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MUCOSAL BIOLOGY

Heregulin- and heregulin- expression is linked to a COX-2-PGE₂ pathway in human gastric fibroblasts

Third Department of Internal Medicine, Nippon Medical School, Bunkyo-ku, Tokyo, Japan

Submitted 1 June 2005 ; accepted in final form 8 December 2005

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We have previously shown heregulin (HRG)- expression in human gastric fibroblasts and its stimulation of gastric epithelial cell growth. Although cyclooxygenase (COX)-2 has also been shown to stimulate growth factor production in these cells, the interaction between COX-2 and HRG remains unknown. Conditioned media (CM) from gastric fibroblasts incubated with PGE₂ or interleukin (IL)-1, a well known COX-2 inducer, were analyzed for their effect on erbB3 tyrosine phosphorylation in MKN28 gastric epithelial cells. HRG protein expression in fibroblast lysates and CM was also examined by western blot. HRG- and HRG- mRNA expression in gastric fibroblasts and human gastric tissue was examined by real-time quantitative PCR. HRG and COX-2 expressions in surgical resections of human gastric ulcer tissue were examined immunohistochemically. CM from fibroblasts incubated with PGE₂, or IL-1, stimulated erbB3 phosphorylation in MKN28 cells. Preincubation of the fibroblasts with celecoxib, a selective COX-2 inhibitor, suppressed CM-induced erbB3 phosphorylation. This inhibition was reversed by exogenous PGE₂. As with erbB3 phophorylation, IL-1 stimulated both HRG- and HRG- mRNA expression, as well as HRG release into gastric fibroblast CM. IL-1-stimulated HRG expression and release were also inhibited by celecoxib, and exogenous PGE₂ restored this inhibitory effect, suggesting the activation of an IL-1-COX-2-PGE₂ pathway that culminates in the release of HRG from fibroblasts. HRG- and HRG- mRNA levels were significantly higher in gastric ulcer tissue than in normal gastric mucosa. HRG immunoreactivity was found in interstitial cells of the gastric ulcer bed and coexpressed with COX-2. These results suggest that HRG might be a new member of the growth factor family involved in the COX-2-dependent ulcer repair process.

cyclooxygenase-2

Cyclooxygenase (COX)-2 is induced in fibroblasts or macrophages by the stimulation of inflammatory cytokines such as IL-1 and catalyzes the reaction of arachidonic acid to PGs (6, 25, 39). COX-2 expressed in fibroblasts and macrophages at the ulcer edge and ulcer bed of the gastric mucosa is thought to play a key role in the ulcer repair process of the stomach, because administration of COX-2 selective inhibitors has been shown to delay ulcer healing (23, 30, 34). PGE₂ derived from COX-2 expressed in fibroblasts and macrophages has been suggested to contribute to growth factor production, similar to hepatocyte growth factor (HGF) and VEGF, via autocrine mechanisms (4, 22, 33). Although both COX-2 and HRG are expressed in gastric fibroblasts and both have been shown to contribute to gastric epithelial cell growth, the interaction between COX-2 and HRG has yet to be determined. Therefore, in the present study, we investigated the possibility that HRG expression in gastric fibroblasts may be linked to the COX-2-PGE₂ pathway. In our study, we used tyrosine phosphorylation of erbB3 as a biological marker of HRG release as well as Western blot analysis of HRG itself, and we examined whether HRG depends on a COX-2-PGE₂ pathway for its expression in gastric fibroblasts. We also examined and compared expression levels of HRG- and HRG- mRNA by real-time PCR analysis. Finally, we determined HRG expression and localization in human gastric ulcer tissue by real-time PCR analysis and immunohistochemistry, respectively.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cell cultures. The MKN28 human gastric cancer cell line was obtained from the Health Science Research Resources Bank (Osaka, Japan); human gastric fibroblasts (Hs 262. St), NIH-3T3 cells, and HT-1080 human fibrosarcoma cells were purchased from the American Type Culture Collection (ATCC; Rockville, MD). MKN28 cells and gastric fibroblasts were cultured in RPMI 1640 medium supplemented with 10% FCS, 50 U/ml penicillin, and 50 µg/ml streptomycin at 37°C in a 5% CO₂ humidified atmosphere. NIH-3T3 cells and HT-1080 cells were cultured in DMEM supplemented with 10% FCS, 50 U/ml penicillin, 50 µg/ml streptomycin, and 4 mM glutamine at 37°C in a 5% CO₂ humidified atmosphere.

Antibodies and reagents. IL-1 and TNF- were purchased from Genzyme-Techne (Minneapolis, MN); human recombinant HRG- EGF domain was from R&D systems (Minneapolis, MN); the selective COX-2 inhibitor celecoxib was from Pharmacia (Skokie, IL); PGE₂ was from Sigma-Aldrich (St. Louis, MO); erbB3 neutralizing antibody (Ab-5), particularly well suited for inhibiting the binding of HRG to erbB3, was from Neomarkers (Fremont, CA). Antibodies for Western blot analysis and immunohistochemical studies were as follows: rabbit anti-human COX-2 polyclonal antibody (C295) from IBL (Gunma, Japan); mouse anti-phosphotyrosine monoclonal antibody (PY-20) from ICN (Costa Mesa, CA); rabbit anti-human polyclonal antibodies against erbB2 (C-18), erbB3 (C-17), and HRG- (C-20), later shown to react with both HRG- and HRG-, were from Santa Cruz Biotechnology (Santa Cruz, CA); mouse anti-human -actin monoclonal antibody was from Sigma; horseradish peroxidase (HRP)-conjugated donkey anti-rabbit immunoglobulin IgG; and sheep anti-mouse IgG was from Amersham Biosciences (Buckinghamshire, UK); mouse anti-HRG monoclonal antibody raised against HRG- was from Novocastra (Newcastle, UK); and mouse anti-vimentin monoclonal antibody raised against DAKO (Carpinteria, CA) and biotinylated anti-mouse IgM and Texas red-conjugated goat anti-rabbit IgG was from Vector Laboratories (Burlingame, CA).

Preparation of cellular extracts and conditioned media and Western blot analysis. Human gastric fibroblasts were grown to confluence in 10-cm dishes and washed with PBS before 24 h of starvation. Cells were then incubated with or without IL-1 (10 ng/ml), TNF- (10 ng/ml), or PGE₂ (10 µM) for an additional 24 h. In some experiments, cells were pretreated with celecoxib at 10 µM for 60 min, a molarity shown in previous studies to selectively inhibit COX-2 activity in vitro. Supernatants were then recovered as conditioned media (CM) and cells lysed in protein lysis buffer [RIPA buffer: 50 mM Tris·HCl buffer (pH 8.0) containing 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 0.5% deoxycholate, 1 mM phenylmethylsulfonyl fluoride, and 5 µg/ml pepstatin A]. Thereafter, lysates containing 50 µg protein were separated in 10% SDS-PAGE gels and proteins were transferred electrophoretically onto polyvinylidene difluoride transfer membranes (Amersham Biosciences). Blots were blocked in 5% nonfat milk and probed with anti-HRG (C-20) or anti-COX-2 antibodies for 2 h at room temperature. Membranes were then washed with 0.05% Tween 20 in Tris-buffered saline and incubated with HRP-conjugated anti-rabbit IgG antibodies. Immunoreactive bands were visualized with an enhanced chemiluminescence (ECL) detection kit (Amersham Biosciences). For immunoblot analysis of CM, CM were concentrated down to 1% of initial volume with a Centricon YM-30 concentrator (Millipore, Bedford, MA) and subjected to Western blot analysis using anti-HRG antibodies as described previously. The protein concentration was measured with BIO-RAD DC protein assay reagent (Biorad, Hercules, CA).

Immunoprecipitation. MKN28 cells were stimulated with gastric fibroblast CM for 5 min and lysed with RIPA buffer containing 1 mM sodium orthovanadate. For inhibition studies, MKN28 cells were pretreated with anti-erbB3 neutralizing antibodies for 60 min. Cleared lysates were incubated with 1 µg anti-erbB2 or anti-erbB3 receptor antibodies coupled with protein G-Sepharose (Amersham Biosciences) for 4 h at 4°C. Immunoprecipitates were washed three times with a solution containing 50 mM HEPES (pH 7.6), 150 mM NaCl, and 0.1% Triton X-100; then they were boiled for 5 min in SDS sample buffer. Samples were fractionated by 7.5% SDS-PAGE, then subjected to Western blot analysis with PY-20 anti-phosphotyrosine antibodies to examine phosphorylation of the receptors, as described previously.

Stable transfection of HRG- into NIH-3T3 cells. Full-length HRG- cDNA was isolated by PCR from HT-1080 cell RNA with oligonucleotides based on the published DNA sequence. PCR products were subcloned into pBlueScript II plasmid vectors (Stratagene, La Jolla, CA) and transformed chemically into JM109 Escherichia coli cells. Nucleotide sequencing was performed by Sequencing Pro (Toyobo, Osaka, Japan). cDNA clones were inserted into SR plasmid vectors (pSR). NIH-3T3 cells were transfected with both 6 mg pSR containing HRG- cDNA and 0.6 mg hph plasmid containing the hygromysin B phosphotransferase gene, following the LipofectAMINE protocol (GIBCO-BRL, Rockville, MD). Cells were selected by hygromycin B (300 mg/ml) following isolation of colonies 14 days after transfection.

Real-time quantitative RT-PCR for HRG- and HRG- isoforms. Gastric tissue samples from 10 subjects (5 males, 5 females; mean age, 60.7; range, 36–82 yr) were obtained from the edge of ulcers by endoscopic biopsy. Ten healthy individuals without Helicobacter pylori infection (5 males, 5 females; mean age, 55.9; range, 32–76 yr) were also included in the study. Patients receiving nonsteroidal anti-inflammatory drugs for medical indications were excluded. All subjects gave informed consent and the project reviewed and approved by the ethics committee of Nippon Medical School, Tokyo, Japan. Total RNA was isolated from human gastric fibroblasts incubated with IL-1 (10 ng/ml) in the presence or absence of celecoxib at 10 µM and from the aforementioned gastric tissue samples with an RNeasy Mini Kit (Qiagen Sciences; Germantown, MD) according to manufacturer's instructions. Reverse transcription (RT) of RNA (2 µg) was performed with an Omniscript RT Kit (Qiagen; Hilden, Germany). Briefly, the reaction was performed in the presence of RT buffer, 0.5 mM each dNTP, 1 µM random hexamers, 0.5 U/µl RNase inhibitor, and 0.2 U/µl Omniscript RT for 60 min at 37°C. After cDNA synthesis from the cellular RNA, real-time PCR was performed for HRG-, HRG-, and -actin to quantitate HRG- and HRG- mRNA expression levels (GenAmp 5700; Applied Biosystems, Foster City, CA). TaqMan probes and primers for HRG- and HRG- were Assay-on-Demand gene expression products (Applied Biosystems, Assay ID Hs01103794_m1 and Hs00247624_m1, respectively). A -actin primer/probe set (Applied Biosystems) was used as the internal control for input cDNA. A serial dilution of cDNA from human gastric fibroblasts stimulated with PGE₂ was used to generate a standard curve by plotting the cycle threshold versus the log of input cDNA. The thermal cycling conditions included 2 min at 50°C and 10 min at 95°C, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. To control variations in the amount of input cDNA, the level of HRG mRNA for each sample was normalized to -actin mRNA levels. All values are the means of triplicates, with <3% variation between triplicates. To verify specificity of HRG primers, the resulting amplification products were separated by electrophoresis on a 1.5% agarose gel and visualized with ethidium bromide (data not shown).

Immunohistochemistry. Gastric ulcer tissue samples with perforation were obtained by surgical resection from patients not undergoing H2 receptor antagonist or proton pump inhibitor treatment at the time of resection. Resected specimens were fixed in 10% formalin, embedded in paraffin wax, and cut into 3-µm sections. Sections were rehydrated and immersed in 0.3% hydrogen peroxide (H₂O₂) in methanol solution for 30 min to block endogenous peroxidase activity. Sections were then microwaved in 0.01 M citrate phosphate buffer (pH 6.0) for antigen retrieval and incubated with 10% normal goat serum for 10 min at 37°C to block nonspecific IgG binding. Thereafter, the sections were incubated for 18 h at 4°C with anti-HRG monoclonal antibodies (dilution 1:25). They were treated for 1 h at 37°C with biotinylated anti-mouse IgM (dilution 1:200), followed by treatment with avidin and biotinylated peroxidase complex (DakoCytomation; Glostrup, Denmark) for 1 h at room temperature. Reaction products were developed by immersing sections in 3,3'-diaminobenzidine tetrahydrochloride solution containing 0.03% H₂O₂. Nuclei were counterstained with Mayer's hematoxylin. Nonimmunized mouse IgM was used for negative control. Double-immunofluorescence staining and confocal laser scanning microscopy were also performed to evaluate the colocalization of immunoreactivity for COX-2 and HRG with a COX-2 polyclonal antibody (dilution 1:10) and HRG monoclonal antibody (dilution 1:5). Sections were incubated overnight at 4°C with a mixture of the two primary antibodies. The antibody against COX-2 was allowed to react with goat anti-rabbit IgG (dilution, 1:100) labeled with Texas red, the antibody against HRG, with biotinylated goat anti-mouse IgM (dilution, 1:100). Antibody binding was detected by streptavidin FITC; followed by nuclear counterstaining with 4',6-diamidino-2-phenylindole (DAPI; Vector) for 15 min to facilitate identification of morphological features. Immunohistochemical control procedures such as those described previously for single labeling were used in conjunction with dual staining methods. All preparations were examined with a confocal microscope (model TCS4D/DMIRBE; Leica, Heidelberg, Germany), equipped with argon and argon-krypton laser sources.

Statistics. The data of triplicate experiments are expressed as the means ± SE and analyzed by unpaired Student's t-tests. P values <0.05 are considered statistically significant. Comparisons among several groups were made using ANOVA with Mann-Whitney U-tests where appropriate. A P value of <0.05 shows a significant statistical difference.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
COX-2 expression in human gastric fibroblasts. We first examined the effects of proinflammatory cytokines IL-1 and TNF- on COX-2 expression in cultured gastric fibroblasts. IL-1 stimulation for 24 h significantly increased COX-2 protein expression in gastric fibroblasts, whereas TNF- stimulation for 24 h had no effect (Fig. 1). There was also no change in COX-1 protein expression levels with or without IL-1 stimulation (data not shown). Thus we selected IL-1 as the proinflammatory cytokine to induce COX-2 expression in gastric fibroblasts for experiments following.

View larger version (51K): [in this window] [in a new window]   Fig. 1. Effects of IL-1 and TNF- on cyclooxygenase-2 (COX-2) expression in human gastric fibroblasts. Gastric fibroblasts were incubated without serum for 24 h and then stimulated with IL-1 (10 ng/ml) or TNF- (10 ng/ml) for another 24 h. Cells were lysed and subjected to 10% SDS-PAGE. The separated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes and immunoblotted with anti-COX-2 antibodies. Transfer membranes were then reprobed with anti--actin antibodies to confirm uniform protein load per lane. Representative blots from 3 independent experiments are shown. St., recombinant COX-2 protein for standard.

View larger version (30K): [in this window] [in a new window]   Fig. 2. Effects of gastric fibroblast conditioned media (CM) on erbB3 and erbB2 phosphorylation in MKN28 cells. Gastric fibroblasts were stimulated with control media or IL-1 (10 ng/ml) for 24 h. The supernatants were recovered as CM. MKN28 cells were then stimulated with CM for 5 min and lysed. ErbB3 and erbB2 in lysates were immunoprecipitated with anti-erbB3 (A) or anti-erbB2 (B) antibodies, respectively. Tyrosine phosphorylation (P) of erbB3 and erbB2 was determined by Western blot analysis with PY20. Transfer membranes were then reprobed with each anti-erbB receptor antibody to confirm equal amounts of receptor protein per lane. Representative blots from 3 independent experiments are shown. CM, control CM; CM + IL-1, CM from fibroblasts incubated with IL-1; HRG-, direct stimulation of MKN28 cells with heregulin-.

View larger version (20K): [in this window] [in a new window]   Fig. 3. A: effects of anti-erbB3-neutralizing antibodies on erbB3 phosphorylation induced by recombinant HRG-. MKN28 cells were incubated for 5 min with recombinant HRG- (50 ng/ml) in the presence or absence of anti-erbB3-neutralizing antibodies. Cells were then lysed and immunoprecipitated with anti-erbB3 antibodies. Immunoprecipitates were analyzed by immunoblotting with PY-20. B: effects of anti-erbB3-neutralizing antibodies on gastric fibroblast-CM-induced erbB3 phosphorylation. MKN28 cells were pretreated with anti-erbB3-neutralizing antibodies for 60 min and then incubated for 5 min with CM from gastric fibroblasts with or without IL-1 (10 ng/ml) stimulation. Cells were then lysed and immunoprecipitated with anti-erbB3 antibodies. Immunoprecipitates were analyzed by immunoblotting with PY-20. ErbB3 Ab, anti-erbB3-neutralizing antibody.

View larger version (32K): [in this window] [in a new window]   Fig. 4. Effect of COX-2 inhibition on erbB3 phosphorylation by gastric fibroblast-CM. After preincubation with celecoxib (10 µM) for 60 min, gastric fibroblasts were stimulated with IL-1 in the presence or absence of PGE2 (10 µM) for 24 h. Where indicated, cells were stimulated with PGE2 (10 µM) alone. Supernatants were collected as CM and MKN28 cells incubated with CM for 5 min. Cells were then lysed and immunoprecipitated with anti-erbB3 antibodies. Immunoprecipitates were analyzed by immunoblotting with PY-20.

View larger version (22K): [in this window] [in a new window]   Fig. 5. A: immunoblot analysis of pro-HRG in NIH-3T3 cells transfected with HRG- cDNA or hph plasmid with anti-HRG antibodies. HRG--transfected NIH-3T3 cells (NIH-HRG) and control cells (NIH-hph) were lysed and subjected to 10% SDS-PAGE. The separated proteins were transferred onto PVDF membranes and immunoblotted with anti-HRG antibodies. B: effect of COX-2 inhibition on pro-HRG protein expression in human gastric fibroblasts. After preincubation with celecoxib (10 µM) for 60 min, gastric fibroblasts were stimulated with IL-1 (10 ng/ml) in the presence or absence of PGE2 (10 µM) for 24 h. Where indicated, cells were stimulated with PGE2 (10 µM) alone. Thereafter, cell lysates, prepared as described in MATERIALS AND METHODS, were subjected to immunoblot analysis using anti-HRG antibodies.

View larger version (27K): [in this window] [in a new window]   Fig. 6. Effect of COX-2 inhibition on mature HRG protein release from human gastric fibroblasts. After preincubation with celecoxib (10 µM) for 60 min, gastric fibroblasts were stimulated with IL-1 in the presence or absence of PGE2 (10 µM) for 24 h. Where indicated, cells were stimulated with PGE2 (10 µM) alone. After collection of CM, the CM were concentrated down to 1% of initial volume and subjected to immunoblot analysis using anti-HRG antibodies.

HRG- and HRG- mRNA expression in human gastric fibroblasts and gastric mucosa.

View larger version (9K): [in this window] [in a new window]   Fig. 7. Real-time PCR analysis of HRG- and HRG- mRNA expression in gastric fibroblasts. After preincubation with celecoxib (10 µM) for 60 min, gastric fibroblasts were stimulated with IL-1 (10 ng/ml) in the presence or absence of PGE2 (10 µM) for 18 h. Where indicated, cells were stimulated with PGE2 (10 µM) alone. Total RNA was isolated and real-time RT-PCR analysis of HRG- mRNA (A) and HRG- mRNA (B) performed as described in MATERIALS AND METHODS. This is a representative of 4 independent experiments performed in duplicate. Data are expressed as means ± SE. The statistical significance of difference (*P < 0.01) was determined by Student's t-test.

View larger version (10K): [in this window] [in a new window]   Fig. 8. HRG- and HRG- mRNA expression in gastric ulcer edge and normal gastric mucosa. Total RNA was isolated from gastric biopsy specimens and real-time RT-PCR analysis of HRG- mRNA (A) and HRG- mRNA (B) performed as described in MATERIALS AND METHODS. Levels of HRG- and HRG- transcript relative to -actin are indicated on the y-axis. The statistical significance of difference (*P < 0.01) was determined by Mann-Whitney U-test.

View larger version (156K): [in this window] [in a new window]   Fig. 9. Immunohistochemical localization of HRG in the human gastric ulcer tissue. A: HRG immunoreactivity is markedly observed in spindle-shaped cells at the ulcer bed. B: higher magnification view of A. C: moderate HRG immunoreactivity is also present in stromal cells at the ulcer edge (inset: higher-magnification view). Arrows indicate stromal cells expressing HRG immunoreactivity in ulcer edge. D: slightly HRG immunoreactivity is present in scattered lamina propria mesenchymal cells at the gastritis mucosa (arrow). Original magnification: x40 (A and C), x60 (B; C, inset; and D).

View larger version (108K): [in this window] [in a new window]   Fig. 10. A: immunohistochemical localization of vimentin in the gastric ulcer bed. Spindle-shaped cells were stained with vimentin in gastric ulcer bed. B–D: confocal microscopic images of human gastric ulcer tissue stained by single- and dual-labeling procedures using Texas red- and FITC-conjugated antibodies (red and green fluorescence, respectively). B: COX-2 in the gastric ulcer bed. Note COX-2 reactivity (red) in many spindle-shaped cells. C: HRG in the gastric ulcer bed. Note HRG reactivity (green) in many spindle-shaped cells. D: nuclei are counterstained with 4,6-diamidino-2-phenylindole. COX-2 (red) and HRG (green) colocalization is shown in yellow. Many spindle-shaped cells exhibit colocalization of these 2 proteins. Original magnifications: x40 (A–D).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

One of the unexpected findings in the present study was that both HRG- and HRG- mRNAs were equally expressed in IL-1-stimulated gastric fibroblasts in vitro and in fibroblasts of the gastric mucosa at the ulcer edge. There are currently four recognized HRG genes: HRG1, HRG2, HRG3, and HRG4, with at least 15 different HRG1 isoforms originating from the single HRG1 gene (12, 21). However, to date, very little is known concerning the function of the HRG2, 3, and 4 proteins. One intriguing recent study shows that the -isoforms of HRG1 and HRG2 are erbB3 agonists, whereas HRG3 and HRG4 are erbB4 agonists (15). In the present study, we focused on the HRG1 gene and tried to quantify HRG1 mRNA expression levels in cultured gastric fibroblasts and gastric mucosa by real-time PCR analysis using primers/probes designed for the detection of mRNA sequences of two well-known isoforms, HRG- and HRG-. We were able to discover for the first time that both HRG- and HRG- mRNAs were in fact expressed in human gastric fibroblasts in vitro and in gastric mucosa. The expression of these two HRG isoforms increased to similar levels in response to IL-1 stimulation, and their mRNA response patterns were exactly the same as those for HRG protein expression in fibroblasts, suggesting that HRG- and HRG- mRNA expression are similarly dependent on the COX-2-PGE₂ pathway.

We have previously shown HRG expression in fibroblasts of lamina propria in the human gastric mucosa (24), where we confirmed both HRG expression in gastric fibroblasts cultured in vitro and HRG expression confined to fibroblasts of the gastric ulcer bed in vivo, where staining patterns paralleled those of COX-2 expression. Recently, a number of HRG studies has shown its contribution to the wound repair process in a variety of tissues. HRG expression was induced in keratinocytes of full-thickness skin wounds in mice, and HRG- was also shown to be extracted from keretinocytes as a secreted motility factor during the wound-healing process (29). In addition, HRG- was shown expressed in apical membranes of differentiated human airway epithelia at the edge of wounds immediately following mechanical injury (37). HRG- is also considered to activate erbB3 and erbB2 in airway epithelial cells, thus contributing to the wound-repair process. Although, in mesenchymal cells, HGF and keratinocyte growth factor (KGF) production were shown to promote keratinocyte growth during tissue development and repair, recent studies (9) have shown that, in fact, HGF and KGF stimulate HRG production as a proximate mediator of signaling events leading to keratinocyte growth. All of these data suggest that HRG expressed in epithelial cells, rather than in mesenchymal cells, is involved in epithelial growth and mobilization through autocrine mechanisms. However, in the present study, HRG expression was observed only in fibroblasts of the ulcer bed and not in epithelial cells at the ulcer edge of the gastric mucosa. We have already shown in a previous study that gastric fibroblasts do not express erbB3 (24). Thus, in the gastric mucosa, HRG released from fibroblasts in the ulcer bed seems to act on epithelial cells expressing erbB3 and erbB2 through mesenchymal-epithelial interactions. Therefore, the mechanism regulating HRG expression in the gastric mucosa, which depends on inflammatory reactions, might well differ from that of the airway epithelia or the skin.

HRGs have been shown expressed in the gastrointestinal tract, bronchial epithelium, skin, and the central nervous system (12). Its expression has also been shown in various types of cancer cells: mammary gland, lung, and colon (13, 14, 19, 36). However, the mechanism through which HRG expression and its release are regulated has yet to be elucidated. The fibroblasts used in this study appear to secrete HRG even under basal serum-free conditions, as we have previously reported (24). In the present study, we found that, in the absence of IL-1 stimulation, there was erbB3 but not erbB2 phosphorylation activity in CM from fibroblasts. Because erbB3 lacks intrinsic tyrosine kinase activity, erbB3 activation without erbB2 phosphorylation suggests that erbB3 formed heterodimeric complexes with a receptor other than erbB2. We have previously shown that recombinant HRG- stimulates heterodimer formation of not only erbB3/erbB2, but also of the erbB3/EGF receptor, whereas neither TGF- nor EGF induced EGF receptor/erbB3 heterodimerization (24). In the present study, erbB3 receptor antibody inhibited CM-induced erbB3 phosphorylation in fibroblasts not stimulated with IL-1, suggesting that HRG in basal CM is actually involved in erbB3 phosphorylation. In addition to such basal HRG release from fibroblasts, we found in the present study that stimulation with IL-1 further increased HRG expression. PGE₂ itself stimulated HRG expression, whereas celecoxib almost totally inhibited IL-1-stimulated increases in HRG expression in gastric fibroblasts. Therefore, these results suggest that IL-1-stimulated HRG expression may be totally dependent on IL-1-induced COX-2 expression. However, when PGE₂ was added in the presence of IL-1 and celecoxib, there was a twofold increase in HRG mRNA expression levels over those stimulated by PGE₂ alone. Being that IL-1 on its own had no effect on HRG expression, results suggest that in addition to PGE₂, other, yet to be determined, IL-1-/COX-2-dependent factors might also be involved in HRG expression in gastric fibroblasts. We have shown in a previous study that, even in the presence of a selective COX-2 inhibitor, IL-l induces arachidonic acid metabolites that are sensitive to MK 886, an inhibitor of the membrane-associated proteins involved in eicosanoid and glutathione metabolism superfamily and that this product might be also involved in VEGF production in gastric fibroblasts (7, 17, 32). Thus further studies on HRG expression in gastric fibroblasts are clearly required to elucidate the interaction between PGE₂ and other, yet to be determined, factors induced by IL-1 and COX-2 inhibitors.

COX-2 expressed in the ulcer bed of the stomach has been shown to be involved in the ulcer repair process, as discussed previously. Thus it could well be that selective COX-2 inhibitors delay ulcer healing by inhibiting PGE₂ release and subsequent growth factor production. In addition, COX-2 expressed in mesenchymal cells of the lamina propria in colonic adenoma, colon cancer, and gastric cancer tissues has been shown to play a key role in the development of tumor cell growth (1, 27, 28, 31, 39). Recently, HRG has also been shown expressed in colon cancer cells and involved in cancer cell growth by autocrine stimulation of erbB2 phosphorylation (36). It has also been shown that erbB2 activation may upregulate COX-2 expression in colon cancer cells (35). In addition, cotherapy with a COX-2 inhibitor and an erbB2 antagonist has been shown to reduce colorectal carcinoma growth more effectively than with either agent alone (20). These findings demonstrate the complexity of the interaction between COX-2 and HRG, which needs much further elucidation. In the present study, we clearly identified the interaction between COX-2 and HRG: COX-2-derived PGE₂ induced autocrine-mediated HRG expression and ectodomain shedding in gastric fibroblasts with subsequent HRG-induced paracrine erbB3 phosphorylation in epithelial cells. We are now testing the possibility that such an interaction might be involved in HRG expression in gastrointestinal cancer cells.

	FOOTNOTES

 

Address for reprint requests and other correspondence: K. Wada, Third Dept. of Internal Medicine, Nippon Medical School, 1–1-5 Sendagi, Bunkyo-ku, Tokyo 113–8603, Japan (e-mail: wadaken{at}nms.ac.jp)

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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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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