analyzed densitometrically with J imaging and the loading difference was corrected by analyzing density of b-actin.
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PGE2 Assay Fibroblasts were seeded in 24-well plastic culture plates at a density of 5 105 cells/cm2 and grown to confluence in DMEM supplemented with 10% FBS. It is well recognized that serum is a potent inducer of COX-2 and, thus, PGE2 [16]. Therefore, 24 h before administering the treatments to fibroblast cultures, medium was replaced with DMEM supplemented with 1% FBS. To determine the effects of LPS, selected cultures were incubated in medium supplemented with LPS. For the final 30 min before harvesting of cultures, medium was replaced with PBS supplemented with 1% gelatin. Test compounds were present during this final incubation period. PGE2 levels in harvested PBS-gelatin were determined using Radioimmunoassay Kit (Amersham, Arlington Heights, IL, USA) according to the manufacturer’s instructions. Protein content in the harvest was determined by the method of Bradford.
Nitric oxide assay Supernatants were harvested after the period of testing from LPS control and cultures supplemented with LPS, (1 mg/ml). Nitrite concentrations ([NO]) in cell-free supernatants were
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measured spectrophotometrically using Nitric Oxide Assay Kit, Colorimetric (Calbiochem, La Jolla, CA, USA).
iNOS and COX-2 inhibitor assays 1400 W (10 mM), a selective inhibitor of iNOS [27] and NS 398 (10 mM), a selective inhibitor of COX-2 activity [28], were combined with LPS in selected fibroblast cultures.
PKC and PKC inhibitor assay Involvement of protein kinase C (PKC) signaling was addressed by measurement of PKC activity. CCD-18Co cells were seeded in 60 mm dishes at a density of 5 105/well and cultured until 70% confluence. Selected cultures were then treated with LPS for periods of 1 to 10 min. Treated and control cultures were washed with PBS and homogenized in buffer containing 50 mM Tris/HCl at pH 7.5, 0.3% b-mercaptoethanol, 50 mg/ml phenylmethylsulphonyl fluoride, and 10 mM benzamidine. The homogenate was centrifuged at 100,000 g. The pellet was resuspended in the same buffer supplemented with 0.2% Triton X-100 and mixed on ice for 60 min before re-centrifugation at 100,000 g. Solubilized particulate fractions were assayed for PKC activity using the
Fig. 1 – LPS Stimulated PGE2 Synthesis in Normal Human Colonic and CCD-18Co Fibroblasts. Cells were cultured without (control) or in the presence of LPS (1 lg/ml) for the time indicated. PGE2 levels in harvested medium were determined by radioimmunoassay (RIA). Bars represent the mean of results from 4 separated cultures7SE. po0.05, po0.01when compared to the control.
Fig. 2 – LPS Elevated NO Synthesis in Normal Human Colonic and CCD-18Co Fibroblasts. Cells were cultured without (control) or in the presence of LPS (1 lg/ml) for the time indicated. NO levels in harvested medium were determined by Nitric Oxide Assay Kit. Bars represent the mean of results from 4 separated cultures7SE. po0.05, po0.001when compared to the control.
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Fig. 3 – LPS-stimulated PGEs Synthesis was through Inducing COX-2 gene expression in the fibroblasts. (A) LPS Induced COX-2 mRNA expression. Cells were incubated for the times indicated with LPS (1 lg/ml). Total RNA isolated was reverse transcribed and analyzed by real-time PCR with corresponding primers and probes. (B) LPS Upregulated COX-2 protein synthesis. Lysates from the cells incubated from times indicated with LPS (1 lg/ml) were electrophoresed and transferred to membranes, which were then incubated with the polyclonal antibody for COX-2. Bound antibody was detected by chemiluminescence. The results were normalized by b-actin. Bars represent the mean of results from 4 separated cultures7SE. po0.05, po0.01when compared to the control.
Fig. 4 – LPS-stimulated NO Synthesis was through promoting iNOS Gene Expression in the Fibroblasts. A. LPS induced iNOS mRNA Expression. Cells were incubated for the time indicated with LPS (1 lg/ml). Total RNA isolated was reverse transcribed and analyzed by realtime PCR with corresponding primers and probes. B. LPS Promoted iNOS Protein Synthesis. Lysates from the cells incubated from times indicated with LPS (1 lg/ml) were electrophoresed and transferred to membranes, which were then incubated with polyclonal antibodies to iNOS. Bound antibodies were detected by chemiluminescence. The results were normalized by b-actin. Bars represent the mean of results from 4 separated cultures7SE. po0.05, po0.01 when compared to the control.
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BiotrakTM PKC Assay System (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The system is based upon PKC-catalyzed transfer of [g-32P]ATP to a PKC-specific peptide and activity is expressed as pmol/min/mg protein (n ¼4). In further experiments, a nonselective protein kinase inhibitor, staurosporine (STA, 20 mM, Sigma), or a selective PKC inhibitor, bisindoylmalemide I (BIM, 10 mM, Calbiochem), was combined with LPS. Their inhibitory effects on PGE2 and NO synthesis were examined as described above.
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Statistics All summary data were reported as means7S.E. calculated for each group and compared using Analysis of Variance test and Student’s unpaired t-test by MicroSoft ExcelTM (MicroSoft, Redmont, WA, USA). Test results were reported as two-tailed p values, where po0.05 was considered statistically significant.
Results Cell viability Cell viability was determined by trypan blue exclusion [18]. Cells were seeded at the same time from a single parent culture. Cultures were incubated without (control) or with LPS (1 mg/ml). Treated and control cultures were harvested at the same time, after careful washing to remove cells that had detached during incubation. Harvested cells were incubated with trypan blue and counted using a hemacytometer. From each culture, four fields of triplicate preparations were counted for the percentage of cells that excluded the dye. Cell viability was 495% after all treatment periods (data not shown).
LPS upregulated PGE2 production in both normal human colonic and CCD18Co fibroblasts The effective dosages of LPS to induce PGE2 and NO expression were reported in a wide range, from 10 ng to10 mg/ml. [29–31] The optimized dosage of LPS for PGE2 and NO induction in normal human colonic and CCD18Co cells was determined at 1 mg/ml. We measured PGE2 levels in both normal human colonic and CCD18Co fibroblasts after exposure to LPS. The mean basal PGE2 level in normal human colonic fibroblasts from 4 separate experiments was 3 ng/mg protein. An increase in PGE2 level was first evident 12 h after exposure to LPS and rose 8-fold to a
Fig. 5 – COX-2 inhibitor NS398 or COX-2 siRNA attenuated LPS-stimulated PGE2 synthesis, but not NO Production in the fibroblasts. (A) NS398 completely blocked LPS-stimulated PGE2 synthesis in the fibroblasts. CCD-18Co cells were incubated with LPS (1 lg/ml) and NS398 (10 lM) for 24 h. PGE2 levels in harvested medium were determined by RIA. Bars represent the mean results from 4 experiments7SE. (B) COX-2 siRNA blocked LPS-stimulated PGE2 production. Lyophilized duplex siRNAs corresponding to the COX-2 sequence and control (scrambled) RNA were used for transfection. After the fibroblast cultures were incubated in the transfection medium mixture for 24 h, fresh medium (control) or fresh medium supplemented with LPS (1 lg/ ml) was added to each well for a further 24 h. PGE2 analysis was performed as we described. Bars represent the mean results from 4 experiments7SE. (C) NS398 did not affect LPS-stimulated NO synthesis. CCD-18Co cells were incubated with LPS (1 lg/ml) and NS398 (10 lM) for 72 h. NO concentrations in harvested medium were determined by nitric oxide assay kit. Bars represent the mean results from 4 experiments7SE. D. COX-2 siRNA did not Alter LPS-stimulated NO production. Lyophilized duplex siRNAs corresponding to the COX-2 sequence and control (scrambled) RNA were used for transfection. After the fibroblast cultures were incubated in the transfection medium mixture for 24 h, fresh medium (control) or fresh medium supplemented with LPS (1 lg/ml) was added to each well for a further 24 h. NO analysis was performed as described above. Bars represent the mean of results from 4 separated cultures7SE. po0.01, po0.001 when compared to the control.
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maximal level of 24 ng/mg protein at 24 h and remained high through 48 h (Fig. 1, npo0.05, nnnpo0.001 when compared to the control). In contrast, the mean basal PGE2 level in CCD-18CO fibroblasts from 4 separate experiments was also 3 ng/mg protein. An increase in PGE2 level was also evident 12 h after exposure to LPS and rose 7-fold at 24 h (22 ng/mg protein) and remained elevated through 48 h (Fig. 1, npo0.05, nnpo0.01 when compared to the control). The results indicated that LPS is a stimulant for the production of PGE2 with similar intensity in both normal human colonic and CCD-18Co fibroblasts, indicating that CCD-18Co fibroblasts is a good cell model for evaluating PGE2 and its related signaling.
LPS induced NO synthesis in both normal human colonic and CCD18Co fibroblasts In the present study, the effect of LPS on NO synthesis was conducted using both normal human colonic and CCD-18Co fibroblasts. Our results indicate that LPS increased NO production in both normal human colonic and CCD18Co fibroblasts. The NO production in normal human colonic fibroblasts increased to 63, 167, 643 nmoles/107 cells after 6, 12, 24 h of LPS stimulation, respectively and remained high after 48 h (Fig. 2, npo0.05, nnn po0.001 when compared to the control). In contrast, the NO production in CCD18Co fibroblasts increased to 37, 161, 596 nmoles/107 cells after 6, 12, 24 h of LPS stimulation
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respectively and remained high after 48 h (Fig. 2, npo0.05, po0.001 when compared to the control), indicating that LPS stimulated similar levels of NO production in the normal human colonic and CCD-18Co fibroblasts. Thus we used CCD-18Co fibroblasts to conduct further mechanistic studies since it was difficult to grow primary human colonic fibroblasts in large numbers ex vivo. nnn
LPS induced PGE2 production was through upregulating COX-2 mRNA and protein expression in CCD-18Co fibroblasts Real-time PCR and Western analysis were performed to analyze basal expression and upregulation of COX-2 mRNA and protein in response to LPS. Expression of COX-2 mRNA, barely detectable in cellular RNA from CCD-18Co fibroblasts, increased 3-fold by 6 h and 5-fold by 12 h and 7-fold by 24 h (Fig. 3A, npo0.05, npo0.01 when compared to the LPS control). In contrast, COX-1 mRNA level was unaffected by LPS treatment (data not shown). Correspondingly, COX-2 protein was low in CCD-18Co cells and upregulated 2-fold by 6 h, 3-fold by 12 h and 6-fold by 24 h respectively after LPS treatment (Fig. 3B, npo0.05, nnnpo0.01 when compared to the control). COX-1 protein levels were also unaffected by LPS treatment (data not shown). These data demonstrated that LPS-triggered PGE2 production was largely through induction of COX-2 mRNA and protein expression.
Fig. 6 – Inhibition of PGE2 production by COX-2 inhibitor NS398 or COX-2 siRNA was through down regulating COX-2 mRNA and protein expression in the fibroblasts. (A) NS398 blocked LPS-stimulated COX-2 mRNA expression. CCD-18Co cells were incubated with LPS (1 lg/ml) and NS398 (10 lM) for 24 h. COX-2 mRNA levels were determined by RT-realtime PCR. Bars represent the mean results from 4 experiments7SE. (B) NS398 blocked LPS-stimulated COX-2 protein expression. CCD-18Co cells were incubated with LPS (1 lg/ml) and NS398 (10 lM) for 24 h. COX-2 protein levels were determined by Western blotting. Bars represent the mean results from 4 experiments7SE. (C) COX-2 siRNA Attenuated LPS-stimulated COX-2 mRNA expression. CCD18Co cells were incubated with LPS (1 lg/ml) and COX-2 siRNA (100 nM) for 24 h. COX-2 mRNA levels were determined by RTrealtime PCR. Bars represent the mean results from 4 experiments7SE. (D) COX-2 siRNA attenuated LPS-stimulated COX-2 protein expression. CCD-18Co cells were incubated with LPS (1 lg/ml) and COX-2 siRNA (100 nM) for 24 h. COX-2 protein levels were determined by Western blotting. The results were normalized by b-actin. Bars represent the mean of results from 4 separated cultures7SE. po0.01 when compared to the control.
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LPS-stimulated NO production was through promoting iNOS mRNA and protein expression in CCD18Co fibroblasts Real-time PCR and Western blotting analysis were used to characterize basal iNOS mRNA and protein levels and their up-regulation in response to LPS. Expression of iNOS mRNA, barely detectable in cellular RNA from CCD-18Co fibroblasts, increased 3-, 4-, and 6fold at 6, 12 and 24 h and remained high at 48 h (Fig. 4A, npo0.05, nn po0.01 when compared to the LPS control). Western blotting analysis demonstrated that iNOS protein expression was almost undetectable in untreated CCD-18Co cells, but increased 2-, 3-, and 5-fold after 6, 12 and 24 h of LPS treatment but only 2-fold at 48 h (Fig. 4B, npo0.05, nnpo0.01 when compared to the control). The results clearly suggest that LPS-stimulated NO production was through induction of iNOS gene expression.
COX-2 inhibitor NS-398 or COX-2 siRNA attenuated PGE2, but not NO production induced by LPS NS-398 is a selective COX-2 inhibitor. Treatment with NS-398 (10 mM for 24 h) caused complete attenuation of the PGE2
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response to LPS in CCD-18Co fibroblasts (Fig. 5A, p40.05 when compared to the LPS control). The above result was confirmed by knockdown of COX-2 by COX-2 siRNA (Fig. 5B, p40.05 when compared to the LPS control). However, NS398 or COX-2 siRNA had no effect on NO synthesis (Fig. 5C and D, p40.05 when compared to the LPSþ control). These data demonstrated that LPS-stimulated PGE2 production is COX-2 specific and LPS enhanced NO formation is not COX-2 dependent.
Inhibition of PGE2 production by NS398 or COX-2 siRNA was through Downregulating COX-2 mRNA and protein expression in the fibroblasts NS398 or COX-2 siRNA caused complete attenuation of COX-2 mRNA (Fig. 6A and C, p40.05 when compared to the LPS control) and COX-2 protein expression (Fig. 6B and D, p40.05 when compared to the LPS control), indicating an inhibition of PGE2 production by NS398 or COX-2 siRNA was through inhibition of COX-2 mRNA and Protein Expression in the CCD18Co fibroblasts.
Fig. 7 – iNOS inhibitor 1400 W or iNOS siRNA reduced LPS-stimulated PGE2 synthesis, but blocked NO production in the fibroblasts. (A) 1400 W reduced LPS-stimulated PGE2 synthesis in the fibroblasts. CCD-18Co cells were incubated with LPS (1 lg/ ml) and 1400 W (10 lM) for 24 h. PGE2 levels in harvested medium were measured by RIA. Bars represent the mean results from 4 experiments7SE. (B) 1400 W completely blocked LPS-stimulated NO synthesis in the fibroblasts. CCD-18Co cells were coincubated with LPS (1 lg/ml) and 1400 W (10 lM) for 24 h. NO concentrations in harvested medium were determined by nitric oxide assay kit. Bars represent the mean results from 4 experiments7SE. (C) iNOS siRNA reduced LPS-stimulated PGE2 production in the fibroblasts. Lyophilized duplex siRNAs corresponding to the iNOS sequence and control (scrambled) RNA were used for transfection. The PGE2 experimental procedures were described above. Bars represent the mean results from 4 experiments7SE. (D) iNOS siRNA blocked LPS-stimulated NO production in the fibroblasts. Lyophilized duplex siRNAs corresponding to the iNOS sequence and control (scrambled) RNA were used for transfection. The NO experimental procedures were described above. Bars represent the mean of results from 4 separated cultures7SE. po0.05, po0.01 when compared to the control. #po0.05 when compared to LPSþ control.
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iNOS inhibitor 1400 W or iNOS siRNA not only attenuated NO production, but also significantly decreased PGE2 production stimulated by LPS Treatment with iNOS inhibitor 1400 W or iNOS siRNA caused complete attenuation of the iNOS and NO response to LPS in CCD-18Co fibroblasts (Fig. 7B and D, p40.05 when compared to that of LPS control), but reduced PGE2 production by approximately 55% and 50%, respectively (Fig. 7A and C, #po0.05 when compared to the LPSþ control). The results suggest that LPSstimulated NO production is fully iNOS signaling-dependent and LPS-stimulated PGE2 production is partially iNOS signalingdependent.
The partial inhibition of PGE2 production by 1400 W or iNOS siRNA was through inhibition of COX-2 mRNA and protein expression in CCD18Co fibroblasts 1400 W or iNOS siRNA caused partial attenuation of COX-2 mRNA (Fig. 8A and C, #po0.05 when compared to the LPSþ control) and COX-2 protein expression (Fig. 8B and D, #po0.05 when compared to the LPSþ control), indicating that an inhibition of PGE2 production by 1400 W or iNOS siRNA was partially through inhibition of COX-2 mRNA and Protein Expression in the CCD18Co fibroblasts.
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The inhibition of NO production by 1400 W or iNOS siRNA was through inhibition of iNOS mRNA and protein expression in CCD18Co fibroblasts 1400 W or iNOS siRNA caused complete attenuation of iNOS mRNA (Fig. 9A and C, p40.05 when compared to the LPS control) and iNOS protein expression (Fig. 9B and D, p40.05 when compared to the LPS control), indicating an inhibition of NO production by 1400 W or iNOS siRNA was through inhibition of iNOS mRNA and Protein Expression in the CCD18Co fibroblasts.
LPS-stimulated PGE2 and NO synthesis was mediated by protein kinase C (PKC), not Ca2þ signaling The PKC superfamily participates in a wide array of cellular responses. Activation of PKC by LPS peaked at 3 min (Fig. 10, n po0.05, nnpo0.01 when compared to the control) and appeared Ca2þ-independent since no intracellular calcium activation was recorded in CCD18-Co cells treated with LPS (data not shown). Immediate addition of protein kinase inhibitor, STA or PKC inhibitor BIM (20 mM and 10 mM respectively) to the cultures treated with LPS (1 mg/ml) for 24 h completely blocked LPSstimulated synthesis of PGE2 and NO (Fig. 11, p40.05 when
Fig. 8 – Decrease of PGE2 production by 1400 W or iNOS siRNA was partially Through downregulating COX-2 Gene Expression in the Fibroblasts. (A) 1400 W reduced LPS-stimulated COX-2 mRNA expression. CCD-18Co cells were incubated with LPS (1 lg/ml) and 1400 W (10 lM) for 24 h. iNOS mRNA levels were determined by RT-realtime PCR. Bars represent the mean results from 4 experiments7SE. (B) 1400 W downregulated LPS-stimulated COX-2 protein expression. CCD-18Co cells were incubated with LPS (1 lg/ml) and 1400 W (10 lM) for 24 h. COX-2 protein levels were determined by Western blotting. Bars represent the mean results from 4 experiments7SE. (C) iNOS siRNA decreased LPS-stimulated COX-2 mRNA expression. CCD-18Co cells were incubated with LPS (1 lg/ml) and iNOS siRNA (100 nM) for 24 h. COX-2 mRNA levels were determined by RT-realtime PCR. Bars represent the mean results from 4 experiments7SE. (D) iNOS siRNA downregulated LPS-stimulated COX-2 mRNA expression. CCD-18Co cells were incubated with LPS (1 lg/ml) and iNOS siRNA (100 nM) for 24 h. COX-2 protein levels were determined by Western blotting. The results were normalized by b-actin. Bars represent the mean of results from 4 separated cultures7SE. n po0.05, nnpo0.01 when compared to the control. #po0.05 when compared to the LPSþ control.
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Fig. 9 – Inhibition of NO production by 1400 W or iNOS siRNA was through downregulating iNOS gene expression in the fibroblasts. (A) 1400 W blocked LPS-stimulated iNOS mRNA expression. CCD-18Co cells were incubated with LPS (1 lg/ml) and 1400 W (10 lM) for 24 h. iNOS mRNA levels were determined by RT-realtime PCR. Bars represent the mean results from 4 experiments7SE. (B) 1400 W blocked LPS-stimulated iNOS protein expression. CCD-18Co cells were incubated with LPS (1 lg/ml) and 1400 W (10 lM) for 24 h. COX-2 protein levels were determined by Western blotting. Bars represent the mean results from 4 experiments7SE. (C) iNOS siRNA attenuated LPS-stimulated iNOS mRNA expression. CCD-18Co cells were incubated with LPS (1 lg/ml) and iNOS siRNA (100 nM) for 24 h. iNOS mRNA levels were determined by RT-realtime PCR. Bars represent the mean results from 4 experiments7SE. (D) iNOS siRNA Attenuated LPS-stimulated iNOS protein expression. CCD-18Co cells were incubated with LPS (1 lg/ml) and iNOS siRNA (100 nM) for 24 h. iNOS protein levels were determined by Western blotting. The results were normalized by b-actin. Bars represent the mean of results from 4 separated cultures7SE. po0.01 when compared to the LPS control.
Discussion
Fig. 10 – LPS activated PKC activity in the fibroblasts. CCD18Co cells were exposed to LPS (1 lg/ml) for periods of 1–10 min. Cell homogenates were pelleted, and particulate fractions were resolubilized. PKC activity of these samples was determined from transfer of [32]ATP to a PKC-specific peptide and were expressed as pmol/min/mg protein. Bars represent the mean of results from 4 separated cultures7SE. po0.05, po0.01 when compared to the control.
compared to the LPS control). The results suggest that LPSstimulated COX-2 and iNOS gene expression is PKC-dependent and Ca2þ-independent.
In this article, we first tested whether CCD-18CO cells resemble primary human colonic fibroblasts producing PGE2 and NO. To our surprise, CCD18Co cells produced similar levels of PGE2 and NO when compared to those of primary human colonic fibroblasts during the entire experimental period (Figs. 1 and 2). LPS stimulated PGE2 and NO production peaked at 24 h and remained high up to 48 h in both primary human colonic and CCD18Co fibroblasts (Figs. 1 and 2), indicating that CCD18Co fibroblasts are a good model for our experimental purpose. To test whether this PGE2 induction induced by LPS is caused by increased gene expression of COX-2, we performed RT-realtime PCR and Western blotting. Indeed LPS induced timedependent COX-2 mRNA and corresponding protein expression in the cells (Fig. 3), similar to our previous reports that IL1b, TNFa and DCA induced COX-2 expression [16–18]. The results clearly indicated that LPS stimulated PGE2 production was through induction of COX-2 mRNA and protein expression. NO produced by iNOS was reported to enhance the activity of COX-2 [32]. These effects may involve ‘‘cross-talk’’ between the two enzyme systems and be responsible for enhanced growth in some tumors [33]. However, it is still largely unknown whether colonic fibroblasts are main sources of COX-2 and iNOS, and if so, whether those fibroblasts are involved in the cross-talk between COX-2 and iNOS pathways, and whether the cross-talk plays a
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Fig. 11 – Protein kinase inhibitors blocked activation of PKC and synthesis of PGE2 and NO. CCD-18Co cells were coincubated with LPS (1 lg/ml) and staurosporine (STA, 20 lM) or bisindoylmalemide (BIM, 10 lM) for 24 h. PGE2 levels in harvested medium were determined by RIA and NO levels in harvested medium were determined by nitric oxide assay kit. Bars represent the mean of results from 4 separated cultures7SE. po0.01, po0.001 when compared to the LPS control.
significant role in inflammation and progression of colonic cancer. As the results indicated, LPS was found not only to induce a time-dependent NO production (peaked at 24 h, Fig. 2), but also a time-dependent iNOS mRNA and protein expression in the fibroblasts (peaked at 24 h, Fig. 4) in CCD18Co fibroblasts. We suggest that the fibroblasts play a significant role in regulating the production of nitric oxide. Cross-talk of COX-2 and NO signalings between tumor spheroids and normal cells has recently been investigated [34]. However, whether cross-talk between COX-2 and NO signalings within the fibroblasts occurs is not known. To investigate whether the cross-talk between COX-2 and iNOS pathways in CCD18Co cells occurs after LPS treatment, we used specific inhibitors of COX-2 and iNOS, NS 398 and 1400 W, and knockdown of COX-2 and iNOS by COX-2 siRNA and iNOS siRNA respectively. Our results demonstrated that NS398 or COX-2 siRNA completely blocked PGE2 induction, but had no effect on NO production induced by LPS, indicating that the products of COX-2 pathway or COX-2 itself did not affect iNOS signaling since a specific COX-2 inhibitor, NS398 or knockdown of COX-2 by COX-2 siRNA did not inhibit NO production (Fig. 5). Further real-time PCR and Western analysis indicated that an inhibition of PGE2 production by NS398 or COX-2 siRNA was through
attenuating COX-2 mRNA and protein expression (Fig. 6). In contrast, activation of NO pathway was contributed to increased COX-2 gene expression and PGE2 production, since specific iNOS inhibitors, 1400 W or knockdown of iNOS by iNOS siRNA reduced PGE2 induction by 55% and 50%, respectively (Fig. 7). The inhibition was due to inhibition of COX-2 gene expression (Fig. 8). In addition, 1400 W or iNOS siRNA completely abolished the effect on NO production induced by LPS (Fig. 7). Such an inhibition is due to inhibition of iNOS gene expression (Fig. 9). We speculate that products generated by activation of iNOS signaling by LPS or activation of iNOS itself were involved in regulation of production of proinflammatory PGE2, which might contribute to inflammatory response and progression of colorectal cancer [19,35]. Up to now, how NO signaling regulates COX-2 expression is not well understood. Two major mechanisms proposed were that NO influences its intracellular targets are stimulation of guanylyl cyclase by direct binding of NO to iron in heme at the active site of guanylyl cyclase [36], or S-nitrosylation of protein targets on appropriate cysteines [37,38]. Because COX-2 has heme at its active site [39] and contains 13 cysteines [40], it was proposed that iNOS binds S-nitrosylates and activates COX-2 [25]. Whether this is the case in our cell model system requires further
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investigation. Interestingly, the interaction of COX-2 and iNOS signalings did not affect the cell viability during the entire study, probably because the dosage of LPS to the cells was well within physiological tolerance. Furthermore, we suggest that protein kinase C, not Ca2þ, was involved in LPS-induced activation of COX-2 and iNOS signalings in CCD-18Co fibroblasts. LPS treatment to the fibroblasts increased cellular PKC activity within 3 min after administration (Fig. 10). The selective PKC inhibitors, STA and BIM, attenuated the PKC activation and induction of PGE2 and NO in the CCD18Co cells (Fig. 11). We speculate that protein Kinase C, not Caþ, plays a major role in mediating COX-2 and iNOS activation in response to LPS, similar to the action mechanisms elicited by deoxycholic acid we previously reported [16–18]. To conclude, colonic fibroblasts are major sources of PGE2 and iNOS. Products from iNOS signaling is involved in cross-talk with COX-2 pathway in human colonic fibroblasts. The schematic diagram showing components and inhibitors has been summarized as follows:
We speculate that drugs targeting COX-2 and iNOS signalings might be useful for the control of colonic inflammation and colorectal cancer.
Acknowledgments This work was supported in part by Arizona Cancer Center (NIH Institutional Research Grant # 7400128) and a small research grant from the University of Arizona. No potential conflict of interest was noted.
references
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