PGE2 In Regulation Research Articles

Cyclooxygenase-2 knockdown by RNA interference in colon cancer

Overexpression of cyclooxygenase-2 (COX-2) is observed early in colon cancer. Treatments with COX-2-specific NSAIDs have been shown to reduce polyp size and polyp number in FAP patients with a predisposition to colorectal adenoma and cancer. However, the use of COX-2-specific NSAIDs in colon cancer patients has recently revealed increased cardiovascular risks. These harmful side effects may be the result of COX-dependent and/or COX-independent mechanisms. RNA interference (RNAi) is a method of post-transcriptional gene silencing intrinsic to cells. This study employed RNAi to specifically knockdown endogenous COX-2 expression in the HT-29 colon cancer cell line, and to observe the apoptotic response as well as 15-hydroxyprostaglandin dehydrogenase (15-PGDH) expression levels. Following treatment with a COX-2 siRNA, we demonstrated a significant knockdown at the protein level of 57% as compared to a non-silencing siRNA control. Protein results were corroborated by concurrent decrease in COX-2 mRNA levels following the same treatment regimen. Despite previous studies using NSAID treatment to implicate COX-2 involvement in apoptosis, we did not observe any alteration in Bcl-2 expression and Caspase-3 activation following COX-2 knockdown in these cells. 15-PGDH, a physiological antagonist of COX-2 in its catabolism of PGE2, showed a modest but significant induction in response to COX-2 knockdown. The precise role of COX-2 in apoptosis and PGE2 regulation remains unclear; however, having shown that down-regulation of endogenous levels of COX-2 can be achieved in colon cancer by RNAi, this strategy should prove to be a valuable tool in revealing the specific function of COX-2 in tumourigenesis.

093 Prostaglandin E2 Inhibits Fibroblast Migration: Implications for Wound Healing

Background: Wound healing is a complex process involving multiple cell types, extracellular matrix components and soluble mediators. Prostaglandin E2 is an important component of the inflammatory response to injury. PGE2 can regulate the fibroblast response to injury via the EP receptor family. Here, we examine PGE2 regulation of fibroblast migration. Our analysis extends to fibroblasts representing a spectrum of wound healing phenotypes. Hypothesis: Prostaglandin E2 mediated inhibition of fibroblast migration is conserved across multiple fibroblast phenotypes. Methods: Primary cultures of human fetal, adult and keloid fibroblasts were used. Analysis of the EP receptor profile for each fibroblast phenotype was conducted using real‐time PCR, Western blot and immunohistochemistry. Fibroblast migration was quantified using a well established in vitro scratch assay. Results: Prostaglandin E2, via EP2/EP4 receptors, inhibits fibroblast migration in all fibroblast phenotypes. Fetal fibroblasts retain a more robust migratory phenotype when compared to normal adult and keloid fibroblasts. Normal adult fibroblasts exhibit a dramatic destabilization of the actin cytoskeleton which accompanies PGE2 inhibition of cell migration. This effect was not observed in fetal or keloid fibroblasts. Conclusions: Fibroblast activity in the wound bed can be altered by inflammatory mediators. The effects of prostaglandin E2 appear to be partially conserved across various fibroblast phenotypes. Variability in the response of these cells, however, indicates that fibroblasts derived from fetal tissue may retain intrinsic altered response mechanisms to endogenous inflammatory mediators.

Time-dependent effect of LPS on PGE2 and TNF-α production by rat glial brain culture: influence of COX and cytokine inhibitors

This study was undertaken to investigate the effect of lipopolysaccharide (LPS) stimulation on the time course of prostaglandin E2 (PGE2) and tumor necrosis factor alpha (TNF-alpha) production by rat glial brain culture. A concentration of 10 microg/ml LPS from Escherichia coli was used as stimulation treatment. The effect of pentoxifylline (PXF), nimesulide (NIM), indomethacin (INDO) and dexamethasone (DEX) on the regulation of PGE2 and TNF-alpha production was tested. Stimulation of rat glial cells with LPS resulted in different time-dependent production patterns of PGE2 and TNF-alpha The time course of TNF-alpha elevation was short, reaching its peak at 6 h post LPS and decreasing to undetectable levels after 24 h. On the other hand, the time course of PGE2 elevation was longer, starting at 6 h post LPS treatment and increasing 100-fold compared with basal levels, 24 h post LPS exposure. The COX inhibitors (NIM and INDO) and DEX were found to inhibit the LPS-induced elevation in PGE2 production, while PXF lacked such an inhibitory effect. Furthermore, NIM, DEX and PXF were found to reduce the LPS-induced elevation in TNF-alpha levels, while INDO caused a greater elevation in TNF-alpha levels. These results may cast further light on the LPS-induced production of PGE2 and TNF-alpha by rat glial cell cultures and the relation between the two systems.

Regulation of chloride secretion across porcine endometrial epithelial cells by prostaglandin E2

1. The objective of this study was to investigate the mechanism of PGE2 regulation of Cl- transport across glandular endometrial cells grown in primary culture. 2. Most of the basal short circuit current (Isc) was inhibited by luminal addition of 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) or glibenclamide, suggesting the presence of a basally active Cl- conductance in the apical membrane. 3. Basolateral addition of 10 microM PGE2 increased Isc by 41 +/- 3 microA. A similar response was observed when cells were treated with 8-(4-chlorophenylthio) adenosine 3',5'-cyclic monophosphate (CPT-cAMP). Pretreatment of monolayers with NPPB and glibenclamide blocked the PGE2 and cAMP-mediated increase in Isc, suggesting that the effects of PGE2 and cAMP were dependent on the activity of an apical NPPB- and glibenclamide-sensitive conductance. 4. Addition of 50 nM antiPGE2 antibody to the basolateral bathing solution decreased basal Isc by 20 % and shifted the threshold response to exogenous PGE2. This result suggests autocrine regulation of electrogenic Cl- transport by PGE2. 5. Experiments with amphotericin B-permeabilized monolayers revealed that the apical PGE2-activated, NPPB- and glibenclamide-sensitive conductance was Cl- dependent and that the current-voltage relationship and anion permeation properties (SCN->Br- > Cl- > I-) were characteristic of the cystic fibrosis transmembrane conductance regulator (CFTR). 6. Cultured porcine endometrial epithelial cells were specifically labelled with an antibody to a peptide sequence within the regulatory domain of CFTR. 7. The effect of PGE2 was blocked by basolateral addition of bumetanide and furosemide at concentrations that are selective for inhibition of Na+-K+-2Cl-cotransport activity. The effect of bumetanide on Isc was Cl- dependent, suggesting a role for the bumetanide-sensitive transport pathway in Cl- secretion. 8. PGE2 and cAMP also activated an outwardly rectifying basolateral K+ channel which presumably sustains the driving force for electrogenic Cl- efflux across the apical membrane. 9. The concentration-conductance and concentration-Isc response relationships for PGE2 showed that basolateral K+ permeability was rate limiting with respect to transepithelial anion secretion and that activation of a basolateral K+ channel by PGE2 was necessary to achieve maximum rates of Cl- secretion.

Bcl-2 expression inhibits prostaglandin E2-mediated apoptosis in B cell lymphomas.

Apoptosis is a critical mechanism in the maturation and maintenance of the immune system. However, the process by which cells die remains poorly understood. The proto-oncogene bcl-2 is considered important in determining whether cells enter an apoptotic pathway or survive. In this report, we first examined the differential sensitivity of immature (CH31) and mature (CH12) B cell lymphomas to growth inhibition by PGE2. The CH31 cell line was growth inhibited and underwent apoptosis in response to PGE2, unlike its mature counterpart, CH12. Furthermore, endogenous levels of the anti-apoptotic protein Bcl-2 in CH31 cells were low compared with CH12. To further investigate the role of Bcl-2 in PGE2- and cAMP-mediated cell death, a retroviral vector bearing the human bcl-2 gene was introduced into CH31. High expression of Bcl-2 in CH31 had no effect on growth inhibition induced by PGE2 or dibutyryl cAMP. In contrast, increased expression of Bcl-2 completely inhibited PGE2- and cAMP-mediated DNA fragmentation and nuclear condensation. Finally, cell cycle analysis of Bcl-2-expressing CH31 cells demonstrated that PGE2 increased the percentage of cells in G1, and analysis of synchronized populations revealed that PGE2 acts at all phases of the cell cycle to delay normal progression. These results support the hypothesis that apoptosis induced through PGE2 and cAMP signaling is sensitive to regulation by Bcl-2 in CH31 B cell lymphomas. Furthermore, unlike apoptosis, regulation of PGE2- and cAMP-mediated growth inhibition in B lineage cells is a distinct and Bcl-2-independent mechanism.

Immune regulation of the L5178Y murine tumor-dormant state. I. In vivo and in vitro effects of prostaglandin E2 and indomethacin on tumor cell growth.

Immunization and intraperitoneal challenge of DBA/2 mice with L5178Y lymphoma cells results in the suppression and maintenance of the L5178Y cells in a tumor-dormant state in the peritoneal cavity for many months. Cell-mediated immune responses involving lymphocytes and macrophages are involved in maintenance of the tumor-dormant state. Macrophages that have increased immunosuppressive activity and that produce increased amounts of PGE2 appear in the peritoneal cavity of tumor-dormant mice before the breakdown of the tumor-dormant state and formation of ascitic tumors. We report here that the tumor-dormant state can be terminated with formation of ascitic tumors by treatment of tumor-dormant mice with PGE2. Treatment with indomethacin results in inhibition of tumor cell growth and elimination of all recoverable tumor cells. Cultures of peritoneal cells (PC) from mice harboring L5178Y cells in a tumor-dormant state were used to analyze the PGE2 and indomethacin effects. Tumor cells did not grow out in the high-cell density PC cultures prepared from many tumor-dormant mice, but addition of PGE2 to these cultures resulted in tumor cell growth. The tumor cell growth that did occur in the PC cultures from some tumor-dormant mice was associated with PGE2 production by the associated host cells, and the addition of indomethacin to these cultures inhibited both PGE2 synthesis and tumor cell growth. Removal of plastic-adherent cells from the PC cultures eliminated the restraint on tumor cell growth. These experiments suggest that L5178Y tumor cells are maintained in a tumor-dormant state by host peritoneal cells, which are under PGE2 regulation.

Regulation of breathing movements in fetal sheep by prostaglandin E2.

In fetal sheep, plasma prostaglandin (PG) E2 concentrations are high, and fetal breathing movements (FBM) occur intermittently, primarily during low-voltage fast electrocortical activity (LVFA). There is evidence suggesting that prostaglandins, specifically PGE2, may regulate FBM. To define the physiological role of PGE2 in regulation of FBM, we infused meclofenamate (0.9 mg X kg-1 X h-1), a prostaglandin synthetase inhibitor, into six fetal sheep to suppress endogenous prostaglandin production. Afterward, PGE2 was added in mean doses of 9, 18, 36, and 90 ng X kg-1 X min-1. Meclofenamate decreased PGE2 concentrations and increased FBM, especially during high-voltage slow electrocortical activity (HVSA). Addition of PGE2 reversed the effects of meclofenamate, increasing PGE2 concentrations and decreasing FBM, especially during HVSA. The response to PGE2 was dose dependent; the overall incidence of FBM and incidences of FBM during HVSA and LVFA were inversely correlated with both the infused PGE2 dose and the mean PGE2 concentration. At higher doses of PGE2, FBM occurred intermittently and only during LVFA; thus PGE2 infusion restored the physiological pattern of FBM. These results indicate that PGE2 regulates FBM by inhibiting FBM during HVSA.

Evidence for a role of calmodulin in cellular cAMP production in response to vasopressin, prostaglandin E2 and forskolin in cultured rat renal papillary collecting tubule cells

In the present study, the role of calmodulin in the cellular action of arginine vasopressin (AVP), prostaglandin (PG) E2 and forskolin on adenosine-3', 5'-monophosphate (cAMP) production was examined in cultured rat renal papillary collecting tubule cells. In the presence of the phosphodiesterase inhibitor, submaximal concentrations of 10(-9) M AVP, 2 X 10(-8) M PGE2 and 2.4 X 10(-7) M forskolin significantly increased cellular cAMP accumulation by 2.3, 6.0 and 8.4-fold, respectively. Two chemically dissimilar inhibitors of calmodulin, namely trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), attenuated the cellular production of cAMP in a dose-related manner in response to all three stimuli. A dose which inhibited the cellular production of cAMP by 50% (ID50) ranged from 1.6 X 10(-5) to 2.8 X 10(-5) M for trifluoperazine and from 3.5 X 10(-5) to 4.4 X 10(-5) M for W-7. Basal accumulation of cellular cAMP was also decreased by treatment with either trifluoperazine or W-7, but an effective dose was relatively higher than that which inhibited agents-stimulated cellular cAMP production. Since forskolin is recognized as activating adenylate cyclase at one step of the catalytic component and the cellular action of AVP to activate adenylate cyclase is mediated through receptor-catalytic component, the present study indicates calmodulin regulation of basal, AVP-, PGE2- and forskolin-activated adenylate cyclase in the papillary collecting tubule cells. Further study demonstrated the inhibition of AVP- or PGE2-induced cellular cAMP production by treatment with either a calcium-free medium or verapamil, a blocker of cellular calcium uptake, before and during the experiment. These findings suggest that an increase in cytosolic calcium, which interacts with calmodulin to form an active complex, is, at least in part, due to the increased cellular influx of calcium from the extracellular space.