Autotaxin expression and its connection with the TNF-alpha-NF-kappaB axis in human hepatocellular carcinoma.

BACKGROUND: Autotaxin (ATX) is an extracellular lysophospholipase D that generates lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). Both ATX and LPA have been shown to be involved in many cancers. However, the functional role of ATX and the regulation of ATX expression in human hepatocellular carcinoma (HCC) remain elusive. RESULTS: In this study, ATX expression was evaluated in tissues from 38 human HCC and 10 normal control subjects. ATX was detected mainly in tumor cells within tissue sections and its over-expression in HCC was specifically correlated with inflammation and liver cirrhosis. In addition, ATX expression was examined in normal human hepatocytes and liver cancer cell lines. Hepatoma Hep3B and Huh7 cells displayed stronger ATX expression than hepatoblastoma HepG2 cells and normal hepatocytes did. Proinflammtory cytokine tumor necrosis factor alpha (TNF-alpha) promoted ATX expression and secretion selectively in Hep3B and Huh7 cells, which led to a corresponding increase in lysophospholipase-D activity. Moreover, we explored the mechanism governing the expression of ATX in hepatoma cells and established a critical role of nuclear factor-kappa B (NF-kappaB) in basal and TNF-alpha induced ATX expression. Further study showed that secreted enzymatically active ATX stimulated Hep3B cell invasion. CONCLUSIONS: This report highlights for the first time the clinical and biological evidence for the involvement of ATX in human HCC. Our observation that links the TNF-alpha/NF-kappaB axis and the ATX-LPA signaling pathway suggests that ATX is likely playing an important role in inflammation related liver tumorigenesis.

Krüppel-like factor 5 mediates cellular transformation during oncogenic KRAS-induced intestinal tumorigenesis.

BACKGROUND & AIMS: Krüppel-like factor 5 (KLF5) is a zinc finger-transcription factor that regulates cell proliferation. Oncogenic KRAS mutations are commonly found in colorectal cancers. We aimed to determine whether KLF5 mediates KRAS functions during intestinal tumorigenesis. METHODS: The effects of KLF5 on proliferation and transformation were examined in IEC-6 intestinal epithelial cells stably transfected with inducible KRAS(V12G). KLF5 expression was examined in intestinal tumors derived from transgenic mice expressing KRAS(V12G) under villin promoter and in human colorectal cancers with mutated KRAS. RESULTS: Induction of KRAS(V12G) in IEC-6 cells resulted in increased expression of KLF5, accompanied by increased rates of proliferation and anchorage-independent growth. Inhibition of KLF5 expression by mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) inhibitors or KLF5-specific small interfering RNA reduced proliferation and anchorage-independent growth despite KRAS(V12G) induction. Human colorectal cancer cell lines with mutated KRAS contained high levels of KLF5 and reduction of KLF5 by MEK inhibitors or KLF5 small interfering RNA also led to reduced proliferation and transformation. In vivo, both intestinal tumors derived from mice transgenic for villin-KRAS(V12G) and human primary colorectal cancers with mutated KRAS contained high levels of KLF5 and increased staining of the proliferative marker Ki67. CONCLUSIONS: Elevated levels of KLF5 protein are strongly correlated with activating KRAS mutations in intestinal tumors in vitro and in vivo. Inhibition of KLF5 expression in tumor cells resulted in significantly reduced rates of proliferation and transforming activities. We conclude that KLF5 is an important mediator of oncogenic KRAS transforming functions during intestinal tumorigenesis.

Prostaglandin E2 and Krüppel-like transcription factors synergistically induce the expression of decay-accelerating factor in intestinal epithelial cells.

The decay-accelerating factor (DAF) prevents the intestinal mucosa from bystander killing by complement. Prostaglandin E(2) (PGE(2)) induces the expression of DAF that may protect the tumour environment from complement attack. In the present study, we demonstrate synergistic actions of PGE(2) and two Krüppel-like factors (KLFs), which are zinc finger-containing transcription factors, in DAF regulation. Overexpression of KLF4 and KLF5 robustly induced transcriptional activity of the DAF promoter. In combination, PGE(2) and either KLF4 or KLF5 increased the expression of DAF in a synergistic fashion. Moreover, cyclooxygenase (COX-1 and COX-2) enzymes, KLF4/5 and DAF protein were coordinately expressed in normal intestinal mucosa as well as in intestinal neoplasm. In radiation-injured mouse intestine, COX-1 was rapidly induced and remained at relatively high levels. While KLF5 was quickly elevated after irradiation, KLF4 exhibited a delayed increase. Interestingly, levels of DAF increased gradually following the induction of COX-1 and KLFs. Mimicking the circumstances in vivo, coexpression of both COX and KLFs resulted in a synergistic or additive induction of DAF transcription in intestinal epithelial cells. Our data suggest that COX-derived PGE(2) may collaborate with KLF4/5 to regulate the activation of the complement system and exert diverse effects on the intestinal epithelium.

Roles of myofibroblasts in prostaglandin E2-stimulated intestinal epithelial proliferation and angiogenesis.

Prostaglandins (PG) are produced throughout the gastrointestinal tract and are critical mediators for a complex array of physiologic and pathophysiologic processes in the intestine. Intestinal myofibroblasts, which express cyclooxygenase (COX) and generate PGE(2), play important roles in intestinal epithelial proliferation, differentiation, inflammation, and neoplasia through secreting growth factors and cytokines. Here, we show that PGE(2) activated human intestinal subepithelial myofibroblasts (18Co) through Gs protein-coupled E-prostanoid receptors and the cyclic AMP/protein kinase A pathway. 18Co cells and primary colonic myofibroblast isolates expressed a number of growth factors; several of them were dramatically regulated by PGE(2). An epidermal growth factor-like growth factor, amphiregulin (AR), which was not expressed by untreated cells, was strongly induced by PGE(2). Expression of vascular endothelial growth factor A (VEGFA) was rapidly increased by PGE(2) exposure. Hepatocyte growth factor (HGF) was elevated in PGE(2)-treated myofibroblasts at both mRNA and protein levels. Thus, PGE(2)-activated myofibroblasts promoted the proliferation and migration of intestinal epithelial cells, which were attenuated by neutralizing antibodies to AR and HGF, respectively. Moreover, in the presence of PGE(2), myofibroblasts strongly stimulated the migration and tubular formation of vascular endothelial cells. Neutralizing antibody to VEGFA inhibited the observed stimulation of migration. These results suggest that myofibroblast-generated growth factors are important mediators for PGE(2)-induced intestinal epithelial proliferation and angiogenesis, which play critical roles in intestinal homeostasis, inflammation, and neoplasia.

Heterozygous disruption of the PTEN promotes intestinal neoplasia in APCmin/+ mouse: roles of osteopontin.

The persistent activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway is oncogenic and involved in colorectal neoplasia. Mutations of both regulatory subunit and catalytic subunit of PI3K have been demonstrated in colon cancers. In the present study, we show that heterozygous disruption of the phosphatase and tensin homolog (PTEN) tumor suppressor gene promoted tumor progression in APC(min/+) mice. Number and size of intestinal tumors were significantly increased in mice bearing both adenomatous polyposis coli (APC) and PTEN mutations. While APC(min/+)PTEN(+/+) mice developed adenomas, invasive carcinomas developed in APC(min/+)PTEN(+/-) mice. Large tumors often resulted in intestinal intussusception and early death of APC(min/+)PTEN(+/-) mice. Targeted array revealed that osteopontin (OPN) was the leading gene whose expression was strongly induced by deficiency of PTEN. In colon cancer cells, gain-of-function mutation of PI3K robustly increased levels of OPN and treatment with OPN reduced growth factor deprivation-induced programmed cell death. Moreover, OPN expression was strongly increased in Ras-induced transformation of intestinal epithelial cells in a PI3K-dependent manner. Inhibition of OPN expression by specific small interfering RNA reduced uncontrolled growth and invasiveness of Ras-transformed intestinal epithelial cells. Thus, our results suggest that the PI3K pathway promotes the transformation of intestinal adenoma to adenocarcinoma. OPN, a downstream effector of PI3K, protects transformed intestinal epithelial cells from programmed cell death and stimulates their anchorage-independent growth.

Roles of phosphatidylinositol 3'-kinase and mammalian target of rapamycin/p70 ribosomal protein S6 kinase in K-Ras-mediated transformation of intestinal epithelial cells.

Phosphatidylinositol 3'-kinase (PI3K) activity is required for Ras- mediated transformation of intestinal epithelial cells (IECs). The mammalian target of rapamycin (mTOR) and its downstream pathways control the translation of specific mRNAs that are required for cell proliferation and transformation. Here, we elucidated the roles of PI3K and mTOR in K-Ras-mediated transformation of IECs (IEC-6). Induction of K-Ras activated PI3K and mTOR in IECs. p70 ribosomal protein S6 kinase activity was induced by K-Ras in a PI3K- and mTOR-dependent manner. K-Ras did not significantly alter the phosphorylation of eukaryotic initiation factor 4E-binding protein 1. Treatment with either LY-294002 or rapamycin inhibited IEC proliferation and resulted in G(1) growth arrest. However, it was noted that inhibition of mTOR enhanced K-Ras-mediated morphological transformation and increased invasiveness of IECs in a mitogen-activated protein/extracellular signal-regulated kinase-dependent manner. Furthermore, inhibition of PI3K or mTOR impaired the growth of an array of colon cancer cells. Spindle transformation, reduced E-cadherin, and increased invasiveness were observed in LY-294002-treated Moser cells. Thus, our results suggest that K-Ras-mediated transformation of IECs involves activation of the PI3K/mTOR pathway. Inhibition of PI3K/mTOR activity leads to G(1) growth arrest of transformed IECs. On the other hand, inhibition of PI3K or mTOR may induce the epithelial to mesenchymal transdifferentiation of IECs under certain circumstances.

Peroxisome proliferator-activated receptors modulate K-Ras-mediated transformation of intestinal epithelial cells.

Activation of peroxisome proliferator-activated receptors (PPARs) exerts diverse effects on neoplastic cells. Recent work has shown that PPARdelta is up-regulated after loss of adenomatous polyposis coli tumor suppressor gene function and that transcriptional activation of the PPARgamma nuclear receptor can lead to inhibition of carcinoma growth. In this study, we elucidate the regulation and functional importance of PPARgamma and delta after K-Ras-transformation of intestinal epithelial cells. In conditionally K-Ras-transformed rat intestinal epithelial cells (IEC-iK-Ras), the level and activity of PPARdelta were markedly increased. PPARdelta up-regulation occurred due to increased mitogen-activated protein kinase activity and receptor activation required the endogenous production of prostacyclin via the cyclooxygenase-2 pathway. We also demonstrate that activation of the PPARgamma nuclear receptor has antineoplastic effects in Ras-transformed cells. Activation of PPARgamma resulted in a delay in transit through the G(1) phase of the cell cycle that was associated with inhibition of phosphatidylinositol 3'-kinase/Akt activity and a reduction of cyclin D1 expression. Therefore, these two PPAR nuclear receptors, which are structurally related, have distinct roles during neoplastic transformation. PPARgamma appears to modulate differentiation and signal growth inhibition, whereas PPARdelta is up-regulated by oncogenic Ras and activated by cyclooxygenase-2-derived prostaglandins.

Prostaglandin E2 stimulates the growth of colon cancer cells via induction of amphiregulin.

Prostaglandin E(2) (PGE(2)), a major product of cyclooxygenase enzymes, is implicated in colorectal carcinogenesis and has been shown to stimulate the growth of human colorectal carcinoma cells. Here, we show that PGE(2) activated the cyclic AMP/protein kinase A pathway, which induced the expression of amphiregulin (AR), an epidermal growth factor family member, through activation of a cyclic AMP-responsive element in the AR promoter. AR exerted a mitogenic effect on LS-174 cells and partially mediated the PGE(2)-induced growth stimulation. In addition, PGE(2), in collaboration with transforming growth factor-alpha or K-Ras oncogene, synergistically induced AR expression and activated receptor tyrosine kinase-dependent signaling pathways. Our results provide novel mechanisms for cyclooxygenase-2 pro-oncogenic activity and suggest that PGE(2) may act with major oncogenic pathways in a synergistic fashion to activate the epidermal growth factor receptor signaling system through a ligand-dependent autocrine pathway.

Intestinal transformation results in transforming growth factor-beta-dependent alteration in tumor cell-cell matrix interactions.

BACKGROUND: An alteration in the expression of and response to transforming growth factor-beta 1 (TGF-beta 1) appears to be an important event during colorectal carcinogenesis. However, the precise role of TGF-beta 1 in colorectal carcinogenesis is not clear. We have previously described in detail the changes in cell proliferation and differentiation caused by chronic exposure to TGF-beta 1. In this study we sought to better characterize the changes in tumor cell-cell matrix interactions seen during TGF-beta 1-mediated intestinal transformation. METHODS: Rat intestinal epithelial cells (RIE) and RIE cells transformed by chronic exposure to TGF-beta 1 (RIE-Tr) were treated with TGF-beta 1 and production of components of the plasmin/plasminogen system measured by ELISA and Western blotting. TGF-beta 1 effects on invasion and adhesion were determined in vitro. The role of urokinase on TGF-beta 1-mediated invasion and adhesion were determined using immunoneutralization. The role of COX-2 was determined using a specific COS-2 inhibitor. RESULTS: TGF-beta 1 had no effect on RIE-1 adhesion to collagen types I and IV, fibronectin, and laminin, or invasion through collagen types I and IV. However, 5 ng/mL TGF-beta 1 significantly increased the invasiveness and decreased the adhesiveness of RIE-Tr. This effect of TGF-beta 1 on RIE-Tr was associated with a significant increase in plasmin activity secondary to increased expression of uPA. TGF-beta 1 had no effect on either uPA receptor or PAI-1 in this system. Antibodies to uPA completely blocked the TGF-beta 1-mediated invasiveness of the RIE-Tr cells and returned their adhesiveness to basement membrane proteins to baseline. Addition of the selective Cox-2 inhibitor SC-58125 resulted in a dose-dependent decrease in TGF-beta 1-mediated invasion and uPA expression. CONCLUSION: This study provides additional evidence for TGF-beta 1 as a tumor promoter during intestinal carcinogenesis and a possible new mechanism for Cox-2-related colon carcinogenesis.

Pro-neoplastic effects of amphiregulin in colorectal carcinogenesis.

PURPOSE: Epidermal growth factor (EGF) family plays critical roles in intestinal epithelial growth and transformation. Amphiregulin (AREG) is a member of the EGF family, and has been suggested to be more important to tumor versus normal growth. The precise roles of AREG in colorectal carcinogenesis have not been thoroughly elucidated. METHODS: AREG expression was analyzed in colon cancer specimens using immunohistochemistry. Genetically disruption of AREG in APC (min/+) mouse was achieved by crossbreeding AREG knockout mouse with APC (min/+) mice. Knockdown AREG expression was accomplished by using plasmid-based RNA interference. Growth-stimulatory effects of AREG were determined using cell co-culture systems. RESULTS: AREG was expressed in both epithelial and stromal compartments in human colon cancer; however, it was regulated by different mechanisms. AREG was predominantly regulated at transcriptional level in colon cancer cells while both transcriptional and post-transcriptional mechanisms were involved in colon cancer derived myofibroblasts. Functionally, knockout of AREG strongly reduced tumorigenicity in APC (min/+) mice. Immunohistochemistry demonstrated the coordinate expression of AREG, EGF receptor activity, and cell proliferation marker in APC (min/+) mouse adenoma, indicating the growth-stimulatory function of AREG signaling in tumor development. Furthermore, we demonstrated that AREG may stimulate tumor cell growth through both autocrine and paracrine pathways in cell culture models. Knockdown of AREG impaired the ability of anchorage-independent growth of transformed intestinal epithelial cells. On the other hand, myofibroblast-produced AREG stimulated the growth of colon cancer cells when co-cultured in extracellular matrix. CONCLUSIONS: AREG plays pro-neoplastic roles in colorectal carcinogenesis and may be targeted for colon cancer prevention and treatment.

Amphiregulin promotes intestinal epithelial regeneration: roles of intestinal subepithelial myofibroblasts.

Epidermal growth factor family plays critical roles in intestinal epithelial proliferation and differentiation. The precise function of amphiregulin (AREG), a member of the epidermal growth factor family, in intestinal biology is largely unknown. The present study attempted to address the functional roles of AREG in intestinal epithelial regeneration. Total body irradiation was performed, and intestinal regeneration was assessed in AREG knockout mice. Genetically disruption of AREG significantly impaired intestinal regeneration after radiation injury. It is known that prostaglandin E(2) (PGE(2)) exerts radio-protective and growth-stimulatory effects on intestinal epithelium. We found that PGE(2) radio-protective action did not involve AREG. However, PGE(2) growth-stimulatory effects required functional AREG. Localization of AREG expression was determined by immunohistochemistry in regenerative intestine. The immunoreactivity of AREG was predominantly localized in intestinal subepithelial myofibroblasts (ISEMF). Primary ISEMF cultures were established, and growth-stimulatory actions of ISEMF-generated AREG were demonstrated in cell coculture system. In addition, we found that the cAMP/protein kinase A pathway robustly induced AREG in cultured ISEMF. These studies suggest that AREG plays critical roles in intestinal epithelial growth. Modulation of levels of AREG by targeting ISEMF represents a novel strategy for treatment of certain intestinal disorders.

NF-kappaB inhibition in human hepatocellular carcinoma and its potential as adjunct to sorafenib based therapy.

Nuclear factor-kappaB (NF-kappaB) has been shown to play an important role in the development and progression of cancer. In this study, we systematically examined NF-kappaBp65 signaling pathway in both human hepatocellular carcinoma (HCC) tissue and HCC cell lines. NF-kappaBp65 signaling pathway is aberrantly expressed and activated in both human HCC tissue and HCC Hep3B cells. Inhibition of NF-kappaB activity significantly reduced proliferation and invasion of Hep3B cells as well as down-regulated the expression of invasion-related molecules including matrix metalloproteinase (MMP)-2, MMP-9, membrane type-1 MMP (MT1-MMP), urokinase plasminogen activator (uPA) and vascular endothelial growth factor (VEGF). Hep3B cells exhibited a dose-dependent increase in apoptosis after receiving sorafenib treatment. Inhibition of NF-kappaB activity strongly sensitized Hep3B cells to sorafenib-induced cell death. Mechanistically, combined treatment of sorafenib and NF-kappaB inhibition enhanced inhibition of MAPK signaling and down-regulation of anti-apoptotic protein Mcl-1 expression. These observations indicate that inhibition of NF-kappaB may be a potential antineoplastic therapy for HCC, especially the combination of NF-kappaB inhibition and sorafenib provides a novel therapeutic strategy for patients with advanced-stage HCC.

Prostaglandin E2 induces the expression of IL-1alpha in colon cancer cells.

PGE(2) has been shown to exert pro-oncogenic effects in colorectal neoplasia through producing autocrine or paracrine growth factors. In the present study, we demonstrate that PGE(2) induced the expression of IL-1alpha in colon cancer cells, which plays critical roles in tumor metastasis and neoangiogenesis in a variety of cancers. PGE(2) increased the levels of both IL-1alpha mRNA and protein, suggesting a positive feedback loop between the IL-1 pathway and PGE(2) signaling. Mechanistically, PGE(2) induced the expression of IL-1alpha at both transcriptional and posttranscriptional levels. PGE(2) stimulated the transcriptional activity of the IL-1alpha promoter and significantly stabilized IL-1alpha mRNA. Moreover, we show that IL-1alpha enhanced colorectal neoplasia, stimulating cell migration and neoangiogenesis. Knockdown of the expression of IL-1alpha by small-interfering RNA resulted in a reduction of vascular endothelial growth factor secretion in colon cancer cells and an inhibition of tube formation by HUVECs. Thus, our results suggest that PGE(2) induces the expression of proinflammatory cytokine IL-1alpha, which may potentially enhance the proneoplastic actions of the cyclooxygenase-2/PGE(2) signaling pathway.

PTEN expression contributes to the regulation of muscle protein degradation in diabetes.

OBJECTIVE: Conditions accelerating muscle proteolysis are frequently associated with defective phosphatidylinositol 3-kinase (PI3K)/Akt signaling and reduced PI3K-generated phosphatidylinositol 3,4,5-triphosphate (PIP(3)). We evaluated the control of muscle protein synthesis and degradation in mouse models of type 1 and 2 diabetes to determine whether defects besides PI3K/Akt activities affect muscle metabolism. RESEARCH DESIGN AND METHODS: We evaluated the expression and activity of PTEN, the phosphatase converting PIP(3) to inactive phosphatidylinositol 4,5-bisphosphate, and studied how PTEN influences muscle protein in diabetic wild-type mice and in mice with partial deficiency of PTEN(+/-). RESULTS: In acutely diabetic mice, muscle PTEN expression was decreased. It was increased by chronic diabetes or insulin resistance. In cultured C2C12 myotubes, acute suppression of PI3K activity led to decreased PTEN expression, while palmitic acid increased PTEN in myotubes in a p38-dependent fashion. To examine whether PTEN affects muscle protein turnover, we studied primary myotubes cultures from wild-type and PTEN(+/-) mice. The proteolysis induced by serum deprivation was suppressed in PTEN(+/-) cells. Moreover, the sizes of muscle fibers in PTEN(+/-) and wild-type mice were similar, but the increase in muscle proteolysis caused by acute diabetes was significantly suppressed by PTEN(+/-). This antiproteolytic response involved higher PIP(3) and p-Akt levels and a decrease in caspase-3-mediated actin cleavage and activation of the ubiquitin-proteasome system as signified by reduced induction of atrogin-1/MAFbx or MurF1 (muscle-specific RING finger protein 1). CONCLUSIONS: Changes in PTEN expression participate in the regulation of muscle proteolytic pathways. A decrease in PTEN could be a compensatory mechanism to prevent muscle protein losses.

Prostaglandin E2 Stimulates the beta-catenin/T cell factor-dependent transcription in colon cancer.

Cyclooxygenase and its derived prostaglandin E2 (PGE2) have been shown to stimulate the growth of cancer cells and promote tumor angiogenesis. Here, we show that PGE2 activated the beta-catenin/T cell factor-dependent transcription in colon cancer cells through the cAMP/protein kinase A pathway. The expression of cyclin D1 and vascular endothelial growth factor was induced by PGE2 in LS-174T cells. Moreover, PGE2 and mutated beta-catenin stimulated the transcription of cyclin D1 and vascular endothelial growth factor in a synergistic fashion. Mechanistically, PGE2 increased the phosphorylation of glycogen synthase kinase-3 and consequently accumulated beta-catenin. In addition, PGE2 induced the expression of T cell factor-4 transcription factor, which formed transcriptionally active complex with beta-catenin. In animal experiments, administration of 16,16-dimethyl PGE2 strongly increased the expression of cyclin D1 and vascular endothelial growth factor in APC(min/+) mouse polyps. Thus, our results provide a novel mechanism, suggesting that cyclooxygenase-2/PGE2 may exert pro-oncogenic actions through stimulating the beta-catenin/T cell factor-mediated transcription, which plays critical roles in colorectal carcinogenesis.

Prostaglandin E2 synergistically enhances receptor tyrosine kinase-dependent signaling system in colon cancer cells.

Cyclooxygenase (COX)-generated prostaglandin E(2) (PGE(2)) plays critical roles in colorectal carcinogenesis. Recently, we have shown that PGE(2) and transforming growth factor-alpha synergistically induces the expression of amphiregulin (AR) in colon cancer cells (Shao, J., Evers, B. M., and Sheng, H. (2003) Cancer Res. 63, 5218-5223). In this study, we demonstrated synergistic actions of PGE(2) and the receptor tyrosine kinase signaling system in AR expression and in tumorigenic potential of colon cancer cells. Activation of the Ras/Raf/MAPK pathway induced AR transcription in colon cancer LS-174 cells that was enhanced by PGE(2) in a synergistic fashion. The cAMP-responsive element within the AR promoter was required for the synergistic activation of AR transcription. An Sp1 element was responsible for the basal transcription of AR and significantly enhanced the synergy between PGE(2) and the epidermal growth factor receptor (EGFR) signaling system. Furthermore, activation of both PGE(2) and EGFR signaling pathways synergistically promoted the growth and migration of colon cancer cells. Our results suggest that COX-2/PGE(2) may exert pro-oncogenic effects through synergistic induction of receptor tyrosine kinase-dependent signaling pathway, thus, provide a novel mechanism for the combinatorial treatment of colonic neoplasms targeting both COX-2/PGE(2) and the EGFR system that has demonstrated remarkable advantages.

Up-regulation of the enzymes involved in prostacyclin synthesis via Ras induces vascular endothelial growth factor.

BACKGROUND AND AIMS: The constitutive activation of Ras is an important step in the development and progression of several different cancers and is known to increase the level of cyclooxygenase 2 (COX-2). Prostaglandins are the downstream bioactive lipid mediators produced by the COX-2 enzyme. We sought to determine the role of Ras-induced up-regulation of the enzymes involved in prostacyclin biosynthesis in nontransformed rat intestinal epithelial cells (IECs). METHODS: Messenger RNA (mRNA) and protein expression were analyzed by Northern and Western analysis, respectively, to determine the level of enzymes induced by Ras. In vitro assays were used to determine the production of vascular endothelial growth factor (VEGF) and prostaglandins as well as the promoter and enzymatic activation of the rate-limiting enzyme in prostaglandin production (phospholipase A(2) [cPLA(2)]). RESULTS: The inducible expression of Ha-Ras(V12) increased the production of prostaglandin (PG)F(2alpha) and prostacyclin by 2- and 13-fold, respectively. The induction of Ha-Ras(V12) also up-regulated the mRNA and protein levels of cPLA(2), COX-2, and prostacyclin synthase, as well as the promoter and enzyme activity of cPLA(2). Furthermore, oncogenic Ras increased the production of the pro-angiogenic factor VEGF. The increase of VEGF was abolished after treatment with celecoxib, a selective COX-2 inhibitor. The addition of PGI 2 alone also induced the expression of VEGF. CONCLUSIONS: Inducible Ha-Ras(V12) increases the production of PGI(2) through the coordinate up-regulation of cPLA(2), COX-2, and prostacyclin synthase (PGIS). The production of PGI(2) leads to an increase in the level of the pro-angiogenic factor VEGF, which is known to play a crucial role in the regulation of tumor-associated angiogenesis.

Cyclin D3 is essential for intestinal epithelial cell proliferation.

Intestinal epithelium is a complex organ that undergoes continuous proliferation. D-type cyclins bind cyclin-dependent kinases (Cdk4 and Cdk6) and are expressed during the transition from G0 into the S phase. Previously, we reported that cyclins D1 and D3 are induced by growth factors in two rat intestinal epithelial cell lines, IEC-6 and RIE-1. However, transforming growth factor beta induces G1 arrest in both intestinal cell lines without inhibiting cyclin D3, suggesting that cyclin D3 may not have essential functions in the gut. In the present study, we determined whether cyclin D3 is required for the transition from G0 into the S phase in intestinal epithelial cells. Microinjection of anti-cyclin D3 antiserum inhibited quiescent IEC-6 and RIE-1 cells from entering the S phase, while cells microinjected with a nonspecific mouse immunoglobin G continued to progress into the S phase. We also examined the expression of cyclin D3 in rat jejunal mucosa after fasting and refeeding. Cyclin D3 levels were not altered by fasting and refeeding; however, Cdk4 expression was suppressed by fasting and returned to control levels after refeeding. Our results suggest that cyclin D3 is essential for intestinal epithelial cell proliferation, although its expression is not regulated by dietary restriction.