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Am J Physiol Gastrointest Liver Physiol 292: G1511-G1519, 2007. First published March 1, 2007; doi:10.1152/ajpgi.00307.2006
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INFLAMMATION/IMMUNITY/MEDIATORS

Decreased MAPK- and PGE2-dependent IL-11 production in Gi{alpha}2–/– colonic myofibroblasts

Brian Hoang, Alice Trinh, and Robert A. Edwards

Department of Pathology, University of California Irvine, Irvine, California

Submitted 11 July 2006 ; accepted in final form 26 February 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mice deficient in the G-protein alpha subunit Gi{alpha}2 spontaneously develop colitis and colon cancer. IL-11 is a pleiotropic cytokine known to protect the intestinal epithelium from injury in animal models of colitis and is produced by subepithelial myofibroblasts in response to inflammatory mediators including TGF-beta, IL-1beta, and PGE2. Arachidonic acid release and subsequent PGE2 production is significantly decreased in the colonic mucosa of Gi{alpha}2–/– mice, and we hypothesized that this would affect mucosal IL-11 production. Mucosal levels of IL-11 were found to be significantly decreased in Gi{alpha}2–/– mice despite the presence of mild colitis. Primary cultures of Gi{alpha}2–/– intestinal and colonic myofibroblasts (IMF and CMF, respectively) produced less basal and TGF-beta or IL-1beta-stimulated IL-11 mRNA and protein than wild-type cells. Inhibitors of ERK or p38 MAPK activation dose dependently inhibited IMF and CMF IL-11 production in response to TGF-beta stimulation, whereas 16,16 dimethyl-PGE2 and prostanoid receptor subtype-selective agonists induced IL-11 production. Treatment of animals with the EP4-specific agonist ONO-AE1-329 resulted in enhanced mucosal levels of IL-11, and increased IL-11 production by ex vivo cultured CMF. Modulation of cAMP levels produced diverging results, with enhancement of TGF-beta-induced IL-11 release in IMF pretreated with 8-Br-cAMP and inhibition in cells treated either with pertussis toxin or the PKA inhibitor H-89. These data suggest a physiological role for prostaglandins, MAPK signaling, and cAMP signaling for the production of myofibroblast-derived IL-11 in the mouse intestinal mucosa.

Gi{alpha}2 knockout mice; myofibroblasts; interleukin-11; ONO-AE1-329; PGE2

Gi{alpha}2–/– MICE SPONTANEOUSLY develop colitis at 6–8 wk of age and progress to develop mucinous, nonpolypoid colon cancers as early as 16 wk of age (24). Studies of immune cell populations isolated from young colitis-free Gi{alpha}2–/– mice reveal widespread evidence of Th1-skewed immune cell activation (12), with elevated colonic IL-12p40 (24), increased anti-CD3-stimulated T cell proliferation, IL2 and IFN-{gamma} production (14), and elevated IL-12 production by macrophages and splenic CD8{alpha}+ dendritic cells (10). This immune activation is present before any histological evidence of disease is present. We have studied the production of Gi{alpha}2-dependent inflammatory mediators in the colonic mucosa and have demonstrated a pronounced defect in arachidonic acid release from subepithelial myofibroblasts, which results in decreased colonic PGE2 levels (6). PGE2 is an anti-inflammatory prostanoid that suppresses Th1 cytokine production from dendritic cells (17) macrophages (30) and T cells (22). Hence decreased mucosal PGE2 appears to exacerbate the development of Th1-skewed colitis, which can be ameliorated by treatment with exogenous PGE2 receptor analogs (6, 15).

IL-11 is a member of the IL-6 cytokine superfamily and has a wide array of biological activities. Its receptor is composed of an effector subunit, gp130 [which is bound by all members of the IL-6 superfamily (28)], and an IL-11-specific subunit, IL-11R{alpha}. In the intestine, there is a paracrine signaling network between the epithelium, which expresses IL-11R{alpha} (4), and subepithelial myofibroblasts, which are a major source of IL-11 (1). Myofibroblasts form a syncytial sheet immediately under the epithelium and are thus positioned to modulate both epithelial and lamina propria mononuclear cell populations, who respond to IL-11 treatment by downregulating production of Th1 cytokines including IL-12 and IFN-{gamma} (3, 18). Importantly, in clinical trials IL-11 has shown promise in the treatment of inflammatory bowel disease (11, 26, 27).

Modulating prostanoid levels has been shown to regulate IL-11 production. In a number of stromal cell lines, indomethacin treatment can block IL-11 production that is rescued by PGE2 treatment (19, 21, 29). We hypothesized that decreased mucosal PGE2 levels in Gi{alpha}2–/– mice would inhibit myofibroblast IL-11 production that might contribute to the Th1-skewed milieu present in the colon of these mice. In this report, we demonstrate decreased levels of IL-11 in the colonic mucosa of Gi{alpha}2–/– mice, which correlates with deficits in Gi{alpha}2–/– myofibroblast IL-11 production in response to TGF-beta or IL-1beta. Inhibition of either ERK or p38 MAPK activity dose dependently inhibited IL-11 production in vitro. ERK activation in response to TGF-beta treatment was modestly decreased in small intestinal myofibroblasts (IMF) and more significantly in colonic myofibroblasts (CMF); however, p38 activation did not appear to be significantly affected. Acutely increasing cAMP levels in myofibroblast cultures significantly enhanced IL-11 production, whereas PKA inhibition and pertussis toxin (PTX) treatment inhibited production. Paradoxically, cAMP levels were elevated in Gi{alpha}2–/– myofibroblasts. We demonstrated the physiological relevance of these observations by treating mice with the EP4-selective prostanoid agonist ONO-AE1-329, which enhanced mucosal levels of IL-11 and stimulated IL-11 production from CMF isolated from treated animals. Together, these data demonstrate a complex role for Gi{alpha}2-dependent cAMP and MAP kinase signaling in the production of anti-inflammatory mediators, which collectively function to suppress excessive Th1 activity in the colonic mucosa.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mice. Wild-type (WT) and Gi{alpha}2–/– mice on a crossbred 129Sv/C57BL6 background, and WT C57/BL6 mice were housed in a specific pathogen-free facility at the University of California, Irvine vivarium. Male Gi{alpha}2–/– mice were bred with heterozygous females to maximize fertility and the yield of knockout (KO) animals. Genotyping was performed on genomic tail DNA by using a multiplex PCR reaction, which generates an 805-bp product for the WT allele and a 509-bp product for the KO allele. All experiments were carried out in accordance with the Institutional Animal Care and Use Committee at the University of California, Irvine, and using protocols approved by the same.

IMF and CMF isolation and culture. The small intestine and colon of 4- to 5-wk-old WT and Gi{alpha}2–/– mice were removed and linearized, and the Peyer's patches were removed. Washed tissues were shaken five times for 15 min in HBSS containing 5 mM EDTA. Each resulting deepithelialized tissue sample was incubated in 20 ml of RPMI-5 containing 10.5 mg of Dispase (GIBCO-Invitrogen, Carlsbad, CA) and 7.2 mg of collagenase D (Roche Diagnostics, Indianapolis, IN) for 2 h in a shaking 37°C incubator. The digested tissue was treated with ACK lysis buffer for 5 min, and the resulting tissue pieces were passed through a 40-µm cell strainer into 100-mm dishes in RPMI-5 [RPMI with 5% FCS (Atlas Biologicals, Fort Collins, CO), 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml Pen-Strep]. After 3 h, the nonadherent cells were washed off; the adherent cells consisted largely of macrophages and myofibroblastic cells. After several days, the macrophages died off and the cultures were composed exclusively of cells with a myofibroblastic phenotype that were consistently smooth muscle actin (SMA) positive and desmin negative. Primary IMF and CMF cultures were used for experiments up to passage 3.

RT and real-time PCR. Reverse transcription (RT) was carried out with a GeneAmp PCR System 9700 (Applied Biosystems). A 25-µl RT reaction included 2 µg of total RNA, 5 µl of M-MLV RT 5 x Buffer (Promega), 0.625 µl of oligo(dT), 0.5 µl of dNTP, 0.313 µl of RNAsin (Promega), 0.625 µl M-MLV RT (Promega), 1.25 µl of 0.1 M dithiothreitol, and 11.69 µl of diethyl pyrocarbonate-treated water. The RT products were incubated at 38°C for 60 min and then at 90°C for 5 min.

Real-time PCR was performed in an OPTICON continuous fluorescence detector (MJ Research). The RT products were diluted 1:20 in deionized, distilled water. PCR was performed with the iQ SYBRgreen Supermix (Bio-Rad). Primer sequences were designed with the help the Primerbank algorithm (31) and are as follows: IL-11, sense 5'-TGGTGTGCTGACAAGGCTTC-3', antisense 5'-ACATCAAGAGCTGTACGGC-3'; SMA, sense 5' GGACGTACAACTGGTATTGTGC-3', antisense 5'-CGGCAGTAGTCACGAAGGAAT-3'; RNA polymerase II alpha subunit (reference gene), sense 5'-ATACCCAGACAACAGAGGG-3', antisense 5'-GCCAGTCCGCTCAATCACC-3'. The PCR mixtures were incubated at 94°C for 5 min and then went through 45 cycles of incubation at 94°C for 15 s, 60°C for 30 s, 72°C for 30 s, and 73°C for 1 s. Then the reactions were incubated at 72°C for 10 min followed by melting curve analysis whereby the temperature was set to 65°C then raised to 95°C and SYBR fluorescence was monitored at every 0.2°C. Semiquantitation was performed by the {Delta}{Delta}Ct method.

Evaluation of IMF/CMF IL-11 protein expression. Cultured IMF and CMF were grown in 96-well plates at 5 x 104 cells/well and treated in duplicate with increasing concentrations (0, 0.01, 0.1, 1, 10, and 50 ng/ml) of IL-1beta and TGF-beta, two cytokines shown to strongly upregulate IL-11 expression (1). After 24 h, culture supernatants were frozen at –80°C until assayed by IL-11 ELISA. For evaluation of the effect of indomethacin and prostaglandin agonists, CMF were pretreated with 20 µM arachidonic acid overnight or with 10 µM 16,16 dimethyl-PGE2 (dmPGE2) 10 µM indomethacin, 1 µM ONO-AE1-329 (EP4-selective agonist, Ono Pharmaceuticals), or 5 µM butaprost (Cayman Chemical) concurrently with 5 ng/ml TGF-beta. For evaluation of the contribution of MAPK signaling to IL-11 release, 5 x 104 cells/well were pretreated with vehicle or with 1–20 µM of PD 98059 or SB202190 for 30 min before the addition of 5 ng/ml TGF-beta for 24 h. For the evaluation of altering intracellular cAMP levels on IL-11 production, cells were pretreated overnight with 100 ng/ml PTX or pretreated with 5 µM H-89 or 0.5 mM 8-bromo-cAMP for 30 min, then with 5 ng/ml TGF-beta for 24 h.

Evaluation of mucosal homogenate IL-11 levels. Colons from groups of five age-matched WT and Gi{alpha}2–/– mice were harvested and flushed with PBS. The distal 1 cm was reserved for histological evaluation. The mucosa was scraped with a razor blade into 1 ml of PBS, and the scrapings were homogenized with a tissue tearer and then sonicated. Homogenates were clarified by spinning at 16,000g for 15 min, and protein concentrations were measured by Bio-Rad protein assay.

Quantification of IL-11 levels. The amount of IL-11 produced by the cells or in mucosal homogenates was determined by sandwich ELISA kits purchased from R&D Systems and performed according to the manufacturer's instructions. The lower detection limit was 6.8 pg/ml for mouse IL-11.

Quantification of myofibroblast cAMP levels. Confluent 100-mm dishes of IMF and CMF were starved overnight, and then treated with either nothing or 10 µM 16,16 dimethyl PGE2 for 20 min. Duplicate plates were harvested in 0.1 M HCl for 20 min and then scraped, and the lysates were cleared by centrifugation. Samples were acetylated and assayed for cAMP content per manufacturer's instructions (Cayman Chemical, Ann Arbor, MI). The data are expressed as picomoles of cAMP per milliliter and are normalized to cell number.

Western blotting. Six-well dishes of subconfluent IMF and CMF were treated with 10 ng/ml TGF-beta for 0, 15, 30, 60, 120, or 240 min and then lysed for 10 min in ice-cold buffer containing 20 mM Tris pH 7.9, 137 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, 1 mM EGTA, 1 mM NaVO4, 10 mM NaF, and Roche Complete-Mini protease inhibitor cocktail (Roche, Nutley, NJ), then scraped, vortexed, and spun at 16,000g for 10 min. Equal amounts of lysate protein were separated by SDS-PAGE and then Western blotting, which were detected by using phospho- or total ERK and p38 MAPK antibodies (1:1,000, Cell Signaling Technology, Danvers, MA) and developed with Pierce Supersignal Pico ECL reagent. For quantitative analysis of band intensities, blots were scanned at 300 dpi. Band intensities were quantified using ImageJ version 1.37 with the Gel Converter plug-in. For the p42/p44 MAPK blots, the intensities of the two bands were summed. The data are expressed as the ratio of phospho-MAPK band to the total MAPK bands and are representative of three separate experiments.

Intrarectal administration of EP4-specific PGE2-Receptor Agonist, ONO-AE1-329. Groups of six age-matched (6–9 wk old) WT and Gi{alpha}2–/– mice were treated intrarectally with either 100 µg/kg ONO-AE1-329 in ethanol (kindly provided by Dr. Takayuki Maruyama, Ono Pharmaceuticals, Osaka, Japan) or ethanol alone, daily over a 4-day period. This dose was selected because it was shown to be effective at suppressing chronic dextran sodium sulfate colitis over a 14-day period (23). After mice were lightly anesthetized with ketamine-xylazine anesthetic, treatments consisted of inserting a lubricated 3.5-Fr silicone catheter (Instech, Plymouth Meeting, PA) 2 cm into the rectum and injecting 50 µl of compound or vehicle. The catheter was then advanced to the 4-cm mark and another 50 µl were injected. At the end of the 4-day period, the mice were killed and the colons were removed and the lumens flushed of fecal material. The mucosa from the distal 4 cm of each tissue was scraped into 250 µl ofPBS and sonicated, and the sonicate was cleared at 16,000g for 15 min. The cleared lysates were subjected to IL-11 ELISA as described above.

To evaluate the effect of in vivo prostanoid treatment on myofibroblast IL-11 production, groups of 10 WT C57BL/6 mice were treated as above with either ONO-AE1-329 or ethanol. Two hours after the final dose, the distal 4 cm of rectum were harvested and myofibroblasts were isolated as described above. Three hours after plating, supernatants were subjected to IL-11 ELISA and the results were normalized to the number of adherent cells present in the culture.

Statistics. P values were calculated by Student's t-test assuming equal variances.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Decreased IL-11 levels in the colonic mucosa of Gi{alpha}2–/– mice. Gi{alpha}2–/– mice spontaneously develop colitis beginning at 6 wk of age. Our previous work demonstrated a defect in arachidonic acid metabolism in the absence of Gi{alpha}2 that results in decreased mucosal levels of PGE2. Since IL-11 production has been shown to be enhanced by PGE2 (5, 19, 21), we hypothesized that defects in prostanoid formation might result in concomitant suppression of IL-11 production. As shown in Fig. 1 (and in the vehicle-treated animals in Fig. 7A), tissue levels of IL-11 were significantly decreased in scraped colonic mucosa of Gi{alpha}2–/– mice (proximal colon: WT 0.136 ± 0.073 ng/mg protein vs. Gi{alpha}2–/– 0.048 ± 0.048 ng/mg protein; distal colon: WT 0.554 ± 0.44 ng/mg protein vs. Gi{alpha}2–/– 0.076 ± 0.104 ng/mg protein). Histological evaluation of the colons from the Gi{alpha}2–/– revealed mild colitis in all animals, consistent with the natural history of colitis in Gi{alpha}2–/– mice.


Figure 1
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  		Fig. 1. Decreased mucosal IL-11 levels in Gi{alpha}2–/– colonic mucosal homogenates. Mucosal scrapes from the proximal and distal groups of 5 age-matched wild-type (WT) and Gi{alpha}2–/– mice (8–10 wk) were assayed for IL-11 content by IL-11 ELISA. Data represent means ± SD and are representative of 3 separate experiments.

 

Figure 7
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  		Fig. 7. PGE2 receptor agonist treatment enhances mucosal IL-11 levels and IL-11 production by ex vivo-cultured colonic myofibroblasts. Age-matched WT and Gi{alpha}2–/– mice (n = 6) were treated for 4 days with daily intrarectal doses of 100 µg/kg ONO-AE1-329 in ethanol (EtOH) or EtOH alone. Mucosal scrapes were taken from the distal 4 cm of the treated colon and assayed for IL-11 content (A). Data represent means ± SD; asterisks denote significant (*P < 0.05, **P < 0.01) enhancement while # denotes significant (P < 0.05) decrease in IL-11 levels of IL-11 levels. B: age-matched WT C57BL/6 (n = 10) mice were treated as above, and the distal 4 cm of colon used to isolate colonic myofibroblasts. Three hours after plating, the culture supernatants were assayed for IL-11 content and the results were normalized to the number of adherent cells in the culture.

 
Small intestine and colonic myofibroblast production of IL-11 mRNA and protein. A major source of IL-11 in the colon is the subepithelial myofibroblast, which is stimulated to release IL-11 most strongly by TGF-beta and IL-1beta (1). We evaluated basal, TGF-beta-stimulated, and IL-1beta-stimulated IL-11 release from primary cultures of WT and Gi{alpha}2–/– CMF. For comparison, we also studied IL-11 production in primary IMF cultures as well. Preliminary time-course experiments with TGF-beta or IL-1beta revealed significant increases in IL-11 production beginning 12 h after initiation of treatment, with peak expression between 12 and 24 h (data not shown), consistent with previous reports (1). We then treated WT or Gi{alpha}2–/– IMF (Fig. 2, A and B) and CMF (Fig. 2, C and D) with increasing concentrations of either TGF-beta or IL-1beta. At all concentrations, Gi{alpha}2–/– IMF produced between 40 and 50% as much IL-11 as WT cells, whereas IL-11 release from Gi{alpha}2–/– CMF was more profoundly affected (Fig. 2, C and D). At higher concentrations of IL-1beta (10–50 ng/ml), there was a reproducible decline in elicited IL-11 compared with 1 ng/ml IL-1beta. On a per-cell basis, IMF produced considerably higher levels of IL-11 than CMF.


Figure 2
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  		Fig. 2. Small intestinal (IMF) and colonic myofibroblast (CMF) IL-11 production is decreased in Gi{alpha}2–/– mice. Primary cultures of IMF (A and B) and CMF (C and D) were plated at 5 x 104 cells/well and cultured in increasing concentrations of TGF-beta or IL-1beta for 24 h. Supernatants were assayed by ELISA. Data represent means ± SD and are representative of 4 iterations of each experiment. **Significant (P < 0.05) differences between equivalently treated WT and Gi{alpha}2–/– cells.

 
To corroborate the ELISA data, we compared IL-11 mRNA expression between IL-1beta and TGF-beta-treated WT and Gi{alpha}2–/– IMF. Serum-starved cells were treated with 10 ng/ml IL-1beta or 25 ng/ml TGF-beta for 8 h, then harvested for total RNA isolation. Pooled data from multiple real-time semiquantitative PCR experiments revealed significant decreases in IL-11 message from Gi{alpha}2–/– IMF (Table 1). Basal IL-11 expression in Gi{alpha}2–/– IMF was 13 ± 13% that of WT cells; 8 h after IL-1beta treatment, IL-11 mRNA levels in Gi{alpha}2–/– cells was 25 ± 27% that of WT, and TGF-beta treatment resulted in 16 ± 14% as much. To demonstrate the specificity with which the loss of Gi{alpha}2 affects IL-11 expression, we also compared the effect of IL-1beta and TGF-beta on expression of {alpha}-SMA, which has been reported in myofibroblasts to be inhibited by IL-1beta (13) and induced by TGF-beta (32). As shown in Table 1, pooled mean basal and cytokine stimulated IMF {alpha}-SMA expression did not vary significantly between WT and Gi{alpha}2–/– cells.


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  		Table 1. qPCR data

 
Involvement of ERK and p38 MAPK activity in CMF IL-11 production. The lack of Gi{alpha}2 results in decreased ligand-dependent activation of ERK activation (20), and previous reports have indicated a role for MAPK signaling in the induction of IL-11 expression in response to TGF-beta or IL-1beta treatment (1). We determined whether pharmacological ERK or p38 MAPK inhibition would differentially inhibit IL-11 production in the presence or absence of Gi{alpha}2. As shown in Fig. 3, pretreatment with either PD 98059 and SB 203580 inhibited IL-11 release from TGF-beta-treated WT and Gi{alpha}2–/– IMF (Fig. 3A) and CMF (Fig. 3B) in a dose-dependent manner. For a given dose, CMF IL-11 release appeared to be more sensitive to either ERK or p38 inhibition than was observed in IMF cultures. The degree of suppression of IL-11 production by either inhibitor was comparable between WT and Gi{alpha}2–/– cells. However, when WT and Gi{alpha}2–/– IMF and CMF were pretreated with between 100 nM and 100 µM of the p38 MAPK activator anisomycin for 30 min before addition of concomitant TGF-beta, no effect on IL-11 production was seen (data not shown).


Figure 3
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  		Fig. 3. Dose-dependent suppression of myofibroblast IL-11 production by ERK and p38 MAPK inhibitors. Serum-starved IMF (A) or CMF (B) were pretreated with 1–20 µM PD 98059 (PD) or SB 203580 (SB) for 30 min, then TGF-beta was added to a final concentration of 5 ng/ml for 24 h, and then their supernatants were collected for sandwich ELISA. **Significantly (P ≤ 0.05) decreased IL-11 production compared with WT or knockout (KO) CMF treated with TGF-beta alone.

 
Loss of Gi{alpha}2 signaling impairs TGF-beta-induced ERK, but not p38 MAPK activation. T cells from mice deficient in Gi{alpha}2 do not respond normally to TGF-beta-induced suppression of proliferation (33), and MAPK activation has been shown to be important in the induction of IL-11 expression (1). We therefore tested whether there were defects in the phosphorylation of ERK or p38 MAPK following TGF-beta treatment of IMF and CMF. As shown in Fig. 4A, treatment of IMF or CMF with 10 ng/ml TGF-beta induced considerably less phosphorylation of ERK1/2 in both WT and Gi{alpha}2–/– IMF. Scanning densitometry of the summed ERK1/2 bands in IMF and CMF (Fig. 4, B and D) indicates consistently higher P-ERK/ERK ratios in WT vs. Gi{alpha}2–/– cells (one representative experiment is shown), although the differences were not statistically significant when all three replicates were averaged, owing to variability in the relative band intensities across experiments. Phospho-p38 MAPK levels were minimally increased 15 min after TGF-beta stimulation in IMF and CMF (Fig. 4, C and E), with no consistent difference between WT and Gi{alpha}2–/– cells at any time point.


Figure 4
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  		Fig. 4. Decreased Gi{alpha}2–/– myofibroblast ERK, but not p38 MAPK, phosphorylation following TGF-beta stimulation. A: serum-starved IMF and CMF were treated with 10 ng/ml TGF-beta for the indicated time points and lysed, and equivalent amounts of protein were separated by SDS-PAGE. In both IMF and CMF, the induction of ERK1/2 phosphorylation was lower in Gi{alpha}2–/– IMF and CMF. Phospho-p38 levels increased marginally in IMF and CMF by 15 min, but no apparent differences between WT and Gi{alpha}2–/– cells were seen. B–E: ratios of phosphorylated vs. total ERK1/2 (B and D) and p38 (C and E) as measured by image analysis confirm the deficit in ERK1/2 phosphorylation in Gi{alpha}2–/– IMF and CMF. The data shown represent 1 representative experiment of at least 3 replicates of each cell type.

 
Complex regulation of myofibroblast IL-11 production by prostaglandin and modulation of cAMP levels. Previous studies have demonstrated a stimulatory role for PGE2 in the production of IL-11 by stromal cells (19). PGE2 acts at EP2 and EP4-type PGE2 receptors by elevating intracellular levels of cAMP. However, whereas Gi{alpha}2–/– embryonic fibroblasts have been shown in previous studies to have elevated intracellular levels of cAMP (20), they clearly have defects in IL-11 production. This suggests a complex relationship between PGE2 signaling, cAMP levels, and IL-11 synthesis. To reconcile these observations, we tested whether the enhancement or blockade of PGE2 and cAMP production would affect IL-11 production in cultured myofibroblasts, and measured cAMP levels in WT and Gi{alpha}2–/– IMF. For both IMF (Fig. 5A) and CMF (Fig. 5B), concurrent treatment with either exogenous 16,16 dimethyl PGE2 or the EP2-specific agonist butaprost significantly potentiated TGF-beta-induced IL-11 release compared with TGF-beta treatment alone, whereas indomethacin significantly inhibited TGF-beta-induced IL-11 release. In Gi{alpha}2–/– CMF, indomethacin decreased the amount of IL-11 produced in response to TGF-beta to a lesser degree, but the difference was not statistically significant. In IMF and in Gi{alpha}2–/– CMF, concurrent treatment with the EP4-selective agonist ONO-AE1-329 enhanced TGF-beta-induced IL-11 production to a significant extent, but less than with butaprost treatment.


Figure 5
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  		Fig. 5. PGE2 analogs enhance TGF-beta-induced IMF and CMF IL-11 production. Serum-starved IMF (A) or CMF (B) were pretreated with 10 µM 16,16-dimethyl PGE2 (dmPGE2), 1 µM ONO-AE1-329 (ONO; EP4 agonist), 5 µM butaprost (EP2 agonist), or 10 µM indomethacin before treatment with 5 ng/ml TGF-beta for 24 h. Data represent means ± SD, and symbols denote significantly (P ≤ 0.05) increased (**) or decreased (#) IL-11 production compared with treatment with TGF-beta alone.

 
Next, we tested the effects of modulating intracellular levels of cAMP on IL-11 release. As shown in Fig. 6, A and B, overnight pretreatment of either WT or Gi{alpha}2–/– cells with 100 ng/ml PTX significantly inhibited TGF-beta induced IL-11 production, as did 30 min pretreatment with 5 µM of the selective PKA inhibitor H-89. In contrast, the cAMP analog 8-Br-cAMP induced significant elevations in IL-11 production compared with TGF-beta treatment alone in IMF and WT CMF, but not in Gi{alpha}2–/– CMF.


Figure 6
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  		Fig. 6. Modulation of cAMP levels regulate TGF-beta-induced IMF and CMF IL-11 production. Serum-starved IMF (A) or CMF (B) were pretreated overnight with 100 ng/ml pertussis toxin (PTX) or for 30 min with 5 µM H-89 or 500 µM 8-Br-cAMP before treatment with 5 ng/ml TGF-beta for 24 h. Asterisks denote significantly (P ≤ 0.05) increased (**) or decreased (*) IL-11 production compared with treatment with TGF-beta alone. C: comparison of cAMP levels between WT and Gi{alpha}2–/– IMF and CMF either serum starved or 16,16 dimethyl PGE2 (10 µM)- treated measured by cAMP enzyme immunoassay. **Significantly (P ≤ 0.05) increased cAMP levels in Gi{alpha}2–/– cells vs. WT cells. Data represent means ± SD of triplicate measurements and are representative of 3 experiments.

 
The finding that PTX treatment (which block all Gi-coupled signaling and should therefore enhance cAMP levels) blocks IL-11 release, yet 8-Br-cAMP treatment enhances IL-11 release prompted an evaluation of intracellular cAMP levels in WT and Gi{alpha}2–/– cells. As shown in Fig. 6C, basal and dmPGE2-stimulated cAMP levels were significantly higher in Gi{alpha}2–/– myofibroblasts than in WT cells, consistent with previous studies demonstrating higher cAMP levels in Gi{alpha}2–/– fibroblasts (20).

In vivo treatment with the PGE2-EP4 receptor agonist ONO-AE1-329 enhances IL-11 levels in the mucosa and in ex vivo-cultured myofibroblasts. To demonstrate a direct, physiological relationship between PGE2 treatment and IL-11 production in vivo, we first treated WT and Gi{alpha}2–/– mice (n = 6) intrarectally with the EP4-selective agonist ONO-AE1-329 and then measured mucosal IL-11 concentrations from the distal 4 cm of colon. As shown in Fig. 7A, 4 days of ONO treatment resulted in significant (WT: P = 0.008, Gi{alpha}2–/–: P = 0.005) increases over basal mucosal IL-11 levels (which, as in Fig. 1, were greater in WT than Gi{alpha}2–/– animals) compared with vehicle-treated mice (P = 0.005). We then tested whether the increase in mucosal IL-11 levels following intrarectal ONO compound correlated with enhanced colonic myofibroblast IL-11 production. WT C57BL/6 mice were given daily intrarectal ONO in ethanol, or vehicle alone, for 4 days. Myofibroblasts were isolated from the treated colonic segments 2 h after the final dose and cultured for 3 h. In Fig. 7B, we demonstrate a significant (P = 0.038, n = 10) increase in IL-11 release from CMF derived from the ONO-treated animals compared with vehicle-treated control mice.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We have previously shown that decreased arachidonate release from intestinal myofibroblasts leads to lower colonic levels of PGE2 in the Gi{alpha}2–/– mouse model of inflammatory bowel disease (6). Defects in TGF-beta signaling (33), ERK activation (20), and altered adenylate cyclase activity (25) have also been described in cells from Gi{alpha}2–/– mice. Because all of these signaling pathways have been reported to impinge on IL-11 production, in this study we investigated the effect of modulating these pathways in vitro and in vivo. We found significant decreases in mucosal IL-11 concentrations in the absence of Gi{alpha}2, which correlated with decreased Gi{alpha}2–/– myofibroblast IL-11 production in vitro and which could be enhanced in vivo by treating animals with intrarectal prostanoid agonists.

There are a number of mechanisms by which decreased IL-11 levels might contribute to the colitis and cancer phenotype of Gi{alpha}2–/– mice. IL-11 has effects on leukocyte populations, inhibiting the Th1-skewing of CD4+ T cells (3) and suppressing macrophage IL-1, TNF-{alpha}, and IL-12 production (18). Since colitic Gi{alpha}2–/– mice have increased numbers of activated CD4+ T cells in the lamina propria (12) and elevated IL-12 and TNF-{alpha} levels in activated splenocytes (10), decreases in mucosal IL-11 levels and thus its suppressive effect on elaboration of these Th1 cytokines may contribute to the skewed cytokine milieu of the colon. In addition, altered epithelial IL-11 signaling may be a significant contributor to the onset of colitis or the failure to resolve mucosal damage in this model, given the paracrine signaling pathway that exists between IL-11-producing CMF and the IL-11 responsive colonic epithelium (16). IL-11 has been shown to be important in mediating repair in damaged epithelia (8). We are in the process of crossing Gi{alpha}2–/– mice with IL-11 receptor-{alpha} KO mice, to determine whether abrogation of IL-11 signaling in the epithelium will worsen the colitis phenotype.

Our data reveal several differences between IL-11 production in the small intestine and the colon. Comparison of the relative production of IL-11 by IMF and CMF revealed significantly lower levels of IL-11 (Figs. 2, 3, 5, and 6) produced by primary cultured CMF on a per-cell basis. However, ELISA results from homogenates of small intestinal mucosal scrapes were all below the sensitivity limits of the assay (6.8 pg/ml), suggesting that the thicker, villiform small intestinal mucosa diluted out the relatively higher per-cell IL-11 production by IMF. This may also explain the relatively higher IL-11 levels in the distal mucosal scrapes shown in Fig. 7, because the mucosa of the distal mouse colon is considerably thinner than that of the proximal colon.

We also found that PGE2 restores TGF-beta-stimulated IL-11 production in Gi{alpha}2–/– CMF cells to the levels observed in WT CMFs treated with TGF-beta alone. This was not the case in Gi{alpha}2–/– IMF cells (Fig. 5B vs. 5A). This difference may be explained by considering the fold increase in IL-11 production induced by concomitant dmPGE2 and TGF-beta treatment. In the WT CMF, dmPGE2 enhanced IL-11 production 3.6-fold, whereas the KO CMF increased 8-fold. In the WT IMF, dmPGE2 enhanced IL-11 release 1.5-fold, whereas the KO IMF release doubled. Thus the CMF appear to be generally more responsive to PGE2 treatment than the IMF. Whether this is related to the relatively higher per-cell production of IL-11 by IMF is not clear.

The data shown in Fig. 6 reveal a complex relationship between PGE2 receptor activation, cAMP levels, and IL-11 production. In both WT and Gi{alpha}2–/– cells, EP2 and EP4 agonists (which elevate cAMP levels) enhanced IL-11 production in short-term (24 h) cultures. Likewise, direct activation of PKA by 8-Br-cAMP led to potentiation of IL-11 release. However, Gi{alpha}2–/– cells clearly have higher basal and PGE2-stimulated cAMP levels than their WT counterparts (Fig. 6C) yet produce less IL-11 under all conditions. Therefore, whereas acute elevations in cAMP levels induce IL-11 production, chronic increases in intracellular cAMP must lead to compensatory changes in signaling pathways. Indeed, the constitutive absence of Gi{alpha}2 protein that is associated with elevated cAMP levels also results in alterations in the expression of other G-protein signaling molecules, most notably an increase in Gi{alpha}3, and a 30–50% decrease in levels of beta{gamma}-subunits (25). Alterations in the expression of adenylyl cyclase isoforms or PKA regulatory subunits in the constitutive absence of Gi{alpha}2 may also be contributory. Since beta{gamma}-subunits are important in the activation of ERK1/2 via a Ras-dependent pathway (9), deficits in beta{gamma} signaling may account for our observed decreases in the duration of ERK activation in Gi{alpha}2–/– IMF. Overnight PTX pretreatment also inhibited TGF-beta-induced IL-11 production, perhaps by immobilizing additional beta{gamma}-subunits that can activate MAPK and subsequent IL-11 synthesis by preventing their dissociation from ADP-ribosylated {alpha}-subunits.

We also noted differences in the efficacy of EP-selective agonists in potentiating TGF-beta-induced IL-11 production, because the EP2-selective agonist butaprost was more effective than the EP4-selective agonist ONO-AE1-329 (Fig. 4). Recent work has also demonstrated divergence in the signaling pathway from EP2 vs. EP4 receptors. EP2 primarily activates PKA, whereas EP4 activation results in PKA activity, as well as ERK activation in a phosphatidylinositol 3-kinase-dependent manner. EP2 activation thus may result in a more direct induction of cAMP formation than EP4 activation (7).

Whereas p38 inhibition clearly inhibited TGF-beta-stimulated IL-11 release (Fig. 3), we did not see any potentiation of TGF-beta-stimulated IL-11 production by pretreatment with anisomycin (data not shown). Likewise, we found minimal increases in the ratio of phosphorylated to total p38 following TGF-beta treatment (Fig. 4, C and E). We conclude that, whereas SB 203580 can block TGF-beta-induced IL-11 release from myofibroblasts, p38 activation does not appear to enhance IL-11 release. This apparent paradox may be explained by the finding that SB 203580 has been shown to reversibly inhibit both cyclooxygenase-1 and cyclooxygenase-2 (2). Therefore, it is possible that SB 203580's inhibitory effect on IL-11 release is due to its interference with arachidonic acid metabolism and prostaglandin production, which is necessary for IL-11 production.

In summary, we have shown that the absence of Gi{alpha}2 leads to basal and cytokine-stimulated defects in the production of IL-11 by primary cultures of murine IMF and CMF, which results in decreased colonic mucosal IL-11 levels. We have shown that PGE2-receptor analogs are capable enhancing TGF-beta-stimulated IL-11 release, whereas indomethacin inhibits IL-11 release, suggesting that myofibroblast PGE2 production serves as an autocrine enhancer of IL-11 release. Finally, we have shown that acute upregulation of intracellular cAMP increases myofibroblast IL-11 release, whereas chronic elevations in cAMP in the absence of Gi{alpha}2 lead to cellular signaling adaptations that lead to decreased IL-11 production. These data suggest that previously recognized defects in arachidonic acid metabolism and concomitant PGE2 production in the absence of Gi{alpha}2 (6) may in turn result in decreased mucosal IL-11 production, providing another mechanism by which Gi{alpha}2–/– mice may be predisposed to the spontaneous development of Th1-skewed colitis.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant no. K08-DK-59816 to R. A. Edwards.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Takayuki Maruyama of Ono Pharmaceuticals, Osaka, Japan for the kind gift of ONO-AE1-329 used in these studies.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. A. Edwards, Dept. of Pathology, D449 Med Sci I, Univ. of California Irvine, Irvine, CA 92697-4800 (e-mail: redwards{at}uci.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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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