The polyclonal antibody against PGIS was purchased from Cayman. The polyclonal antibody against Erk-1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The monoclonal antibody against GAPDH was purchased from Ambion Inc. (Austin, TX).

The paraffin specimens of 3 cases of colon cancer were supplied from Department of Surgical Oncology Kagoshima University Faculty of Medicine. This study was performed with the approval of the Ethics Committee for Epidemiological Studies in Kagoshima University Graduate School of Medical and Dental Sciences.

WI-38 and TIG-3-20 human fibroblasts were purchased from Health Science Research Resources Bank (HSRRB), human embryonic lung fibroblasts (HEL) and human neuroblastoma cells (NB-1) cell lines were provided from Professor Y. Eizuru (Kagoshima University, Kagoshima, Japan) and Professor A. Tomoda (Tokyo Medical University, Tokyo, China), respectively. Other cell lines were stocked in our laboratory. All of the cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FCS, 2 mM glutamine and 100 U/ml penicillin at 37°C in a 5% CO₂ humidified atmosphere. Hypoxic condition was induced by incubating the cells in a Personal Multigas Incubator (Astec) at 37°C with humidified 1% O₂.

cDNA and biotinylated cRNA were synthesized according to the standard protocols provided by Affymetrix (Santa Clara, CA). Biotinylated cRNA were purified using the Sample Cleanup Module (Affymetrix), and subsequently hybridized, according to the manufacturer’s standard protocols, to Affymetrix HGU133A GeneChips (which contain 22283 probe sets). Arrays were scanned using an Affymetrix confocal laser scanner. A gene expression analysis software program, GeneSpring, version 7.1 (Agilent), was used to perform statistical analysis.

1 μg first-strand cDNA was used for each polymerase chain reaction (PCR). The human PGIS primers were as follows: forward, 5′-ctggctcctgc tcttccttctcaag-3′; reverse, 5′-caccacgtcgcaggttgaattcttg-3′. The human VEGF primers were: forward, 5′-tgtcttgggtgcattggag ccttgc-3′; reverse, 5′-gttgtgctgtaggaagctcatctctc-3′. The human GAPDH primers for internal control were: forward, 5′-tgcacc accaactgcttag-3′; reverse, 5′-gaggcagggatgatgttc-3′. The human PPARδ primers were: forward, 5′-actgagttcgccaagagcatc-3′; reverse, 5′-aacgttcatgaggcctggccg-3′.

The PCR amplification mixture was adjusted to a final volume of 20 μl. Twenty-eight cycles were performed at 94°C for 30 sec for denaturation, 58°C for 30 sec for annealing, and 72°C for 30 sec for extension. The PCR products were separated on 1.5% agarose gel and then stained with ethidium bromide.

Total cellular RNA was extracted using TRIzol reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). One microgram of RNA was reverse transcribed using a first-strand cDNA synthesis kit (ReverTra Ace-α-, Toyobo, Osaka, Japan). The set of SYBR Premix Ex Taq kit was purchased from Takara Bio (Shiga, Japan). Human PGIS, VEGF, GAPDH gene expression levels were assayed by real-time reverse PCR (PRISM 7900HT, Applied Biosystems, Foster City, CA) according to the technical brochure of the company. Human GAPDH was used for normalization. Quantification of target gene expression was obtained with the comparative cycle threshold method according to the instructions of the manufacturer (Applied Biosystems). All experiments were performed in triplicate for each data point. Each quantitation was performed with the standard curve method.

Cells were harvested and lysed with RIPA buffer [50 mM Tris-HCI (pH 7.4), 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 20 mM NaF, 100 mM Na₃VO₄, 0.5% Nonidet P-40 (NP-40), 1% Triton X-100, 1 mM PMSF, DTT, aprotinin, APMSF and protease inhibitor cocktail (Sigma)]. The lysates were passed through a 21-gauge needle to break up the cell aggregates and were cleared by centrifugation at 14,000 × g for 15 min at 4°C; the supernatant (total cell lysate) was immediately used or stored at −80°C until use.

Lysates were subjected to 9.4% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and then were transferred to polyvinylidene difluoride membranes (Immobilon-P transfer membrane; Millipore, Bedford, MA). The membrane was treated with buffer A [350 mM NaCl, 10 mM Tris-HCl (pH 8.0), and 0.05% Tween-20] containing 3% skimmed milk for 1 h and incubated with the indicated antibody (1:1,000) in buffer A containing 3% skimmed milk for 1 hour. Following four washes with buffer A (10 min each), the membrane was incubated with a peroxidase-conjugated horse anti-mouse IgG diluted 1:1,000 in buffer A containing 3% skimmed milk for 1 hour. The membrane was washed with buffer A and developed using the enhanced chemiluminescence western blot analysis detection system (Amersham Biosciences, Buckinghamshire, UK).

Cells were washed with PBS and then resuspended in 3 volumes of buffer B [10 mM HEPES-KOH (pH 7.8), 10 mM KCl, 0.1 mM EDTA (pH 8.0), 1 μg/ml aprotinin and 1 mM p-aminophenyl methanesulfonyl fluoride]. The cells were disrupted by dounce homogenizer. The nuclear fractions were centrifuged at 13,000 rpm for 5 min at 4°C. The supernatant was used as cytosolic fractions.

Specific small interfering RNA (siRNA) of PGIS was purchased from Santa Cruz Biotechnology. SiRNA duplexes of PPARδ were synthesized using the Silencer™ siRNA construction kit (Ambion Inc.). The siRNA used in this study consisted of a 21-nucleotide sense strand and a 21-nucleotide antisense strand with a two-nucleotide overhang at 3′-end. The sequences were: PPARδ-siRNA(1) sense 5′-CAUGGAGUGCCGGGUGUG CUU-3′ starting at nucleotide 211 from AUG start codon of human PPARδ coding sequence. PPARδ-siRNA(1) antisense, 5′-GCACACCCGGCACUCCAUGUU-3′. PPARδ-siRNA(2) sense 5′-GCUGGAGUACGAGAAGUGUUU-3′ starting at nucleotide 313 from AUG start codon of human PPARδ coding sequence. PPARδ-siRNA(2) antisense, 5′-ACACUUCUCG UACUCCAGCUU-3′.

Transfections were accomplished using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol. After transfection, the cells were incubated under normoxic condition for 5 h, then changed with fresh DMEM and incubated under hypoxic condition until harvested at different time points. The effect of the siRNA on the expression of PGIS and VEGF was assessed using RT-PCR and real-time PCR, as described above.

6-Keto-prostaglandin F1α (stable hydrolysis product of PGI₂) was analyzed by specific enzyme immunoassay kit (EIA) (Assay Designs), following the manufacturer’s instructions. Lysates were added to every well of the kit plate. The resulting values of 6-keto-prostaglandin F1α concentrations in the supernatant were normalized for lyses buffer.

WI-38 and HEL cells were cultured under normoxic or hypoxic condition for different time, and fixed with 4% paraformaldehyde (PFA) in PBS for 30 min at room temperature. After blocking with 5% normal goat serum in PBS/0.02% Tween-20, the cells were incubated with an antibody against PGIS (1:100) at 4°C overnight. After washing thrice in PBS/0.02% Tween-20, the cells were incubated with secondary antibodies (anti-rabbit-FITC 1×100). Nuclei were stained by incubating cells with 6 μmol/l 4′6-diamidino-2-phenylindole (DAPI). The cells were observed using confocal fluorescence microscopy (FV500, Olympus Corporation).

Immunohistochemical studies for PGIS and HIF-1α were performed on formalin-fixed, paraffin-embedded surgical sections obtained from patients with colorectal cancer. Slides were deparaffinized in xylene and rehydrated in a graded alcohol series. To retrieve antigen, sections were heated in the citrate buffer pH 6.0 (Target retrieval solution, S1699, Dako, Carpinteria, CA) for 5 min at 121°C by means of an autoclave. For detection and visualization, the retrieved antigens were reacted with the appropriate specific primary antibodies solution (diluted to 1:100) for 1 h and were visualized by means of a polymer method (ChemMate Envision, K5027, Dako) and colorimetric diaminobenzidine (DAB)-peroxide (H₂O₂) reaction (DAB+ liquid system, K3468, Dako). Nuclear counterstaining was hematoxylin (ChemMate hematoxylin, S2020, Dako). These procedures were performed by an autostainer (Autostainer, Dako) with rinsing buffer warmed at 35°C (14).

GeneChip analysis was used for identifying the modulation of the gene expression in fibroblasts under hypoxic condition. A total of 234 genes were differentially expressed between fibroblasts under normoxic or hypoxic condition in this study. Relative to normoxic condition data profiling, 172 genes were upregulated and 62 genes were downregulated (3-fold change). Of 172 upregulated genes, genes related to tumor metastasis, endothelial cell biology and TGF-β signaling pathway etc. were included. Table I lists some upregulated genes in fibroblasts under hypoxic condition. Among these genes, we focused on PGIS, which showed 13.5-fold increased expression levels.

As shown in Fig. 1A, the expression levels of PGIS gene were significantly enhanced in fibroblasts such as WI-38, TIG-3-20 and HEL time-dependently under hypoxic condition. To quantitate the mRNA expression levels of PGIS in fibroblasts, real-time quantitative RT-PCR was performed (Fig. 1B). Moreover, the expression levels of PGIS in some human cancer cells were also investigated in this study. The time-dependently enhanced expression levels of PGIS under hypoxic condition were detected in human neuroblastomas (NB-1) (Fig. 1A and B) and human melanomas (G361), but not in glioblastomas (U251MG), pancreas cancer (MiapacaII), non-small cell lung cancer (A549), KB/TP and EJ/TP cells (15) (data not shown). PGIS mRNA was not detected in human macrophages (U937 and THP1) under normoxic or hypoxic condition (data now shown). Since the enhanced expression levels of PGIS were not detected in fibroblasts cultured in the medium containing 1% FCS (data not shown), the induction of PGIS could not be attributed to the low nutrient condition. These results suggested that the enhanced expression of PGIS mRNA in fibroblasts and some cancer cells was induced by hypoxic condition.

We next examined expression levels of PGIS in fibroblasts (HEL) and neuroblastomas (NB-1) by western blot analysis (Fig. 1C). Expression levels of PGIS protein in both cell lines were also enhanced time-dependently under hypoxic condition consistent with mRNA level. To identify the subcellular localization of PGIS induced by hypoxia, PGIS was observed using confocal fuorescence microscopy. As shown in Fig. 2A, the expressions of PGIS were mainly localized in the cytoplasm of WI-38 cells, and translocated to nuclei under hypoxic condition. Immunoblot analysis also showed that PGIS was translocated from cytoplasm to nuclei when the cells were cultured under hypoxic condition (Fig. 2B).

PGI₂ is known to increase the expression levels of the pro-angiogenic factor VEGF (16,17). To confirm that PGIS is involved in the enhanced expression of VEGF under hypoxic condition in fibroblasts, PGIS siRNA was used to knockdown the expression of PGIS. The induction of PGIS mRNA expression by hypoxia was considerably suppressed by PGIS siRNA (Fig. 3A). PGIS knockdown resulted in the decreased expression of VEGF mRNA (Fig. 3B). These results show that PGIS is required for the hypoxic induction of VEGF.

Since PGI₂ is unstable, the level of the stable hydrolysis metabolite of PGI₂, 6-keto-prostaglandin F1α was analyzed in HEL and NB-1 cells under hypoxic condition. The level of 6-keto-prostaglandin F1α was also significantly increased in a time-dependent manner under hypoxic condition compared with that under normoxic condition (p<0.05, triplicate determinations; Fig. 3C).

It was previously reported that endogenous PGI₂ generation by co-expression of COX-2 and PGIS activated PPARδ (18). Because the expression of COX-2 was not induced under hypoxic condition in fibroblasts and tumor cells (data not shown), we considered that PGI₂ produced by the hypoxia-induced PGIS entered the nuclei and interacted with PPARδ.

To examine whether PPARδ regulates the expression of VEGF, WI-38 cells transfected with PPARδ siRNA or green fluorescent protein (GFP) siRNA as a control were cultured under hypoxic condition. As shown in Fig. 4, downregulation of PPARδ suppressed the expression of VEGF mRNA. These results indicate that PPARδ regulates transcription of VEGF in WI-38 cells.

To determine whether PGIS is expressed in fibroblasts and cancer cells in clinical human tumor tissues, we examined 3 cases of human colon cancer tissues. As shown in Fig. 5, PGIS was expressed in cancer cells and fibroblasts which are located in the deep layers of the intestinal wall but not in the surface layers (Fig. 5a–b). Immunohistochemical staining of PGIS validated the induced expression of PGIS in fibroblasts under hypoxic condition in tumors (Fig. 5d).