Roles of Myofibroblasts in Prostaglandin E2–Stimulated Intestinal Epithelial Proliferation and Angiogenesis

Research Article

Jinyi Shao,1 George G. Sheng,2 Randy C. Mifflin,3 Don W. Powell,3 and Hongmiao Sheng1

1Department of Surgery and Cancer Center, Indiana University School of Medicine, Indianapolis, Indiana; 2Department of Surgery, University of Cincinnati, Cincinnati, Ohio; and 3Department of Medicine, University of Texas Medical Branch, Galveston, Texas

Abstract advantage to intestinal neoplasms (6, 7). In contrast, genetic dis- Prostaglandins (PG) are produced throughout the gastroin- ruption of the cyclooxygenase-2 (COX-2) gene or the E-prostanoid testinal tract and are critical mediators for a complex array receptor 2 (EP2) results in a substantial reduction of polyps in of physiologic and pathophysiologic processes in the intestine. APC knockout mice (8, 9). Further evidence shows that PGE2 Intestinal myofibroblasts, which express cyclooxygenase promotes intestinal neoplasia through enhancing tumor angiogenesis (9–11). Knockout of the EP2 receptor or inhibition of COX-2 (COX) and generate PGE2, play important roles in intestinal D716 epithelial proliferation, differentiation, inflammation, and enzyme results in a reduction of neoangiogenesis in APC neoplasia through secreting growth factors and cytokines. mouse tumors (9, 12). Understanding the precise mechanisms by Here, we show that PGE activated human intestinal sub- which PGE2 promotes intestinal epithelial growth and angiogenesis 2 remains a significant challenge. It has been shown that PGE epithelial myofibroblasts (18Co) through Gs protein–coupled 2 E-prostanoid receptors and the cyclic AMP/protein kinase A directly stimulates the proliferation of transformed intestinal pathway. 18Co cells and primary colonic myofibroblast iso- epithelial cells (6, 13, 14) and increases the expression of lates expressed a number of growth factors; several of them proangiogenic growth factors in colon cancer cells (15, 16). However, the effects of PGE2 on cell growth and angiogenesis were dramatically regulated by PGE2. An epidermal growth factor–like growth factor, amphiregulin (AR), which was not in vivo are considerably complex. Interactions between intestinal epithelial cells and stromal cells, which include fibroblasts, expressed by untreated cells, was strongly induced by PGE2. Expression of vascular endothelial growth factor A (VEGFA) myofibroblasts, endothelial cells, and other cell types, may dramatically influence the homeostasis and transformation of the was rapidly increased by PGE2 exposure. Hepatocyte growth factor (HGF) was elevated in PGE -treated myofibroblasts at intestinal epithelium (17). 2 A large body of studies has shown that intestinal subepithelial both mRNA and protein levels. Thus, PGE2-activated myofibroblasts promoted the proliferation and migration of in- myofibroblasts (ISEMF) play crucial roles in intestinal organo- testinal epithelial cells, which were attenuated by neutralizing genesis (18–21), proliferation, and differentiation of intestinal antibodies to AR and HGF, respectively. Moreover, in the epithelial cells (19), mucosal protection, and wound healing (22). ISEMFs are located in the lamina propria throughout the presence of PGE2, myofibroblasts strongly stimulated the migration and tubular formation of vascular endothelial cells. gastrointestinal tract (23, 24) and act through the secretion of Neutralizing antibody to VEGFA inhibited the observed growth factors, cytokines, and chemokines. ISEMFs express and stimulation of migration. These results suggest that myofibro- produce a large number of growth factors, including hepatocyte blast-generated growth factors are important mediators for growth factor (HGF; ref. 25), insulin-like growth factor (26), PGE -induced intestinal epithelial proliferation and angio- basic fibroblast growth factor (bFGF; ref. 27), platelet-derived 2 growth factor (PDGF; ref. 28), transforming growth factor-h genesis, which play critical roles in intestinal homeostasis, h inflammation, and neoplasia. (Cancer Res 2006; 66(2): 846-55) (TGF- ; ref. 29), colony stimulating factor (30), nerve growth factor (30), and stem cell factor (31). Furthermore, immunohistochemical studies reveal that fibroblasts are the predominant Introduction cell type in the lamina propria of normal colon; however, in Prostaglandins (PG) are generated throughout the gastrointes- both hyperplastic and neoplastic polyps, interstitial fibroblasts are tinal tract and play critical roles in an array of physiologic and replaced by myofibroblasts, suggesting that myofibroblasts play pathophysiologic processes (1, 2). PGs exert a trophic effect on critical roles in colorectal neoplasia (32). small intestinal mucosa and stimulate intestinal epithelial cell 18Co cells were derived from human colonic mucosa and exhibit proliferation (3). Short-term administration of PGE2 causes many properties of intestinal subepithelial myofibroblasts (33). significant stimulation of DNA synthesis; prolonged PGE2 treat- Expression of COX-1 is constitutive in 18Co cells, whereas COX-2 ment markedly increases the weight and DNA content of the can be induced by a variety of stimuli (34). Interleukin-1-activated intestinal mucosa (4). PGE2 and prostacyclin stimulate intestinal 18Co cells produce a significant amount of PGE2 (35). Given the epithelial cell migration and therefore promote intestinal restitu- critical functions of both ISEMF and PGE2 in the intestine, we tion (5). Moreover, PGE2 exerts growth-stimulatory effects on hypothesized that PGE2 may induce the production of certain intestinal tumors, and administration of PGE2 provides a growth growth factors by ISEMF, which, in turn, stimulate the growth and transformation of intestinal epithelium. In the present study, we show that PGE2 exposure increased the expression and secretion of amphiregulin (AR), HGF, and vascular endothelial growth factor Requests for reprints: Hongmiao Sheng, Department of Surgery, Indiana (VEGF) in 18Co cells and as well as in primary human colonic University, Indianapolis, IN 46202. Phone: 317-274-2630; E-mail: [email protected]. I2006 American Association for Cancer Research. myofibroblasts. PGE2-activated 18Co cells stimulated the prolifer- doi:10.1158/0008-5472.CAN-05-2606 ation and migration of intestinal epithelial cells. Conditioned

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media from PGE2-activated 18Co cells promoted the migration and RNA extraction and Northern blot analysis. Extraction of total cellular tubular formation of vascular endothelial cells. Thus, our study RNA was carried out as previously described (39). RNA samples were separated on formaldehyde-agarose gels and blotted onto nitrocellulose suggests that myofibroblasts may play critical roles in PGE2- induced intestinal growth and transformation. membranes. Blots were hybridized with cDNA probes labeled with [a-32P]dCTP by random primer extension (Stratagene, La Jolla, CA). After hybridization and washes, the blots were subjected to autoradiography. Materials and Methods Reverse transcription-PCR. Expression of EP receptors in 18Co cells Cell culture and reagents. 18Co cells were purchased from American was determined using reverse transcription-PCR (RT-PCR) as described Type Culture Collection (Manassas, VA) and grown in MEM supplemented previously (14). Human HGF and VEGF primer pairs were purchased from with 10% fetal bovine serum (FBS) and nonessential amino acids. 18Co cells R&D Systems. RT-PCR was carried out using ProStar RT-PCR system used for this study were passages 12 to 14. Primary colonic myofibroblast (Stratagene) according to the manufacturer’s instructions. (CMF, passages 4-10) cultures were established from histologically normal ELISA. Levels of human HGF, AR, and VEGF proteins in cell culture margins of surgically resected colonic tissue using the outgrowth method media were quantified using ELISA kits (R&D Systems). Cells were seeded in described by Mahida et al. (36, 37). The myofibroblast phenotype was 24-well plate, and serum was deprived for 24 hours before PGE2 treatment. verified by immunohistochemistry and flow cytometry. All are positive for Culture media were collected and stored at À80jC until assays. a-SMA, myosin heavy chain, and vimentin but are negative for cytokeratin Transient transfection and luciferase assay. Assays to determine (epithelial cell marker), desmin (smooth muscle cell marker), factor VIII transcriptional activity were described previously (39). Briefly, 18Co cells (endothelial cell marker), CD45 (bone marrow–derived hematopoetic cell were transfected with 0.5 Ag of AR reporter plasmid (À850 to À87) or VEGF marker), CD83 and ILT3 (dendritic cells), lysozyme and MAC 387 (both for reporter plasmid (À2279 to +54) along with 0.1 Ag of the pRL-SV plasmid, macrophages), and other markers of dendritic cells, B cells, or endothelia. containing the Renilla luciferase gene (Promega, Madison WI), using the Rat intestinal epithelial (RIE) cells were a generous gift from Dr. Susan FuGENE 6 procedure (Roche, Indianapolis, IN) as described in the Kirkland (University of London) and grown in DMEM with 10% FBS. Human manufacturer’s protocol. Transfected cells were lysed at indicated times umbilical vein endothelial cells (HUVEC) were purchased from Cascade for luciferase assay. Firefly and Renilla luciferase activities were measured Biologics (Portland, OR) and grown in Medium 200 supplemented with using a Dual-Luciferase Reporter assay system (Promega) and a lumin- low serum growth supplement. PGE2, 17-phenyl-trinor-PGE2, Butaprost, ometer. Firefly luciferase values were standardized to Renilla values. Sulprostone, and PGE1 alcohol were purchased from Cayman Chemical Immunoblot analysis. Immunoblot analysis was done as previously (Ann Arbor, MI). H-89, LY-294002, and PD-98059 were purchased from described (14). Anti–phosphorylated extracellular signal-regulated kinase Calbiochem (San Diego, CA). AR, HGF, and neutralizing antibodies were (pERK) and anti-phosphorylated Akt (pAkt) antibodies were purchased purchased from R&D Systems (Minneapolis, MN). from Cell Signaling Technology (Beverly, MA). Growth factor array. To determine the relative expression levels of Data analysis. All statistical analyses were done on a personal computer growth factors, GEArray Q Series Human Growth Factor Gene Array with the StatView 5.0.1 software (SAS Institute, Inc., Cary, NC). Analyses (SuperArray Bioscience Corporation, Frederick, MD) was carried out between two groups were determined using the unpaired Student’s t test. according to the manufacturer’s instructions. Biotin-labeled probe was Differences with P < 0.05 were considered as statistically significant. synthesized from total RNA and hybridized with a nylon membrane printed with cDNAs of 96 growth factors and cytokines. The array image was captured with chemiluminescence detection and analyzed using the Results software of GEArray Expression Analysis Suite. PGE2 induced stellate transformation of 18Co myofibro- RIE cell-18Co cell coculture system and DNA synthesis. RIE cells blasts. In response to increased levels of intracellular cyclic AMP (5 Â 103) suspended in 400 AL complete medium were placed in Transwell (cAMP), myofibroblasts undergo stellate transformation (40). chambers (0.4 Am, Corning Costar Co., Cambridge, MA) and then grown in serum-free medium for 24 hours. Separately, confluent 18Co cells were Agents that increase cAMP levels including forskolin, cholera toxin, and PGE2 induce stellate morphology in 18Co cells (33). grown in a 24-well plate and treated with 0.5 Amol/L PGE2 for 24 hours. Subsequently, Transwell chambers containing RIE cells were inserted into Confluent 18Co cells were grown in serum-deprived medium for 3 the 24-well plate and grown for 24 hours. [ H]thymidine (1 ACi) was added 24 hours and then treated with 0.5 Amol/L PGE2. Cells acquired a to the lower chambers 5 hours before harvest. The Transwell chambers stellate shape with dendritic-like processes by 2 hours following the were washed thrice with 10% trichloroacetic acid times, and the filters addition of PGE2 (Fig. 1A). By 24 hours, most stellate-transformed 3 were collected from the chambers. Incorporation of [ H]thymidine was cells returned to their regular fibroblastoid morphology. A selective determined using a scintillation counter. protein kinase A (PKA) inhibitor, H-89 (5 Amol/L), completely Cell migration assay. 18Co cells were grown in 24-well plates, serum attenuated the PGE -induced stellate transformation of 18Co cells. starved for 24 hours, and treated with vehicle or PGE . RIE or HUVEC cells 2 2 PGE acts via specific transmembrane G protein-coupled receptors; suspended in 400 AL serum-free McCoy’s 5A medium were placed in 2 uncoated Transwell chamber (8 Am, Corning Costar). The Transwell four E type prostaglandin (EP) receptor subtypes have been chambers were then inserted into the 24-well plate containing 18Co cells. identified (41). EP2 and EP4 are known to increase intracellular After an incubation period of 5 hours at 37jC, cells on the upper surface of cAMP levels and activate the PKA pathway. Expression of all of the filter of Transwell chambers were removed with a cotton swab. The four EP receptors was detected in 18Co cells by RT-PCR (Fig. 1B). filters were fixed and stained with 0.5% crystal violet solution. Three To determine which EP receptor mediated the PGE2-induced microscope fields (Â200) from each Transwell chamber were randomly stellate transformation, selective agonists were employed. Butap- selected, and cells adhering to the undersurface of the filter were counted. rost, a selective EP2 agonist, strongly transformed 18Co cells; HUVEC tube formation. HUVECs were suspended in 0.1 mL of almost all cells acquired a stellate appearance by 2 hours after indicated conditioned media and placed on growth factor reduced Matrigel the treatment. Activation of EP receptor modestly induced (Collaborative Biomedical Products, Bedford, MA) in 96-well plates. 4 stellation; a small portion of 18Co cells was transformed by PGE Morphology of the cells was documented using a digital camera attached 1 to an inverted microscope. Three photographs from random fields of each alcohol (Fig. 1B). As expected, EP1 and EP3 agonists (17-phenyl- microtiter well (quadruplicate wells for each group) were analyzed. Tubes trinor-PGE2 and Sulprostone) did not induce any morphologic were defined as straight cellular extensions joining two cell masses (38). transformation of 18Co cells (data not shown). Tube formation was assessed by the numbers of tubular structures and the The functional role of PGE2-induced stellation of 18Co cells is length of tubes. complex (42). We found that PGE2 did not alter the proliferation of www.aacrjournals.org 847 Cancer Res 2006; 66: (2). January 15, 2006

Figure 1. PGE2 induction of stellate transformation of 18Co cells. A, confluent 18Co cell cultures were serum deprived for 24 hours before addition of vehicle (V )or 0.5 Amol/L PGE2 (E2). H-89 (5 Amol/L) was added 15 minutes before PGE2 treatment. Cells were photographed at the indicated times. B, expression of EP receptors were analyzed by RT-PCR (top). Butaprost or PGE1 alcohol at 0.5 Amol/L was added to serum-deprived 18Co cells. Morphologic alterations were documented using a digital camera attached to an inverted microscope (bottom). C, 2 Â 105 18Co cells were suspended in serum-free medium and seeded in 8-Am Transwell chambers. Vehicle or 0.5 Amol/L PGE2 were added into the lower chambers. After a 24-hour incubation, filters were fixed and stained with 0.5% crystal violet solution. Cells adhering to the undersurface of the filter were photographed (left and middle) and counted (right). Columns, mean of cell numbers done in triplicate; bars, SD. *, P < 0.05. Cell migration assays were done at least three times independently.

18Co cells in the absence of serum (data not shown); however, (Fig. 2C). The stimulatory action of PGE2 on AR production required PGE2 significantly stimulated the migration of 18Co cells, activation of the cAMP/PKA pathway; inhibition of PKA by H-89 determined by a modified Boyden chamber assay (Fig. 1C). completely attenuated the PGE2-induced AR (Fig. 2D). In contrast, Numbers of migrating 18Co cells increased f1-fold in the presence a mitogen-activated protein (MAP)/ERK kinase (MEK) inhibitor of 0.5 Amol/L PGE2 compared with the cells treated with vehicle. (PD-98059) and a phosphatidylinositol 3-kinase (PI3K) inhibitor 18Co cells expressed an array of growth factors. To (LY-294002) did not block PGE2-induced AR expression. Activation determine which growth factors were expressed by 18Co cells, we of either EP2 receptor or EP4 receptor increased the production of carried out targeted cDNA arrays using GEArray Human Growth AR; however, the EP2 signaling seemed to be the predominant Factor Array. 18Co cells were treated with either vehicle or PGE2 pathway mediating AR induction (Fig. 2E). To determine the for 1, 2, or 4 hours. The expression profile of 96 growth factors, regulatory mechanism mediating PGE2 induction of AR, an AR cytokines, and chemokines was analyzed. Changes in gene promoter-driven reporter plasmid was introduced into 18Co cells. f expression z2-fold were shown. As summarized in Table 1, 18Co PGE2 induced the activity of the AR promoter 7-fold, which was cells expressed a number of growth factors, which belong to the completely blocked by H-89 (Fig. 2F). Although Butaprost exerted epidermal growth factor (EGF), FGF, PDGF, HGF, TGF-h, and a similar stimulatory effect on the AR promoter, PGE1 alcohol did neuronal growth factor families. Expression of several growth not have any effect on AR transcription in 18Co cells. factors was significantly regulated by PGE2, including amphiregulin PGE2 induced the expression of HGF. The Growth Factor (AR), HGF, neuregulin 1, and VEGFA. Although AR was not cDNA Array showed that HGF was constitutively expressed by expressed under regular circumstances, AR expression was strongly 18Co cells and significantly increased by 4 hours after PGE2 induced by PGE2. A 27-fold increase was detected after 18Co cells exposure. RT-PCR analysis revealed that PGE2 did not significantly were exposed to PGE2 for 1 hour. HGF was constitutively expressed change the expression of HGF at 1 hour; however, levels of HGF by 18Co cells, which was increased 5.8-fold at 4 hours after PGE2 mRNA were robustly increased at 2 and 4 hours after PGE2 exposure. VEGFA was expressed at low levels in vehicle-treated treatment (Fig. 3A). HGF protein was detected in 18Co culture f 18Co cells, which was increased 5-fold at the earliest time point. media at a concentration of 0.4 ng/mL. Addition of PGE2 f PGE2 increased the expression of AR. To validate the findings increased the levels of HGF 5-fold by 24 hours (Fig. 3B). made from the Growth Factor cDNA Array, we first investigated the Inhibition of the PKA pathway by H-89 attenuated the regulation of AR by PGE2. AR mRNA was not detected in vehicle- PGE2-induced production and secretion of HGF. Addition of either treated 18Co cells; however, PGE2 rapidly increased the levels of AR Butaprost or PGE1 alcohol increased HGF production, suggesting mRNA, noted by Northern blot (Fig. 2A) and real-time RT-PCR the involvement of both EP2 and EP4 signals in PGE2 induction analysis (Fig. 2B). The PGE2-induced expression of AR remained at of HGF. least 24 hours. Secretion of AR was not detected in untreated 18Co PGE2-activated 18Co cells stimulated intestinal epithelial cell culture medium. AR secretion, however, was robustly increased proliferation and migration. To determine whether PGE2- f in PGE2-stimulated 18Co cells, reaching 100 pg/mL by 24 hours activated 18Co cells were able to stimulate the proliferation of

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a cuboidal appearance. Placing 18Co cells in the bottom chamber Table 1. PGE2 regulation of 18Co-expressed growth factors significantly increased the motility of RIE cells. However, when activated by PGE2, 18Co cells strongly stimulated the migration f Gene Gene name Fold change of RIE cells; the number of migrating cells increased by 100% symbol (Fig. 5A, right). Additionally, it was noted that the migrating RIE 1h 2h 4h cells acquired a widely stretched morphology when cocultured with PGE2-activated 18Co cells (Fig. 5A, left, d, arrows). To AR Amphiregulin 27.0 3.2 2.0 determine which growth factor mediated the promigratory action BDNF Brain-derived neurotrophic factor 2.8 CSF1 Colony stimulating factor 1 2.3 CTGF Connective tissue growth factor 2.8 FGF2 Fibroblast growth factor 2 2.3 FGF13 Fibroblast growth factor 13 FGF7 Fibroblast growth factor 7 À3.0 À2.7 FIGF c-fos-induced growth factor 2.0 2.7 GDNF Glial cell derived neurotrophic factor À2.4 HGF Hepatocyte growth factor 5.8 IGF2 Insulin-like growth factor 2.1 NRG1 Neuregulin 1 2.3 15.6 NRP1 Neurophilin 2 NRP2 Neurophilin 2 PDGFA Platelet-derived growth factor A PDGFC Platelet-derived growth factor C PDGFD Platelet-derived growth factor D PTN Pleiotrophin TGF-b1 Transforming growth factor-h1 TGF-b2 Transforming growth factor-h2 VEGFA Vascular endothelial growth factor-A 5.0 VEGFB Vascular endothelial growth factor-B 2.5

NOTE: Listed are the growth factors expressed by 18Co cells. Results of growth hormone, cytokines, and chemokines are not shown. Fold

changes of differential expression are expressed as PGE2/vehicle– treated 18 Co cells.

intestinal epithelial cells, we carried out experiments using nontransformed RIE cells. Previous studies have shown that the EGF receptor (EGFR) is restricted to the basolateral compartment of intestinal epithelial cells (43, 44). Therefore, when EGFR ligands are added to the apical compartment, no mitogenic response is observed. In contrast, basolateral administration of EGFR ligands to intestinal epithelial cells grown on Transwell filters results in proliferation (44). RIE cells were insensitive to the growth- stimulatory effect of AR and HGF when grown on plastic dishes. AR Figure 2. PGE2 induction of AR expression. A, 18Co cells were serum deprived slightly increased RIE cell proliferation only at relatively high for 24 hours before PGE2 treatment. Levels of AR mRNA were analyzed by concentrations (10-100 ng/mL; Fig. 4A). However, when RIE cells Northern blot. B, 18Co cells were serum deprived for 24 hours before PGE2 treatment. Levels of AR mRNA were analyzed by real-time RT-PCR. C, 18Co A were plated on Transwell filters (0.4 m); addition of AR to the cells were serum deprived for 24 hours and treated with vehicle (V ) or PGE2 lower chamber strongly stimulated the proliferation of RIE cells (E2) for the indicated times. Levels of AR protein in cell culture media were determined by ELISA assay. Columns, mean of AR content done in triplicate; (Fig. 4B). Based on these results, RIE cells and 18Co cells were bars, SD. *, P < 0.05. ELISA assays were done at least three times cocultured in a similar system, in which RIE cells were grown in the independently. D, 18Co cells were treated with vehicle, 5 Amol/L H-89 (H), upper chamber and 18Co cells were grown in the bottom chamber. 25 Amol/L PD-98059 (P), or 10 Amol/L LY-294002 (L) for 15 minutes before the addition of 0.5 Amol/L PGE2. After a 24-hour incubation, levels of AR in cell Neither PGE2 nor 18Co cells stimulated the proliferation of RIE culture media were determined by ELISA assay. Columns, mean of AR done in cells; however, PGE2-activated 18Co cells increased DNA synthesis triplicate; bars, SD. *, P < 0.05. E, 18Co cells were serum deprived for 24 hours A A of RIE cells by f100% (Fig. 4C). Addition of anti-AR–neutralizing before treatments (V = ethanol, E2 = 0.5 mol/L PGE2, EP1/3 = 0.5 mol/L 17-phenyl-trinor-PGE2, EP2 = 0.5 Amol/L Butaprost, EP3 = 0.5 Amol/L antibody significantly attenuated the growth advantage of RIE cells Sulprostone, and EP4 = 0.5 Amol/L PGE1 alcohol). After 24 hours, levels of AR that were stimulated by PGE2-treated 18Co conditioned media, protein were determined by ELISA assay. Columns, mean of AR content done in triplicate; bars, SD. *, P < 0.05. F, 18Co cells were transiently transfected indicating the mitogenic effect of 18Co cell–generated AR (Fig. 4D). with an AR promoter reporter vector. Cells were then treated with ethanol (V), We next investigated if PGE2-stimulated 18Co cells modulate the 0.5 Amol/L PGE2 (E2), 0.5 Amol/L Butaprost (But), or 0.5 Amol/L PGE1 alcohol (E1) motility of intestinal epithelial cells. In a modified Boyden chamber (E1) along with 5 Amol/L H-89 or DMSO (V) for 6 hours. Firefly and Renilla luciferase activities were measured and standardized. Columns, mean of Renilla assay, PGE2 did not stimulate the migration of RIE cells and the adjusted luciferase activity done in quadruplicate; bars, SD. *, P < 0.05. cells that migrated through the polycarbonate membrane retained Representative of three separate experiments. www.aacrjournals.org 849 Cancer Res 2006; 66: (2). January 15, 2006

However, PGE2-activated 18Co conditioned media exerted significantly stronger effects on activation of both MEK/ERK and PI3K/ Akt pathways (Fig. 5D). PGE2 increased the production of VEGF in 18Co cells. COX- 2/PGE2 mediates hypoxic induction of VEGF in hepatic stellate cells (46). Our results of Growth Factor Array showed that PGE2 exposure induced the expression of VEGFA, suggesting that VEGFA is a PGE2 target gene in 18Co cells. Exposure to PGE2 rapidly increased the levels of VEGFA mRNA in 18Co cells, noted by Northern analysis (Fig. 6A). Similar results were observed using real-time RT-PCR; levels of VEGFA mRNA increased f3.5-fold after the 18Co cells were treated with PGE2 for 1 hour (Fig. 6B). Moreover, PGE2 treatment increased the production and secretion of VEGFA protein. Levels of VEGFA protein were elevated f3.5- fold in PGE2-stimulated 18Co culture media (Fig. 6C). Induction of Figure 3. Expression of HGF in 18Co cells. A, 18Co cells were serum deprived VEGFA production was mediated by both EP2 and EP4 receptors, for 24 hours before addition of ethanol (V ) or 0.5 Amol/L PGE2 (E2). Total RNA was extracted at the indicated time points, and the expression of HGF because both Butaprost and PGE1 alcohol increased the levels of mRNA was analyzed by RT-PCR. B, 18Co cells were serum deprived for VEGFA mRNA (Fig. 6D) and protein (Fig. 6E). Addition of H-89 24 hours before treatments (V = ethanol, E2 = 0.5 Amol/L PGE2, EP1/3 = completely attenuated the PGE2-induced expression of VEGFA at 0.5 Amol/L 17-phenyl-trinor-PGE2, EP2 = 0.5 Amol/L Butaprost, EP3 = 0.5 Amol/L Sulprostone, and EP4 = 0.5 Amol/L PGE1 alcohol). Supernatants of the cell both mRNA and protein levels. Furthermore, PGE2 stimulated cultures were collected after 24 hours, and levels of HGF protein were VEGF transcription through both EP2 and EP4 signaling. Addition determined by ELISA assay. Columns, mean of HGF content done in triplicate; bars, SD. *, P < 0.05. ELISA assay was repeated at least three times. of PGE2, Butaprost, and PGE1 alcohol increased the activity of VEGF promoter f1-fold (Fig. 6F). PGE2-activated 18Co cells enhanced angiogenesis. Because of PGE2-activated 18Co, RIE cells were stimulated with AR or HGF. PGE2 induced the expression of VEGF in 18Co cells, it was of We found that AR did not increase RIE cell migration, but HGF interest to determine whether PGE2-activated 18Co cells enhanced significantly stimulated the migration of RIE cells (Fig. 5B). In angiogenesis. Proangiogenic factors, including VEGF, stimulate agreement with this observation, anti-HGF–neutralizing antibody neoangiogenesis by inducing endothelial cell proliferation, migra- completely blocked the PGE2/18Co–induced RIE cell migration tion, and tubular organization. The effects of 18Co cells on the (Fig. 5C). migration of endothelial cells were evaluated in a coculture system Proliferation and migration of intestinal epithelial cells require similar to the system described in Fig. 4D. HUVECs were seeded in activation of the MAP kinase and the PI3K pathways (45). To the Transwell; 18Co cells were placed in the bottom chamber. determine whether PGE2-stimulated 18Co cells activated these Addition of PGE2 into the bottom chamber without 18Co cells signaling pathways in intestinal epithelial cells, levels of pERK and slightly stimulated the migration of HUVECs. The presence of 18Co pAkt in RIE cells were analyzed. Addition of PGE2 did not change cells in the bottom chamber significantly increased the motility of the levels of pERK and pAkt in RIE cells (data not shown). 18Co HUVECs. When cocultured with PGE2-activated 18Co cells, conditioned media rapidly increased the levels of pERK and pAkt. HUVECs acquired a widely stretched morphology (Fig. 7A), and

Figure 4. RIE cell proliferation in coculture with 18Co cells. A, RIE cells (2.5 Â 104) were seeded in 24-well plates and subjected to serum deprivation for 24 hours. AR or HGF at indicated concentrations were added. After a 24-hour incubation, DNA synthesis was analyzed by [3H]thymidine incorporation. Columns, mean of CPM done in quadruplicate; bars, SD. Representative of three separate experiments. B, 4 Â 103 RIE cells were seeded in a Transwell (0.4 Am) and serum deprived for 24 hours. AR or HGF at the indicated concentrations were added to the lower chambers. After 24 hours, [3H]thymidine (1 A Ci) was added. The filters containing RIE cells were collected, and [3H]thymidine incorporation was measured using a scintillation counter. C, in a coculture system, 4 Â 103 RIE cells were seeded in the Transwell (0.4 Am) and serum deprived for 24 hours before being inserted into a 24-well plate, where serum-starved 18Co cells were stimulated with vehicle (V)or 3 0.5 Amol/L PGE2 for 24 hours. [ H]thymidine incorporation in RIE cells was determined. D, 18Co cells were treated with vehicle (V) or PGE2 (E2) for 24 hours, and then 18Co conditioned media were collected. Normal goat IgG (IgG,20Ag/mL), anti-AR–neutralizing antibody (aAR,20Ag/mL), or anti-HGF–neutralizing antibody (aHGF,20Ag/mL) was added to the 18Co conditioned media and incubated at 4jC for 30 minutes. RIE cells (4 Â 103) were seeded in the Transwell (0.4 Am) and serum deprived for 24 hours before being inserted into a 24-well plate, which contained pretreated 18Co conditioned media as indicated. [3H]thymidine incorporation in RIE cells was determined after a 24-hour incubation and a 5-hour pulse.

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Figure 5. RIE cell migration in coculture with 18Co cells. A, 2 Â 105 RIE cells suspended in serum-free medium were seeded in Transwell chambers (8 Am). The chambers were then inserted into 24-well plates, where 18Co cells were grown and stimulated with vehicle (V ) or 0.5 Amol/L PGE2 for 24 hours. After a 5-hour incubation, filters were fixed and stained with 0.5% crystal violet solution. Cells adhering to the undersurface of the filter were photographed (left). Cell numbers in three microscope fields (Â200) from each Transwell were counted. Columns, mean of migrating cells done in triplicate; bars, SD. B, 2 Â 105 RIE cells suspended in serum-free medium were seeded in Transwell chambers (8 Am). Vehicle (V), 100 ng/mL AR or 10 ng/mL HGF were added to the lower chambers. RIE cell migration was determined after a 5-hour incubation as above. C, 2 Â 105 RIE cells suspended in serum-free medium were seeded in Transwell chambers (8 Am). Vehicle (V) or PGE2 (E2)–stimulated 18Co conditioned media that were pretreated with normal IgG (IgG)or 20 Ag/mL anti-HGF–neutralizing antibody (aHGF) were added to the lower chambers. RIE cell migration was determined after a 5-hour incubation. D, 18Co cells were serum deprived for 24 hours and then stimulated with vehicle (V) or 0.5 Amol/L PGE2 (E2) for 24 hours before collecting the conditioned media. To eliminate the direct effect of PGE2, 0.5 Amol/L PGE2 was added to vehicle-treated conditioned media. 18Co conditioned media were then added to serum-deprived RIE cells, and cellular protein was extracted at the indicated time points. Levels of pERKs and pAkt were determined by Western analysis.

Figure 6. Expression of VEGF in 18Co cells. A and B, 18Co cells were serum deprived for 24 hours before addition of ethanol (V ) or 0.5 Amol/L PGE2 (E2). Total RNA was extracted at the indicated time points, and levels of VEGF mRNA were analyzed by Northern blot (A) and real-time RT-PCR (B). C, 18Co cells were serum deprived for 24 hours before PGE2 exposure. After 24 hours, conditioned media were collected, and levels of VEGF protein were determined by ELISA assay. Columns, mean of VEGF content done in triplicate; bars, SD. *, P < 0.05. D and E, 18Co cells were serum deprived for 24 hours before treatments (V = ethanol, E2 = 0.5 Amol/L PGE2) in the presence of a PKA inhibitor (5 Amol/L H-89) or DMSO (V ). Cells were also treated with EP agonists (EP1/3 = 0.5 Amol/L 17-phenyl-trinor-PGE2, EP2 = 0.5 Amol/L Butaprost, EP3 = 0.5 Amol/L Sulprostone, and EP4 = 0.5 Amol/L PGE1 alcohol). D, levels of VEGF mRNA were determined by RT-PCR after the cells were treated for 2 hours. E, after a 24-hour incubation, levels of VEGF protein in culture media were determined by ELISA assay. Columns, mean of VEGF content done in triplicate; bars, SD. *, P < 0.05. F, 18Co cells were transiently transfected with a VEGF promoter reporter vector. Cells were subjected to the indicated treatments for 6 hours (V = ethanol, E2 = 0.5 Amol/L PGE2, But = 0.5 Amol/L Butaprost, and E1 = 0.5 Amol/L PGE1 alcohol). Firefly and Renilla luciferase activities were measured and standardized. Columns, mean of Renilla-adjusted luciferase activity done in quadruplicate; bars, SD. *, P < 0.05. Representative of three separate experiments. www.aacrjournals.org 851 Cancer Res 2006; 66: (2). January 15, 2006

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2006 American Association for Cancer Research. Cancer Research the number of migrating cells was robustly increased (Fig. 7B, left), may actually occur in the human intestine, the induction of growth which, however, was significantly attenuated by the addition of factors by PGE2 was evaluated in human primary subepithelial anti-VEGFA–neutralizing antibody (Fig. 7B, right). CMF. Three CMF primary isolates (4, 5, and 7) were treated with To determine the effects of 18Co cell–released growth factors on PGE2; levels of AR, HGF, and VEGFA were determined by RT-PCR. tubular organization, HUVECs were placed on growth factor– PGE2 exposure robustly increased the expression of AR mRNA in all reduced Matrigel. HUVECs spontaneously form tubular structures CMF cultures, which normally did not express AR (Fig. 8A). HGF on extracellular matrix. Addition of PGE2 stimulated HUVEC tube and VEGFA mRNAs were induced by PGE2 in a majority of CMF formation by 8 hours, as quantitated by the numbers and the isolates. Moreover, ELISA assay revealed that whereas HGF and f length of tubes (Fig. 7C). Conditioned media that collected from VEGF proteins were increased 1-fold in PGE2-stimulated CMF PGE2-stimulated 18Co cells robustly increased the number and culture media, the presence of PGE2-induced protein levels of AR length of tubes of HUVECs. Moreover, HUVEC-formed tubes were >10-fold in all CMF isolates (Fig. 8B). dissociated by 24 hours (Fig. 7D). Addition of PGE2-treated 18Co conditioned media prevented HUVEC tube dissociation. Interest- ingly, the presence of anti-VEGFA–neutralizing antibody did not Discussion significantly reduce the PGE2/18Co–induced HUVEC tube forma- COX-2 is not expressed in normal intestinal mucosa; its activity tion (data not shown), suggesting the involvement of a complex increases dramatically in inflammation, injury, and neoplasia of the mechanism. intestine (47). In studies of human colorectal cancer, COX-2 levels f PGE2 induction of growth factors in primary myofibro- are increased in about 90% of cancers and 50% of premalignant blasts. To determine whether the observations from 18Co cells colorectal adenomas, but the enzyme is not usually detected in

Figure 7. HUVEC migration and tubular formation in coculture with 18Co cells. A and B, 1 Â 105 HUVECs suspended in serum-free medium were seeded in Transwell chambers (8 Am). The chambers were then inserted into 24-well plates, where 18Co cells were grown and stimulated with vehicle (V ) or 0.5 Amol/L PGE2 for 24 hours. After a 5-hour incubation, filters were fixed and stained with 0.5% crystal violet solution. A, cells adhering to the undersurface of the filter were photographed. B, numbers of migrating cells in three microscope fields (Â200) from each Transwell were counted. Columns, mean of migrating cells done in triplicate (left); bars, SD. HUVEC migration was assessed when vehicle (V ) or PGE2 (E2)–stimulated 18Co conditioned media that were pretreated with normal IgG (IgG) or anti-VEGF–neutralizing antibody (aVEGF,20Ag/mL) were added to the lower chambers (right). C, 1 Â 104 HUVEC suspended in serum-free medium containing vehicle (V ), 0.5 Amol/L PGE2 (E2), vehicle-treated 18Co conditioned media, or PGE2-activated 18Co conditioned media were placed onto growth factor–reduced Matrigel. After an 8-hour incubation, cells were photographed (left, Â100). Numbers of tubes were counted (middle), and the relative length of the tubular structure was measured (right). D, 5 Â 103 HUVEC cells suspended in serum-free medium containing vehicle (V ), 0.5 Amol/L PGE2 (E2), vehicle-treated 18Co conditioned media, or PGE2-activated 18Co conditioned media were placed onto growth factor–reduced Matrigel. After a 24-hour incubation, cells were photographed (left, Â100).

Cancer Res 2006; 66: (2). January 15, 2006 852 www.aacrjournals.org

Figure 8. PGE2 induction of growth factors in human colonic myofibroblasts. A, human primary colonic myofibroblast cell isolates CMF4, CMF5, and CMF7 were serum deprived for 24 hours before PGE2 treatment. Total RNA was extracted at the indicated time points. Levels of AR, HGF, VEGF, and h-actin were analyzed by RT-PCR. B, CMF4, CMF5, and CMF7 cells were serum deprived for 24 hours before vehicle (V ) or PGE2 (E2) treatment. After a 24-hour incubation, protein levels of AR, HGF, and VEGFA in conditioned media were measured by ELISA assay.

adult intestinal tissues (48, 49). Although there are conflicting data with a dramatic down-regulation of AR. In contrast, an EP2-specific regarding which cell types express COX-2 in intestinal tumors, agonist strongly increases the expression of AR in mammary cancer COX-2 expression increases in both the epithelial and the stromal cell lines, suggesting that AR is a mediator for COX-2/PGE2–induced compartments (50). Indeed, PGs can be produced by a variety of mammary gland hyperplasia. In another study, Moraitis et al. (58) cell types, including normal and transformed intestinal epithelial reported that tobacco smoke stimulates the expression of COX-2 cells, myofibroblasts, and macrophages (34, 51, 52). PGs serve as through activating the EGFR signaling system in human oral autocrine or paracrine lipid mediators to signal changes within mucosa. Overexpression of AR and TGF-a was determined to be the their immediate environment, suggesting that PGE2 may mediate mechanism for the tobacco smoke–induced EGFR activity and interactions between intestinal epithelial cells and stromal cells COX-2 expression, suggesting a positive loop between the COX-2/ through both autocrine and paracrine mechanisms. PGE2 derived PGE2 pathway and AR/EGFR signaling. from both stromal and epithelial compartments may stimulate The growth of solid tumors requires a blood supply that is stromal cells to release growth factors, which, in turn, provide a achieved through neoangiogenesis. VEGF is one of the major pro-neoplastic environment for the intestinal epithelium. In the regulators for neoangiogenesis, which induces endothelial cell present study, we found that exogenous PGE2 induced the proliferation, migration, and tubular organization (19). PGE2 expression and secretion of several pro-proliferative and proangio- induces the expression of VEGF in colon cancer cells and in min/+ genic growth factors in intestinal subepithelial myofibroblasts, APC polyps (16). EP2-mediated PGE2 signaling plays critical providing the evidence that myofibroblasts may be a critical roles in neoangiogenesis. Homozygous deletion of the EP2 receptor mediator for COX-2/PGE2–mediated intestinal epithelial growth, significantly reduces the number and size of intestinal polyps in transformation, and neoangiogenesis. APCD716 mice that is associated with a reduction of VEGF Our results show that exogenous PGE2 induced the expression of expression, suggesting that PGE2/EP2 signaling is critical for AR at both mRNA and protein levels in 18Co cells. AR is a member increased levels of VEGF in intestinal neoplasm (9). Our data show of the EGF growth factor family and a ligand of the EGFR. It has that PGE2 increased the expression, production, and secretion of been shown that AR is a primary mitogen for hepatocytes and VEGF in 18Co cells and that PGE2-activated 18Co cells promoted critical in the early steps of liver regeneration; AR-null mice display the migration and tubular formation of HUVEC. This suggests that an impaired proliferative response after partial liver resection (53). myofibroblasts may provide proangiogenic factors for intestinal Moreover, AR exerts tumor-promoting effects on colorectal remodeling and transformation. In addition to VEGF, a number of carcinomas. AR mRNA is expressed in 60% to 70% of primary and members of the FGF family and the TGF-h family were expressed metastatic human colorectal carcinomas but in only 2% to 7% of by 18Co cells and regulated by PGE2; their functional roles in PGE2 normal colonic mucosa samples studied (54). AR plays critical roles proangiogenic actions are still under investigation. in colon cancer cell proliferation and transformation that are HGF is a known myofibroblast-derived growth factor that required for the growth of human colon carcinoma xenografts (55). regulates epithelial cell proliferation, differentiation, motility, and We have reported that in response to PGE2 exposure, the expression morphology (59). HGF is expressed in the stomach, small intestine, of AR is significantly increased in transformed intestinal epithelial and colon (60). The biological functions of HGF in the small cells, which stimulates the growth of colon cancer cells via an and large intestines are not clear, but it is likely that HGF exerts autocrine mechanism (39, 56). Recent studies have further stressed growth-stimulatory effects on intestinal epithelial cells. When the critical role of AR in the transformation of a variety of epithelial colon cancer T84 cells are grown in type I collagen gel, HGF does cell types. Chang et al. (57) showed that COX-2 overexpression in the not induce their differentiation but stimulates their growth (61). mammary gland of transgenic mice induced mammary cancer. Our results showed that PGE2-activated myofibroblasts increased Interestingly, genetic deletion of the EP2 receptor significantly the production of HGF, which predominantly stimulated the reduced the COX-2-induced mammary cancer, which is associated migration of intestinal epithelial cells. www.aacrjournals.org 853 Cancer Res 2006; 66: (2). January 15, 2006

PGE2 signals through specific receptors, including EP1,EP2,EP3, In summary, our studies suggest that myofibroblasts are a and EP4. Our results showed that PGE2-induced expression and potential mediator of the growth-stimulatory effect and pro- production of growth factors was mediated by the EP2,4/cAMP/ neoplastic action of PGE2 in the intestine. Myofibroblasts may PKA pathway, because both Butaprost and PGE1 alcohol repro- receive PGE2 stimulation via autocrine and paracrine pathways. duced PGE2 actions on induction of HGF, AR, and VEGF. In Upon activation by PGE2, myofibroblasts increasingly produce and support of these findings, a selective PKA inhibitor attenuated the secrete growth factors, which stimulate intestinal epithelial cell stimulatory effects of PGE2. Although both EP2 and EP4 pathways growth and promote angiogenesis. Given the special localization of acted quite similarly, differences were observed. For example, AR myofibroblasts in normal intestine and intestinal neoplasia, the was regulated by PGE2 at the transcriptional level, which was interaction between myofibroblasts and intestinal epithelial cells mediated by the EP2/cAMP/PKA pathway only. PGE1 alcohol did may play important roles in intestinal epithelial growth and not stimulate the transcription of AR but modestly increased the transformation. levels of AR protein in 18Co culture media, suggesting the involvement of a post-transcriptional regulation. Interestingly, Acknowledgments activation of both EP and EP increased the transcription of 2 4 Received 7/25/2005; revised 10/10/2005; accepted 11/8/2005. VEGF. Because PGE1 alcohol also binds to other EP receptors (62), Grant support: NIH grants DK-065615, DK-064593 (H. Sheng), and DK-55783 (D.W. Powell). its specificity to EP4 is relative. Further experiments are required The costs of publication of this article were defrayed in part by the payment of page to determine the precise function of EP4 in PGE2 activation of charges. This article must therefore be hereby marked advertisement in accordance 18Co cells. with 18 U.S.C. Section 1734 solely to indicate this fact.

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2006 American Association for Cancer Research. Roles of Myofibroblasts in Prostaglandin E2−Stimulated Intestinal Epithelial Proliferation and Angiogenesis

Jinyi Shao, George G. Sheng, Randy C. Mifflin, et al.

Cancer Res 2006;66:846-855.

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