Upregulation of prostaglandin receptor EP1 expression involves its association with cyclooxygenase-2.

While many signals cause upregulation of the pro-inflammatory enzyme cyclooxygenase -2 (COX-2), much less is known about mechanisms that actively downregulate its expression. We have recently shown that the prostaglandin EP1 receptor reduces the expression of COX-2 in a pathway that facilitates its ubiquitination and degradation via the 26S proteasome. Here we show that an elevation of COX-2 intracellular levels causes an increase in the endogenous expression of prostaglandin EP1. The increase in EP1 levels does not occur at the transcriptional level, but is rather associated with complex formation between the receptor and COX-2, which occurs both in vitro and in mammalian tissues. The EP1-COX-2 complex is disrupted following binding of arachidonic acid to COX-2 and accompanied by a parallel reduction in EP1 levels. We propose that a transient interaction between COX-2 and EP1 constitutes a feedback loop whereby an increase in COX-2 expression elevates EP1, which ultimately acts to downregulate COX-2 by expediting its proteasomal degradation. Such a post translational mechanism may serve to control both the ligand-generating system of COX-2 and its reception system

Overexpression of COX-2 raises endogenous levels of EP₁.

<p><i>A</i>, HEK 293 cells do not express detectable levels of COXs. Representative immunoblot of cell lysates transfected with 0.5 µg empty vector (pcDNA3.1, Mock) or wt COX-1 (upper panel) or COX-2 (lower panel). <i>B</i>, Effect of COX-1 and COX-2 overexpression on EP receptor levels. Representative immunoblots of cells were transfected with 0.5 µg mock, COX-1 or COX-2. Endogenous expression of EP receptor levels was determined using specific antibodies. <i>C</i>, Summary graph of the effect of COX overexpression on EP levels n = 7–9 different experiments. Shown are mean + SEM *p<0.05 vs. Mock transfection. <i>D</i>, Bovine aortic endothelial cells (BAEC) were treated with 1 µM LPS for the indicated times. Lysates were collected and probed for COX-2 and EP₁ levels. <i>E</i>, HEK 293 cells were transfected with 0.5 µg COX-2 or mock and samples were analyzed for content of endogenously expressing EP₁ or EP₂ mRNA using real-time PCR. n = 3 independent experiments performed in triplicates. P = 0.386 vs. mock.</p

The interaction between COX-2 and EP₁ depends upon the conformation of COX-2.

<p><i>A</i>, Cells were transfected with either wild type (wt) or G533A COX-2 and exposed to 10 µM AA for 19 h. While wt COX-2 levels were lowered in the presence of AA, G533A COX-2 was not affected. n = 4 independent experiments *p<0.05 vs. untreated. <i>B</i>, Summary graph of n = 5 independent experiments showing a reduction in the levels of EP₁ co-immunoprecipitated with COX-2 following treatment with AA. *p<0.05 vs. Mock transfection. HEK 293 cells, transfected with mock or G533A COX-2 were treated with either vehicle (ethanol) or AA (50 µM) for 15 min. Blots were probed for COX-2 and EP₁. <i>C</i>, Representative immunoblot of n = 3 independent experiments depicting the dose-dependent effect of AA on the interaction of G533A COX-2 with EP₁. Cells were transfected with either mock or G533ACOX-2 DNA, and 16 h post-transfection treated with 2.5 or 50 µM AA for 15 min prior to immunoprecipitation. Immunoprecipitate were probed for COX-2 and EP₁ levels. Total lysates from the same samples were probed first for phosho-ERK followed by total ERK levels. <i>D</i>, prolonged exposure to AA, does not affect G533A COX-2 but reduces EP₁ levels. Cells overexpressing G533A COX-2 were treated with 10 µM AA for 1.5–2 h, samples were collected and probed for levels of COX-2 and EP₁. n = 3 independent experiments, *p<0.05 vs. untreated.</p

Limited Proteolysis of Cyclooxygenase-2 Enhances Cell Proliferation

Accumulating evidence suggests that the cyclooxygenase-2 (COX-2) enzyme has additional catalytic-independent functions. Here we show that COX-2 appears to be cleaved in mouse and human tumors, which led us to hypothesize that COX-2 proteolysis may play a role in cell proliferation. The data presented herein show that a K598R point mutation at the carboxyl-terminus of COX-2 causes the appearance of several COX-2 immunoreactive fragments in nuclear compartments, and significantly enhances cell proliferation. In contrast, insertion of additional mutations at the border of the membrane-binding and catalytic domains of K598R COX-2 blocks fragment formation and prevents the increase in proliferation. Transcriptomic analyses show that K598R COX-2 significantly affects the expression of genes involved in RNA metabolism, and subsequent proteomics suggest that it is associated with proteins that regulate mRNA processing. We observe a similar increase in proliferation by expressing just that catalytic domain of COX-2 (&Delta;NT- COX-2), which is completely devoid of catalytic activity in the absence of its other domains. Moreover, we show that the &Delta;NT- COX-2 protein also interacts in the nucleus with &beta;-catenin, a central regulator of gene transcription. Together these data suggest that the cleavage products of COX-2 can affect cell proliferation by mechanisms that are independent of prostaglandin synthesis

Requirement for direct cross-talk between B1 and B2 kinin receptors for the proliferation of androgen-insensitive prostate cancer PC3 cells.

Stimulation of endogenous kinin receptors promotes growth of androgen-independent prostate cancer PC3 cells via activation of the mitogenic extracellular-signal-regulated kinase (ERK) pathway. In the present study, we show that kinin-mediated mitogenic signalling and prostate-cell growth involves two subtypes of bradykinin (BK) receptors, B1R and B2R. Specific stimulation of either B1R or B2R by their respective agonists des-Arg(9)-BK and Lys-BK promoted ERK activation and cell growth, whereas selective blockade with specific antagonists des-Arg(9)-[Leu(8)]BK and Hoe 140 respectively obliterated this effect, indicating the presence of both receptor subtypes. However, blockade of B1R also inhibited B2R-mediated ERK activation and cell growth, and, similarly, antagonism of B2R inhibited the B1R-mediated response. Furthermore, both B1R and B2R agonists promoted internalization of B1R, whereas both receptor antagonists blocked this effect. The B1R ligands des-Arg(9)-BK and des-Arg(9)-[Leu(8)]BK had no effect on the binding of BK to B2R, as demonstrated by radioligand competitive binding studies. However, blockade of either B1R or B2R impaired the ability of the reciprocal receptor to produce inositol phosphates, suggesting that the interaction between B1R and B2R is proximal to activation of phospholipase C. These results provide evidence for the existence of B1R-B2R complexes in prostate cancer PC3 cells and demonstrate that antagonism of one receptor interferes with the signalling ability of the other, possibly at the level of receptor-Galpha(q) protein coupling. Selective inhibition of B1R, which is up-regulated in injured and cancerous tissue, may be beneficial for the treatment of advanced prostate cancer

DNA methylation of specific CpG sites in the promoter region regulates the transcription of the mouse oxytocin receptor.

Oxytocin is a peptide hormone, well known for its role in labor and suckling, and most recently for its involvement in mammalian social behavior. All central and peripheral actions of oxytocin are mediated through the oxytocin receptor, which is the product of a single gene. Transcription of the oxytocin receptor is subject to regulation by gonadal steroid hormones, and is profoundly elevated in the uterus and mammary glands during parturition. DNA methylation is a major epigenetic mechanism that regulates gene transcription, and has been linked to reduced expression of the oxytocin receptor in individuals with autism. Here, we hypothesized that transcription of the mouse oxytocin receptor is regulated by DNA methylation of specific sites in its promoter, in a tissue-specific manner. Hypothalamus-derived GT1-7, and mammary-derived 4T1 murine cell lines displayed negative correlations between oxytocin receptor transcription and methylation of the gene promoter, and demethylation caused a significant enhancement of oxytocin receptor transcription in 4T1 cells. Using a reporter gene assay, we showed that methylation of specific sites in the gene promoter, including an estrogen response element, significantly inhibits transcription. Furthermore, methylation of the oxytocin receptor promoter was found to be differentially correlated with oxytocin receptor expression in mammary glands and the uterus of virgin and post-partum mice, suggesting that it plays a distinct role in oxytocin receptor transcription among tissues and under different physiological conditions. Together, these results support the hypothesis that the expression of the mouse oxytocin receptor gene is epigenetically regulated by DNA methylation of its promoter

The Heparanase Inhibitor PG545 Attenuates Colon Cancer Initiation and Growth, Associating with Increased p21 Expression

Heparanase activity is highly implicated in cellular invasion and tumor metastasis, a consequence of cleavage of heparan sulfate and remodeling of the extracellular matrix underlying epithelial and endothelial cells. Heparanase expression is rare in normal epithelia, but is often induced in tumors, associated with increased tumor metastasis and poor prognosis. In addition, heparanase induction promotes tumor growth, but the molecular mechanism that underlines tumor expansion by heparanase is still incompletely understood. Here, we provide evidence that heparanase down regulates the expression of p21 (WAF1/CIP1), a cyclin-dependent kinase inhibitor that attenuates the cell cycle. Notably, a reciprocal effect was noted for PG545, a potent heparanase inhibitor. This compound efficiently reduced cell proliferation, colony formation, and tumor xenograft growth, associating with a marked increase in p21 expression. Utilizing the APC Min+/− mouse model, we show that heparanase expression and activity are increased in small bowel polyps, whereas polyp initiation and growth were significantly inhibited by PG545, again accompanied by a prominent induction of p21 levels. Down-regulation of p21 expression adds a novel feature for the emerging pro-tumorigenic properties of heparanase, while the potent p21 induction and anti-tumor effect of PG545 lends optimism that it would prove an efficacious therapeutic in colon carcinoma patients

Methylation of specific CpG sites in the <i>Oxtr</i> minimal promoter inhibits transcription.

<p><b>A</b>) A schematic depiction of the distinct <i>Oxtr</i> promoter/<i>EYFP</i> constructs used; An <i>EYFP</i> gene was coupled to a minimal promoter (positions −1417 to +46) of the mouse <i>Oxtr</i> gene (<i>Unmodified</i>). This construct was modified by a C to A mutation at CpG sites 1 (<i>Mut 1</i>) or site 7 (<i>Mut 7</i>) or by deleting the ∼400 bp amplicon region (<i>Del</i>). <b>B,C</b>) EYFP mean fluorescence intensity measured in GT1-7 cells that were transfected with either (<b>B</b>) untraeatd or (<b>C</b>) methyltransferase-treated <i>Oxtr</i> promoter/<i>EYFP</i> constructs. No significant differences were found among the untreated plasmids whereas a highly significant difference was found among the methyltransferase-treated plasmids. <b>D</b>) A comparison between the treatedand untreated versions of all plasmids. Highly significant differences were found only in the cases of the <i>Unmodified</i> and <i>Mut 1</i> plasmids. <b>Statistics</b>: <b>B</b>) one-way ANOVA F₍₃₎ = 2.32, <i>p</i>>0.1. <b>C</b>) - one-way ANOVA F₍₃₎ = 9.4, <i>p</i><0.01, * differ from <i>Unmodified</i>, <i>p</i><0.05, n = 5. <b>D</b>) t-test, *<i>p</i><0.05/4.</p

<i>Oxtr</i> mRNA levels correlate with <i>Oxtr</i> promoter methylation in cell lines.

<p><b>A</b>) The relative mRNA levels in GT1-7 are significantly higher than in 4T1 cells. <b>B</b>) Methylation of the seven CpG sites is higher in 4T1 cells than in GT1-7 cells. Each row represents a single clone and each column represents one of the seven CpG sites. The total percentage of methylation was calculated from the fraction of black spots (methylated CpG sites). <b>C</b>) Representative gel showing greater ERK phosphorylation in GT1-7 cells stimulated with 1 µM OT for 10 min compared vehicle-treated cells Graph is summary of three independent experiments. <b>D</b>) Quantities of <i>Oxtr</i> mRNA in GT1-7 cells following 24 h treatment with 1 µM OT. An OT-stimulated increase in <i>Oxtr</i> mRNA was documented in three independent experiments. <b>Statistics</b> (t-test): <b>A</b>) *<i>p</i><0.001, n = 4. <b>C</b>) *<i>p</i><0.05, n = 3. <b>D</b>) *<i>p</i><0.01 n = 3.</p