Department of Pharmacology, University of Nevada School of Medicine, Reno, Nevada 89557-0270
Submitted 30 October 2003 ; accepted in final form 26 April 2004
ABSTRACT
Intestinal mucosal cells and invading leukocytes produce inappropriate levels of cytokines and chemokines in human colitis. However, smooth muscle cells of the airway and vasculature also synthesize cytokines and chemokines. To determine whether human colonic myocytes can synthesize proinflammatory mediators, strips of circular smooth muscle and smooth muscle cells were isolated from human colon. Myocytes and muscle strips were stimulated with 10 ng/ml of IL-1, TNF-
, and IFN-
, respectively. Expression of mRNA for IL-1
, IL-6, IL-8, and cyclooxygenase-2 (COX-2) was induced within 2 h and continued to increase for 812 h. Regulated on activation, normal T cell-expressed and -secreted (RANTES) mRNA expression was slower, appearing at 8 h and increasing linearly through 20 h. Expression of all five mRNAs was inhibited by 0.1 µM MG-132, a proteosome inhibitor that blocks NF-
B activation. Expression of IL-1
, IL-6, IL-8, and COX-2 mRNA was reduced by 30 µM PP1, an Src family tyrosine kinase inhibitor, and by 25 µM SB-203580, a p38 MAPK inhibitor. MAPK/extracellular regulated kinase-1 inhibitor PD-98059 (25 µM) was much less effective. In conclusion, human colonic smooth muscle cells can synthesize and secrete interleukins (IL-1
and IL-6) and chemokines (IL-8 and RANTES) and upregulate expression of COX-2. Regulation of cytokine, chemokine, and COX-2 mRNA depends on multiple signaling pathways, including Src-family kinases, extracellular regulated kinase, p38 MAPKs, and NF-
B. SB-203580 was a consistent, efficacious inhibitor of inflammatory gene expression, suggesting an important role of p38 MAPK in synthetic functions of human colonic smooth muscle.
cyclooxygenase; cytokine synthesis; mitogen-activated protein kinase-activated protein kinase; nuclear factor-B; p38 mitogen-activated protein kinase
Studies of patients with inflammatory bowel disease (24, 26) have demonstrated increased mucosal levels of a variety of cytokines and chemokines. IL-1, -2, -6, and -12 have been observed in the inflamed mucosa along with several chemokines, TNF-
, and IFN-
(see Ref. 3). Chemokines present in inflammatory bowel diseases include IL-8, macrophage inhibitory proteins 1
and 1
, and monocyte chemotactic proteins 1, 2, and 3 (4). The chemokines are thought to play a critical role in recruiting neutrophils and monocytes to sites of inflammation. Current evidence supports the notion that several cell types, such as epithelia, myofibroblasts, and leukocytes in the intestinal mucosa, produce complex combinations of mediators that promote persistent inflammation and ultimately mucosal degradation, which causes the symptoms of Crohn's disease and ulcerative colitis.
In contrast to extensive studies of the mucosa, there is less evidence for synthesis and secretion of inflammatory signaling proteins from intestinal smooth muscle cells. There is some evidence that IL-1 is expressed by human colonic smooth muscle in patients with ulcerative colitis (36), and there is evidence for fibroblast growth factor and stem cell factor synthesis by intestinal smooth muscle (8, 38). In addition, IL-1
, norepinephrine, vasoactive intestinal polypeptide, and calcitonin gene-related peptide can induce synthesis of IL-6 by rat intestinal smooth muscle cells (19, 35). There is extensive literature on the potential for immune mediators to modify colonic motility (reviewed in Ref. 7), but relatively few studies directly address the capacity for human colonic smooth muscle to synthesize mediators. This relatively sparse data set is in sharp contrast to abundant in vitro evidence for cytokine and chemokine synthesis by smooth muscle cells in the vasculature and airways, which are known to be metabolically dynamic cells able to express and secrete many highly active signaling proteins.
In this study, we tested the hypothesis that smooth muscle cells of the colon respond to proinflammatory stimuli by synthesizing interleukins, chemokines, and cyclooxygenase-2 (COX-2) using a cultured human smooth muscle cell model. A proinflammatory mixture of cytokines (IL-1, TNF-
, IFN-
) was then used to test for synthesis of several interleukins, RANTES, and COX-2 using real-time quantitative-PCR (Q-PCR), Western blot analysis, and immunocytochemical techniques. This cytokine mixture was chosen because each component has been reported to be present in inflamed mucosa in patients with inflammatory bowel disease. Additionally, the combination has been used successfully in studies (16, 31) of other smooth muscles to elicit cytokine and chemokine synthesis. The mixture is used empirically in this study to elicit a profound synthetic response from cultured colonic myocytes and to test the working hypothesis. There was significant upregulation of IL-1
, IL-6, IL-8, RANTES, and COX-2 transcripts in response to the cytokine stimulus. Upregulation of these signaling molecules could be blocked to varying degrees by inhibitors of Src family kinases, NF-
B, and p38 MAPK signal transduction pathways. The results show that human colonic smooth muscle cells in culture synthesize cytokines, chemokines, and COX-2 and that intact human colonic smooth muscle strips upregulate COX-2. This supports prior observations of Vrees et al. (36) and suggests that human colonic smooth muscle might synthesize a broad range of mediators and signaling proteins that might contribute to inflammation in vivo.
MATERIALS AND METHODS
Materials.
IL-1, TNF-
, IFN-
, and all other tissue culture reagents were purchased from Sigma (St. Louis, MO). SB-203580, PD-98059, and MG-132 were purchased from Calbiochem, (La Jolla, CA). TRIzol, Superscript II, and PCR reagents were purchased from Invitrogen (Carlsbad, CA). Thermus aquaticus (Taq) polymerase was purchased from Promega (Madison, WI). Antibodies for COX-1, COX-2, p38 MAPK, and I
B
were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against smooth muscle myosin, calponin, and smooth muscle tropomyosins were obtained from Sigma. Anti-caldesmon antibodies were a generous gift from Dr. Leonard P. Adam (Bristol-Myers-Squibb, Pennington, NJ). Phosphorylated p38 MAPK-selective antibody was purchased from Cell Signaling Technology (Beverly, MA). The STAT-prostaglandin E2 enzyme immunoassay was purchased from Cayman Chemical (Ann Arbor, MI). Synthetic Src tyrosine kinase peptide substrate p34cdc2(620) was obtained from Upstate Biotechnology (Lake Placid, NY). The antibody against ERK1/2 was purchased from Upstate Biotechnology and anti-phosphorylated ERK1/2 was purchased from Cell Signaling Technology.
Isolation and treatment of cultured human colonic smooth muscle cells.
Colonic smooth muscle cells were isolated from the circular smooth muscle layer of human colon. Human colon tissue was obtained by a protocol approved by the University of Nevada, Reno Biomedical Institutional Review Board. Human colon tissue was obtained from the Cooperative Human Tissue Network-Western Division (Vanderbilt University, Nashville, TN). Tissue strips of circular smooth muscle 0.5 x 0.5 x 0.5 mm were cut by sharp dissection, and cells were dispersed by treatment with 0.6 mg/ml type II collagenase (cat. no. 4176; Worthington) and 0.07 mg/ml protease (cat. no. A7511; Sigma) in calcium-free Hanks' balanced salt solution. Cell dispersions were plated on tissue culture plastic and allowed to grow to near confluence in humidified 5% CO2 atmosphere at 37°C in medium 199 supplemented with 10% newborn calf serum, 3 mg/ml glutamine, 0.5 ng/ml epidermal growth factor, 2 ng/ml fibroblast growth factor, 200 U/ml penicillin G, and 200 µg/ml streptomycin. Cells from passages 4 through 7 retained expression of smooth muscle isoforms of myosin, actins, calponin, caldesmon, and tropomyosins as determined by immunocytochemical staining (see Fig. 1) and Western blot analysis (not shown). To stimulate expression of inflammatory mediators, cells were growth arrested for 24 h in medium 199 supplemented with (in µg/ml) 6.25 insulin, 6.25 transferrin, 6.25 selenious acid, and 5.35 linoleic acid, and 0.1% serum. Growth-arrested cells were treated with 10 ng/ml each of IL-1
, TNF-
, and IFN-
for 0, 2, 4, 8, 12, or 20 h. In selected experiments, cultures were pretreated for 15 min with signaling pathway inhibitors [25 µM SB-203580, 25 µM PD-98059, 30 µM PP1 (an Src family tyrosine kinase inhibitor), or 0.1 µM MG-132] or with 0.1% DMSO as a solvent control. Inhibitors were present before and during exposure to the cytokine mixture. At the appropriate times after cytokine stimulus, the medium was aspirated, cells were washed with PBS, and then processed for immunocytochemistry, RNA, or protein analysis.
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RNA analysis. Total cellular RNA was extracted from confluent cells with 1 ml TRIzol reagent/10 cm2 according to the manufacturer's protocol (Invitrogen). The RNA fraction was collected and used to synthesize cDNA for RT-PCR analysis of gene expression. First-strand cDNA synthesis was performed at 42°C from 1 µg total RNA using 250 ng random hexamers and (in mM) 50 Tris·HCl (pH 8.3), 75 KCl, 3 MgCl2, 10 DTT, and 0.125 each of deoxyadenosine triphosphate, deoxythymidine triphosphate, deoxyguanidine triphosphate, and deoxycytidine triphosphate, and 1 unit SuperScript II RT. RNAse H (20 units) was added to remove RNA complementary to the cDNA.
Real-time Q-PCR was used to assess changes in gene expression induced by the combination of IL-1, TNF-
, and IFN-
. Confluent cells were treated with IL-1
, TNF-
, and IFN-
(10 ng/ml each) with or without inhibitors (in µM: 25 SB-203580, 25 PD-98059, 30 PP1, or 0.1 MG-132). Total RNA was isolated and first-strand cDNA was synthesized from 1 µg of total RNA. Primers were designed to yield amplicons of 125200 bp using Vector NTI software, version 8.0 (Informax; Bethesda, MD). All reactions were performed in triplicate in 25 µl total volume containing a 1x concentration of SYBRgreen PCR Master Mix (Applied Biosystems, Foster City, CA), 2.5 pmol of each forward and reverse primer, and 2 µl cDNA. Reactions were carried out in a MicroAmp 96-well optical plate and amplified in a GeneAmp 5700 Sequence Detection System (Applied Biosystems) for one cycle at 94°C for 10 min, 40 cycles at 94°C for 15 s followed by 60°C for 1 min, and one cycle at 72°C for 7 min. Gene-specific primer pairs (sense and antisense, respectively) used in these studies were COX-2: 5'-TCCCTTGAAGTGGGTAAGTATGTAGTGC-3', 5'-AAA-ACCGAGGTG-TATGTATGAGTGTGG-3; IL-1
: 5'-TTGTTGCTCCATATCCTGTCCCTG-3', 5'GAGCACCTTTCCCTTCATCTTTG-3'; IL-6: 5'-GAGGTGAGTGGCTGTCTGTGTGG-3', 5'-CAAGCGCCTTCGGTCCAGTT-3'; IL-8: 5'-CAAAAACTTCTCCACAACCCTC-TGC-3', 5'-CTCCAAACCTTTCCACCCCAAA-3'; RANTES: 5'-CCCCATATTCCTCGGACACCA-3', 5'-CACACTTGGCGGTTCTTTCGG-3'; and 18S: 5'-GCTCGTCGGCATGTATTAG-3', 5'-ACAGTGAAACTGGGAATG-3'. Fluorescence values, representing the amount of product amplified at that point in the reaction, were recorded for each cycle in the reaction. The point at which the fluorescence signal was statistically significant above background was calculated for each amplicon in each experimental sample using GeneAmp 5700 software. This value was then used to determine the relative amount of amplification in each sample by interpolating from the standard curve. Standard curves for each amplicon were generated from a dilution series of cDNA synthesized from stimulated human airway smooth muscle cells (10 ng/ml IL-1
, TNF-
, and IFN-
for 20 h). Gene-specific amplification was verified as a single amplification peak in first derivatives of the amplification plot showing fluorescence vs. cycle number. Transcript levels calculated from standard curves were normalized to 18S ribosomal RNA (rRNA) amplification and reported as a relative expression value. Experiments were performed on duplicate cultured cells from three different human subjects.
Protein analysis by Western blotting.
Total protein was extracted from human colonic smooth muscle cells to measure intracellular IL-1, ERK1/2, phosphorylated ERK1/2, p38 MAPKs, phosphorylated p38 MAPKs, and COX-1 and -2 expression. Cytokine-stimulated cells were washed with cold PBS and immediately lysed with extraction buffer containing (in mM) 60 Tris·HCl, 1 EGTA, 1 Na2EDTA, 1 AEBSF, and 5 NaF, plus 2% SDS, 10% glycerol, and 1 µM leupeptin. Cellular extracts were centrifuged at 10,000 g for 20 min at 4°C, and the supernatants were used to assay for protein expression. Protein concentrations were determined by the bicinchoninic acid method using bovine serum albumin as the standard. Total protein extracts were separated by 10% SDS-PAGE. Proteins were transferred to nitrocellulose paper and detected by labeling with anti-human IL-1
(AF-201, diluted 1:500; Research Diagnostics), COX-2 [cat. no. SC-1745, diluted 1:3,000 for tissue and 1:1,000 for cells; Santa Cruz Biotechnology], COX-1 (cat. no. SC-1752, diluted 1:2,000), ERK1/2 (cat. no. 06-182, diluted 1:1,000; Upstate Biotechnology), phosphorylated ERK1/2 (cat. no. 9101, diluted 1:1,000; Cell Signaling Technology) primary antibodies. For IL-1
, COX-1 and -2, anti-goat, anti-rabbit, or anti-mouse IgG alkaline phosphatase secondary antibodies (diluted 1:10,000; Promega) were used to visualize and quantitate immunoreactive proteins. Immunoreactive bands were scanned by using Umax Powerlook flatbed scanner, and densitometry was performed by using Molecular Analyst software (version 1.5) Bio-Rad (Hercules, CA). For ERK and phosphorylated ERK1/2, anti-rabbit IgG IR 800 secondary antibodies (Licor Biosciences, Lincoln, NE) were visualized by using the Odyssey Infrared Imaging System (Licor Biosciences). Preliminary studies established the amount of cell or tissue extract (micrograms of total protein) needed to generate signals in the linear range of detection of each antigen-antibody pair.
ELISA.
Supernatants obtained from cells treated with 10 ng/ml IL-1, TNF-
, and IFN-
in the absence or presence of inhibitors [(in µM) 25 SB-203580, 25 PD-98059, 30 PP1, or 0.1 MG-132] or with 0.1% DMSO were used for detection of secreted IL-6, IL-8, and RANTES by ELISA following the manufacturer's protocol (R&D Systems, Minneapolis, MN). The minimum detectable concentration was <0.70 pg/ml for IL-6, 1.57.5 pg/ml for IL-8, and 1.746.63 pg/ml for RANTES.
Secreted PGE2 was also assayed by enzyme-linked immunoassay. Conditioned medium was collected 8 h after stimulation of cultured colonic myocytes with IL-1, TNF-
, and IFN-
(10 ng/ml each). The assay was performed according to the manufacturer's instructions (Cayman Chemical). Minimum detectable levels of PGE2 with this assay were
95 pg/ml. Constitutive and inducible PGE2 levels in this study ranged from 50 to 250 ng/ml of undiluted culture medium.
Protein kinase assays.
Src tyrosine kinase activity was assayed to verify the efficacy of PP1 in cultured cells. Whole cell lysates were prepared from all treatments by homogenization and centrifugation in (in mM) 50 HEPES, 150 NaCl, 1 sodium orthovanadate, 10 NaF, and 0.1 AEBSF, plus 1 µM leupeptin, 0.5% Triton X-100, 0.5% NP-40, and 10% glycerol. Protein concentrations were determined by using the bicinchoninic acid method with bovine serum albumin as the standard. Ten micrograms of total protein were incubated for 60 min at 30°C in kinase assay buffer, 10 µCi [-32P]ATP, and 150 µM of the synthetic peptide substrate p34cdc2 (620) (KVEKIGEGTYGVVYK) as directed by the manufacturer (Upstate Biotechnology). Reactions were then spotted onto P81 phosphocellulose, washed in 0.75% H3PO4, followed by scintillation counting.
To verify the efficacy of SB-203580 in inhibiting p38 MAPK, we assayed the activity of MAPKAPK 2, which is activated by p38 MAPK. MAPKAPK 2 was assayed by extracting the enzyme from cultured cells in (in mM) 30 MOPS, 80 -glycerophosphate, 2 EGTA, 0.1 Na3VO4, 25 MgCl2, 40 KCl, 0.1 NaF, and 1 AEBSF, plus 1 µM leupeptin. Kinase activity was assayed at 30°C for 30 min in 60 µl final volume containing (in mM) 25 MOPS (pH 7.2), 25
-glycerophosphate, 15 MgCl2, 1 EGTA, 0.1 NaF, 1 Na3VO4, and 4 dithiothreitol, plus 10 µg protein from cell extracts, 10 µCi of [
-32P]ATP (250 µM), and 0.15 mg/ml recombinant human heat shock protein 27 (HSP27). Recombinant human HSP27 was expressed in Escherichia coli and purified by DEAE chromatography as previously described (20). Phosphorylated HSP27 was resolved by 12% acrylamide SDS-PAGE, and phosphorylation levels were visualized and quantitated with a Bio-Rad GS-525 Molecular Imager and Molecular Analyst software.
Immunocytochemistry.
Expression of smooth muscle isoforms of contractile proteins was assessed by immunofluorescence microscopy and confocal microscopy. Cells were plated on glass coverslips coated with 0.75 mg/ml type I bovine dermal collagen and cultured in medium 199 with 10% newborn calf serum for 13 days. When cells were 5070% confluent, they were serum starved for 1 or 7 days, fixed 10 min with 4% paraformaldehyde, and washed with PBS. Coverslips were blocked by incubation in PBS with 1% bovine serum albumin for 1 h at room temperature. Cells were washed three times with PBS and 0.5% Tween-20 and then incubated overnight at 4°C in primary antibodies. Coverslips were washed three times with PBS and 0.5% Tween-20 and then incubated for 1 h at room temperature with secondary antibodies conjugated to either Alexa Flour 488 or Alexa Flour 594 (Molecular Probes, Eugene, OR). In all studies, coverslips incubated only with secondary antibodies served as negative controls. The primary antibodies against contractile proteins were all selective for smooth muscle isoforms. The source of antibodies and dilutions used were: anti-
-actin (1:100; cat. no. 069D, Biomedica), anti-
/
-actin (1:100; cat. no. 69133, ICN), anti-tropomyosin (1:400; cat. no. T02780
[GenBank]
Sigma), anti-caldesmon [1:100; Franklin et al. (11)], anti-calponin (1:400; cat. no. C-2687, Sigma), and anti-COX-2 (1:100; cat. no. SC-1745, Santa Cruz Biotechnology). Immunoreactive proteins were visualized, and images were recorded by using either a Nikon TE-200 epifluorescence microscope with Pixera model 120es camera or with a Bio-Rad MRC-600 confocal microscope.
Statistical analysis. One-way ANOVA was performed followed by post hoc testing with the Student-Newman-Keuls method or a paired Student's t-test using SigmaStat software (Jandel Scientific, San Rafael, CA). Time-dependent changes in signaling protein expression were analyzed by Student's t-test.
RESULTS
Characterization of cultured human smooth muscle cells.
Smooth muscle cells from the circular muscle layer of the human colon were isolated by enzymatic digestion and subcultured through 46 passages (812 population doublings). A homogeneous culture of muscle cells was achieved by differential plating and repeated passage of cells under conditions favoring growth of smooth muscle similar to methods described by Brittingham et al. (6). To verify the smooth muscle origin and homogeneity of the cultures, we tested for expression of smooth muscle restricted isoforms of contractile proteins. Cells were cultured to near confluence, growth arrested for 24 h by reducing the medium serum concentration from 10 to 0.1% (vol/vol). All cells showed immunoreactivity for smooth muscle myosin heavy chain (Fig. 1A), smooth muscle -actin (Fig. 1B), smooth muscle
-actin (Fig. 1C),
-calponin (Fig. 1E), caldesmon (Fig. 1D), and smooth muscle tropomyosins (Fig. 1F). A more detailed analysis of contractile proteins demonstrated that filaments were formed in some but not all cells after 24 h of growth arrest (not shown). We also tested the effect of longer term serum deprivation, because others (5, 6, 12) have shown that extended serum deprivation (>57 days) upregulates expression of contractile proteins and favors resumption of the contractile phenotype. After 7 days in 0.1% serum, most cells showed filamentous distribution of actin, caldesmon, and tropomyosin immunoreactivity, as visualized by confocal microscopy (Fig. 1, GI). The immunofluorescence data strongly support the contention that the cultures are of smooth muscle origin.
Proinflammatory gene expression.
A limited survey of cytokine and chemokine transcripts was conducted to test for expression of signaling proteins commonly induced in vascular and airway smooth muscle tissues by proinflammatory stimuli. The hypothesis was that similar sets of signaling molecules are produced by colonic smooth muscles in response to inflammation. The stimulus used in this study was a combination of IL-1, TNF-
, and IFN-
(10 ng/ml each), which is known to induce synthesis of interleukins, chemokines, TNF-
, and COX-2 in other types of smooth muscles (31, 16). A combination of agents was used to obtain maximum stimulus intensity because combinations of proinflammatory signals are very likely to be present in situ during inflammation of the colon.
Cultured colonic myocytes were grown to near confluence and growth arrested for 24 h before treatment with the cytokine mixture. Cells from three human subjects were prepared in duplicate for total RNA extraction and cDNA synthesis. Q-PCR was performed in triplicate on both sets of cells from each of three donors. Expression levels of each gene-specific amplicon were normalized to 18S rRNA. Stimulation of cells with cytokine mixture did not change levels of 18S rRNA (Fig. 2A) validating the use of 18S rRNA transcript as a reference. Figure 2, B and C shows that transcripts for cytokines IL-1 and -6 were significantly increased within 4 h and that the levels continued to increase for up to 12 h after stimulation. The levels of IL-1
transcript relative to 18S rRNA were significantly greater than IL-6 and were highly variable among the three cell lines used in this study. Although the levels of IL-1
varied among cell lines, the time course of upregulation was very consistent among cell lines. A similar pattern of induction was observed for the two chemokines investigated, IL-8 and RANTES (Fig. 2C). IL-8 was induced within 4 h, and reached a steady state at 8 h. RANTES induction was somewhat slower, requiring 8-h stimulation before a significant increase in transcript levels was observed. These results demonstrate induction of several cytokine and chemokine transcripts in cultured human colonic myocytes stimulated with a proinflammatory cytokine mixture.
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To test for p38 MAPK dependence of cytokine gene expression, cells were treated with the p38 MAPK inhibitor SB-203580 and then stimulated with IL-1, TNF-
, and IFN-
. SB-203580 (25 µM) inhibited synthesis of transcripts for IL-1
, -6, and -8, but not RANTES (Fig. 3). However, pretreatment with SB-203580 only reduced IL-
and -8 protein expression (Fig. 4, A and C). IL-6 protein level was not affected by SB-203580 (Fig. 4B). Interestingly, RANTES transcript and protein expression was significantly increased by inhibition of the p38 MAPK pathway (Figs. 3D and 4D). Efficacy of SB-203580 was verified by testing the effect of 25 µM SB-203580 on MAPKAPK2 activation in cells treated with IL-1
, TNF-
and IFN-
. MAPKAPKs were extracted from activated cells, and the extent of phosphorylation of recombinant human HSP27 was assayed. The crude MAPKAPK extract from cells treated with SB-203580 inhibited HSP27 phosphorylation by
55% (Fig. 5C).
In many cell types, transcription of interleukin and chemokine genes depend on activation of the NF-B signaling pathway (reviewed in Ref. 22). To determine whether NF-
B activation was necessary for cytokine gene induction in human colonic myocytes, MG-132 was used to inhibit catabolism of I
B, which is necessary for activation of NF-
B. Under steady-state conditions, I
B is bound to NF-
B in the cytoplasm. On activation, I
B is phosphorylated by IKK, ubiquitinated and targeted for degradation by the 26S proteasome and therefore allowing NF-
B to translocate to the nucleus and activate gene transcription (34). MG-132 (0.10 µM) blocked I
B degradation stimulated by IL-1
, TNF-
, and IFN-
(Fig. 5D) and partly inhibited induction of IL-1
, IL-6, IL-8, and RANTES transcripts and all except IL-1
protein (Figs. 3 and 4). The results suggest each of these genes depends on NF-
B activation for full expression in human colonic smooth muscle cells.
COX-2.
COX-2 is an inducible enzyme that participates in inflammation via synthesis of prostanoids including PGE2. To investigate mechanisms of induction of COX-2 in human colonic myocytes, cultured cells were stimulated for various times with IL-1, TNF-
, and IFN-
, and changes in COX-2 mRNA and protein levels were analyzed. COX-2 transcript levels increased significantly within 4 h of stimulation and continued to increase reaching a steady-state level at 1220 h (Fig. 6A). We noticed that there was measurable COX-2 mRNA in unstimulated cells, suggesting some constitutive expression of COX-2. Immunocytochemical staining of unstimulated cells with antibodies to COX-2 verified this observation (Fig. 6B) and demonstrated upregulation of COX-2 immunoreactivity after 8 h cytokine stimulation (Fig. 6C) consistent with induction of COX-2 mRNA.
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Multiple interleukins, chemokines, peptide growth factors, and autacoids are present at elevated levels in the gastrointestinal wall during colitis (3, 4, 25, 26, 29). The cellular source of proinflammatory signaling proteins include mucosal epithelium, myofibroblasts, and various leukocytes in and around the intestinal mucosa. Our new results support the notion that in addition to these established cellular sources of proinflammatory signals, colonic smooth muscle cells are also active secretory cells. It is well known that vascular, airway, and urogenital smooth muscle cells secrete cytokines and growth factors. IL-1, -5, -6, -8, and -11, the CC and CXC chemokines, INF-
, TNF-
, and a variety of peptide growth factors have been reported to be synthesized by smooth muscle cells in culture (15, 23, 27). Cytokines produced by smooth muscle in the vasculature may stimulate vessel wall remodeling during atherosclerosis and after injury induced by balloon angioplasty. A similar argument is made for airway smooth muscle cells that are thought to contribute to signaling proteins present in asthmatic airways. Chronic airway hyperreactivity and smooth muscle remodeling through hyperplasia and hypertrophy may be due, in part, to excessive, persistent production of multiple signaling proteins in the airway wall. Although the functional effects of persistent proinflammatory signals is quite different in each organ system when comparing gastrointestinal vs. airway vs. vascular, there may be a common feature. Smooth muscle cells of the organ wall in each case might respond to proinflammatory signals such as IL-1
and TNF-
by synthesizing multiple soluble signaling proteins that then act on muscle cells, fibroblasts, endogenous leukocytes, and epithelia or endothelia to induce synthesis of even more mediators.
To test this hypothesis in smooth muscle cells from the gastrointestinal system, we used cultured circular smooth muscle cells from the adult human colon. These cells were obtained from excess tissue removed during bowel resections and were cultured by using standard methods of mammalian smooth muscle cell culture, similar to that described by Brittingham et al. (6), in which the authors defined the phenotypes of cultured rat duodenal smooth muscle. Because our cultures might be a mixture of cell types with variable phenotypes, the identity of the cultured cells were verified by surveying expression of a panel of contractile proteins characteristic of smooth muscle cells. Expression of smooth muscle isoforms of - and
-actin, myosin heavy chain, caldesmon, calponin, and tropomyosins were verified in both short (1 day) and long (57 days) growth-arrested cultures. In all cases, we observed filamentous distribution of smooth muscle contractile proteins. Long-term growth arrest increased the percentage of cells expressing contractile proteins consistent with previous reports (5, 12, 33) of contractile protein upregulation in growth-arrested smooth muscle cultures.
One of the limitations of cultured smooth muscle cells is that the phenotype is altered by culture conditions. Exposure to serum and adherence to culture plates in a monolayer is different than the condition of native smooth muscle cells surrounded in three dimensions by a basement membrane and a fibrous collagen and elastin matrix. Serum and matrix composition alter contractile protein, ion channel, and receptor expression, which are hallmarks of the contractile phenotype. Recent advances in understanding the cell biology of smooth muscles from several tissues suggest that culturing at high density in low serum causes reversion from a proliferating, secretory phenotype to a state similar, but not identical to contractile cells. This is sometimes referred to as dedifferentiation but is more likely phenotypic modulation of a plastic, multipotent cell type (13, 33). We purposefully used short-term growth-arrested cells to mimic the situation in which proliferating smooth muscle cells are stimulated by growth factors and cytokines. Presumably, colonic smooth muscle cells in vivo are exposed to similar conditions during episodes of inflammation and respond, in part, by upregulating expression of cytokines, growth factors, and COX-2. Support for this assumption comes from in situ expression of basic fibroblast growth factor in colonic smooth muscle and IL-1 in the muscularis of patients with colitis (8, 26).
Our results confirm the capacity of colonic myocytes to express IL-1 and IL-6 and extend the literature to include expression of IL-8, RANTES, and COX-2 (Figs. 2 and 6). The mRNA for each of these proteins increased within a few hours of exposure to a complex proinflammatory cytokine mixture. Peak transcript levels occurred at
10 h with the exception of RANTES, which continued to increase up to 20 h. We did not measure later time points, so the time of peak expression is unknown, but it is clearly slower than the other transcripts investigated. We addressed the issue of phenotypic modulation affecting the synthetic capacity of colonic smooth muscle by assessing COX-2 expression in intact strips of circular smooth muscle from human colon biopsies. The colon samples were obtained within 48 h of surgery, and muscle strips were cultured for 20 h in serum-free medium before stimulation with the mixture of IL-1
, TNF-
, and IFN-
. COX-2 mRNA and protein expression were induced during 20 h of stimulation at a somewhat slower rate than in cultured cells (Figs. 6 and 9), but the tissue clearly responded to the complex proinflammatory stimulus in a qualitatively similar fashion. From this limited study, we cannot conclude that intact tissues synthesize the same range of cytokines and chemokines as the cultured cells, but it is clear that COX-2 is inducible.
When we tested the effects of drugs that block common signal transduction pathways, we found Src family tyrosine kinases, NF-B pathway, and p38 MAPK were necessary for full induction of mRNA and protein in most cases. MG-132, the proteosome inhibitor, significantly decreased IL-1
mRNA but not protein expression. Although the drug did not significantly decrease protein expression, the
20% reduction in expression would suggest a decreasing trend (Fig. 4A). It is possible that 8 h after stimulation when we measured pro-IL-1
levels is too early to detect any significant changes in protein expression.
Although IL-6 mRNA expression (Fig. 3B) was inhibited 50% by SB-203580, the p38 MAPK pathway inhibitor, IL-6 protein expression was not affected by pretreatment with SB-203580 (Fig. 4B). This is in contrast to what occurs in cultured airway smooth muscle cells (16). Our observations suggest that IL-6 secretion may be modulated by posttranscriptional mechanisms that regulate IL-6 mRNA stability. Andoh et al. (2) showed that IL-6 mRNA stability was regulated and enhanced by activation of the ERK1/2 signaling pathway on stimulation with IL-17 and TNF-
. Because ERK1/2 is also activated in the colon when stimulated with cytokine mixture, it is possible that this pathway may also regulate IL-6 mRNA stability in our system.
RANTES mRNA and protein induction was blocked only by MG-132. The lack of effect of the ERK and p38 MAPK pathway inhibitors was unexpected but is not unprecedented. Two prior studies of cultured human airway smooth muscle indicate that the effect of MAPK pathway inhibitors on RANTES expression may depend on stimulus conditions. Amrani et al. (1) used TNF- as the stimulant and found that inhibiting ERK MAPK activation had no effect, but inhibiting p38 MAPK activity reduced RANTES expression. Hallsworth et al. (14) using IL-1
as the stimulus found the exact opposite results; inhibiting ERK MAPK activation reduced RANTES expression but inhibiting p38 MAPK did not. In the present study, a combination of IL-1
, TNF-
, and IFN-
was used as the stimulus, and we found no effect of either MAPK pathway inhibitor on RANTES mRNA expression (Fig. 3D). In addition, these inhibitors did not have any effect on RANTES protein expression (Fig. 4D). We also noted a difference in the effect of signal transduction inhibitors on COX-2 mRNA and protein expression. SB-203580, PP1, and MG-132 each partially inhibited COX-2 mRNA induction, but only SB-203580 reduced COX-2 protein expression significantly (Fig. 7). The consistent inhibitory effects of SB-203580 suggests that inhibiting p38 MAPK signaling in intestinal smooth muscle cells may contribute to the beneficial effects of inhibiting stress-response pathways noted previously in patients with Crohn's disease (17). The profound inhibition of COX-2 expression (Fig. 7) and PGE2 synthesis (Fig. 8) by SB-203580 also suggests that the p38 MAPK pathway is a valid target for developing new therapy. However, variability in drug effects on signaling protein expression reinforces the need to consider both mRNA and protein in expression studies as well as the need to carefully consider the nature of the stimulus. Although stimulating with individual agents such as TNF-
or IL-1
is a simple experimental design, the results might not faithfully reflect the effects of combinations of agents expected to occur in situ and in vivo.
Another interesting issue raised by our results is what purpose is served by synthesis and secretion of interleukins and cytokines by colonic myocytes. Mechanical effects of inflammatory mediators on colonic motility have been carefully studied and found to be primarily due to effects on neurotransmission (7, 30). Other functional effects might include regulation of lymphocyte and mast cell influx to the muscularis and vasculitis of the inflamed colon (9). RANTES and IL-8 are promigratory chemokines that attract monocytes and neutrophils to sites were they are produced. Neutrophils and monocytes which differentiate into tissue macrophages might then contribute to innate immune responses in the colon.
Most smooth muscle containing organs are also populated by tissue mast cells. Mast cell survival and tissue location depend on several signals including IL-6, stem cell factor, and monocyte chemoattractant proteins 1 and 2. In this study, we demonstrated that human colonic smooth muscle could be stimulated to synthesize IL-6 (Fig. 3B), which might contribute to mast cell survival in and around the muscularis. Gastrointestinal smooth muscle cells also produce stem cell factor, the ligand for c-Kit (37, 38). Stem cell factor is also synthesized by smooth muscle cells of the airway and the vasculature (18, 28). In the airways, stem cell factor has been suggested to increase recruitment and promote survival of mast cells. A similar function for smooth muscle-derived IL-6 and stem cell factor in the gastrointestinal tract seems likely. Chemokines and peptide growth factors secreted by colonic smooth muscle might favor retention of mast cells, which might participate in pathogenesis of inflammatory bowel diseases.
In summary, our results support the hypothesis that circular smooth muscle cells from the human colon can synthesize multiple proinflammatory signaling proteins including cytokines (IL-1 and IL-6), chemokines (IL-8 and RANTES), and COX-2. A limited study of intact tissue verified that smooth muscle also upregulates COX-2 in response to a complex proinflammatory stimulus. Analysis of signaling pathways necessary for induction of cytokine, chemokine, and COX-2 expression suggests colonic smooth muscle cells employ multiple signaling pathways including Src-family kinases, NF-
B, and p38 MAPKs. Inhibiting the p38 MAPK pathway was the most consistent and effective strategy. p38 MAPKs may play an important role in pathogenesis of inflammatory bowel diseases and may be a potential target for novel anti-inflammatory drug therapy.
GRANTS
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-41315.
ACKNOWLEDGMENTS
The technical assistance of Mariam Ba, Shanti Rawat, and Michelle Deetken is gratefully acknowledged. Helpful discussion and editing of the manuscript was provided by Kimberly Baker, Liza Rechetnik, and Lisa Hanson.
Address for reprint requests and other correspondence: W. T. Gerthoffer, Dept. of Pharmacology, Univ. of Nevada School of Medicine, Reno, NV 89557-0270 (E-mail: wtg{at}med.unr.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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