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Richard M Breyer

Publications and source records attributed to Richard M Breyer.

32 records · Page 2Linked to original sources
PubMed2003

Disrupted function of tumor necrosis factor-alpha-stimulated gene 6 blocks cumulus cell-oocyte complex expansion.

During ovulation, the oocyte and surrounding somatic cumulus cells contained within a specialized, mucoid matrix are released from the ovary. One matrix component, TNF-alpha-stimulated gene 6 (TSG-6), is a hyaluronan binding protein induced in cumulus cells of preovulatory follicles by the LH surge and is decreased in cumulus cells of COX-2 and prostaglandin E2 (PGE2) receptor subtype EP2 null mice that exhibit impaired ovulation and cumulus expansion. To determine if TSG-6 was hormonally induced in cumulus cells in vitro and was functional during the formation of the expanded matrix, we established a cumulus cell-oocyte complex (COC) culture system. This system was used to analyze the effects of FSH, PGE2, EP2 receptor, and selected protein kinase inhibitors on TSG-6 production as well as specific antibodies to the TSG-6 link module on TSG-6 function. We document that TSG-6 message and protein are induced by cAMP/protein kinase A/MAPK signaling pathways and that blocking these cascades prevents expansion and the production of TSG-6. FSH but not PGE2 rescued expansion and production of TSG-6 in the EP2 null COCs, indicating that generation of a cAMP signal is essential. Furthermore, disruption of the functional interactions between TSG-6, inter-alpha trypsin inhibitor, and hyaluronan with specific antibodies severely altered matrix formation and cumulus expansion, as recorded by time-lapse imaging. Collectively, these results indicate that TSG-6 mRNA is induced in cumulus cells in culture by cAMP and that the secreted TSG-6 protein is a key structural component of the mouse COC matrix.

Animals↗
PubMed2003

Expression and molecular pharmacology of the mouse CRTH2 receptor.

Prostaglandin D2 (PGD2), the predominant prostanoid produced by activated mast cells, is implicated in a variety of allergic diseases. PGD2 exerts its effects through two G-protein coupled receptors, DP and CRTH2. PGD2 mediates chemotaxis of eosinophils, basophils, and Th2 cells via CRTH2-evoked signaling, suggesting a role for this receptor in allergic disease. To characterize the mouse CRTH2 ortholog (mCRTH2), we amplified the mCRTH2 receptor gene and expressed it in HEK293 cells. Saturation ligand binding isotherms demonstrated high-affinity binding of [3H]PGD2, with a Kd of 8.8 +/- 0.8 nM. Competition binding assays with a panel unlabeled prostanoids demonstrated an order of affinity of 13,14-dihydro-15-keto-PGD2 (DK-PGD2) >or= 15-deoxy-Delta12,14-PGJ2 (15d-PGJ2) >or= PGD2 >or= PGJ2. [3H]PGD2 binding was also displaced by the nonsteroidal anti-inflammatory drug indomethacin, with a Ki value of 1.04 +/- 0.13 microM. No [3H]PGD2 displacement was detected using fluribrofen, ibuprofen, or aspirin as competitors at concentrations of up to 30 microM. PGD2, DK-PGD2, 15d-PGJ2, and indomethacin each inhibited intracellular cAMP generation in stable transfectant ER293/mCRTH2 cells through a pertussis toxin (PTX) sensitive pathway, consistent with mCRTH2 coupling to a Gi heterotrimeric G-protein. Activation of mCRTH2 elicited chemotaxis of ER293/mCRTH2 cells in response to PGD2, indomethacin, and 15d-PGJ2. mCRTH2-dependent chemotaxis was inhibited by PTX and wortmannin, indicating dependence on Gi and PI 3-kinase signal transduction pathways. These data provide the first pharmacological and functional characterization of the mouse CRTH2 receptor.

Animals↗
PubMed2003

Expression of the prostaglandin F receptor (FP) gene along the mouse genitourinary tract.

PGF(2alpha) is one of the major prostanoids produced by the kidney. The cellular effects of PGF(2alpha) are mediated by a G protein-coupled transmembrane receptor designated the FP receptor. Both in situ hybridization and beta-galactosidase knocked into the endogenous FP locus were used to determine the cellular distribution of the mouse FP receptor. Specific labeling was detected in the kidney, ovary, and uterus. Abundant FP expression in ovarian follicles and uterus is consistent with previous reports of failed parturition in FP-/- mice. In the kidney, coexpression of the mFP mRNA with the thiazide-sensitive cotransporter defined its expression in the distal convoluted tubule (DCT). FP receptor was also present in aquaporin-2-positive cortical collecting ducts (CCD). No FP mRNA was detected in glomeruli, proximal tubules, or thick ascending limbs. Intrarenal expression of the FP receptor in the DCT and CCD suggests an important role for the FP receptor regulating water and solute transport in these segments of the nephron.

Animals↗
PubMed2003

Cancer-associated immunodeficiency and dendritic cell abnormalities mediated by the prostaglandin EP2 receptor.

Prostaglandin E(2) (PGE(2)), a major COX metabolite, plays important roles in several facets of tumor biology. We characterized the contribution of the PGE(2) EP2 receptor to cancer-associated immune deficiency using EP2(-/-) mice. EP2(-/-) mice exhibited significantly attenuated tumor growth and longer survival times when challenged with MC26 or Lewis lung carcinoma cell lines as compared with their wild-type littermates. While no differences in T cell function were observed, PGE(2) suppressed differentiation of DCs from wild-type bone marrow progenitors, whereas EP2-null cells were refractory to this effect. Stimulation of cells in mixed lymphocyte reactions by wild-type DCs was suppressed by treatment with PGE(2), while EP2(-/-)-derived DCs were resistant to this effect. In vivo, DCs, CD4(+), and CD8(+) T cells were significantly more abundant in draining lymph nodes of tumor-bearing EP2(-/-) mice than in tumor-bearing wild-type mice, and a significant antitumor cytotoxic T lymphocyte response could be observed only in the EP2(-/-) animals. Our data demonstrate an important role for the EP2 receptor in PGE(2)-induced inhibition of DC differentiation and function and the diminished antitumor cellular immune responses in vivo.

Animals↗
PubMed2003

Decreased expression of tumor necrosis factor-alpha-stimulated gene 6 in cumulus cells of the cyclooxygenase-2 and EP2 null mice.

Ovulation, the release of fertilizable oocytes from mature follicles, involves tissue remodeling and increased prostaglandin (PG) signaling. Cyclooxygenase (COX)-2 is the rate-limiting enzyme during PG synthesis. Female mice null for either COX-2 or the PGE(2) receptor EP2 are infertile, show decreased ovulation, and exhibit abnormal cumulus expansion. Cumulus expansion is the production of a complex extracellular matrix surrounding the cumulus-oocyte complex (COC). Matrix components consist of hyaluronan, proteoglycans, and proteins with hyaluronan binding domains. One such hyaluronan binding protein is TNFalpha-stimulated gene 6 (TSG-6). By various methods, we show induction of TSG-6 and hyaluronan synthase-2 mRNA in ovaries of mice treated with pregnant mare serum gonadotropin and human chorionic gonadotropin. By in situ hybridization, we show that both genes are expressed in periantral mural granulosa cells and cumulus cells of the mouse ovary. Notably, RT-PCR and in situ hybridization show that TSG-6 mRNA but not hyaluronan synthase-2 mRNA expression is selectively reduced in cumulus cells of COX-2 and EP2 null mice. Western analysis further confirms that TSG-6 protein is reduced in isolated COCs but remains covalently associated with inter alpha-trypsin inhibitor in COX-2 null mice. These observations identify TSG-6 as a target of PG action and show that its production in ovulatory follicles is associated with proper formation of the cumulus-derived extracellular matrix.

Alpha-Globulins↗
PubMed2003

Prostanoid receptors and the urogenital tract.

Prostanoids are the metabolic product of cyclooxygenase-mediated metabolism of arachidonic acid. These prostanoids interact with a family of eight G protein-coupled transmembrane receptors, which are abundantly expressed along the genitourinary tract and mediate the critical physiological actions of the prostanoids. These actions include modulation of renal glomerular hemodynamics, promotion of renal salt excretion, and maintenance of uroepithelial function and integrity. Normal functioning of prostanoid receptors is also a prerequisite for fertility and reproduction. Prostanoid receptor selective agonists and antagonists are likely to affect these processes quite differently from the simultaneous and global inhibition of all prostanoid synthesis that is achieved through the use of non-steroidal anti-inflammatory drugs.

Animals↗
PubMed2002

Prosurvival and antiapoptotic effects of PGE2 in radiation injury are mediated by EP2 receptor in intestine.

The biological activities of PGE(2) are mediated through EP receptors (EP(1)-EP(4)), plasma membrane G protein-coupled receptors that differ in ligand binding and signal-transduction pathways. We investigated gastrointestinal EP(2) receptor expression in adult mice before and after radiation injury and evaluated intestinal stem cell survival and crypt epithelial apoptosis after radiation injury in EP(2) null mice. EP(2) was expressed throughout the gut. Intestinal EP(2) mRNA increased fivefold after gamma-irradiation. Crypt survival was diminished in EP(2)-/- mice (4.06 crypts/cross section) compared with wild-type littermates (8.15 crypts/cross section). Radiation-induced apoptosis was significantly increased in EP(2)-/- mice compared with wild-type littermates. Apoptosis was 1.6-fold higher in EP(2) (-/-) mice (5.9 apoptotic cells/crypt) than in wild-type mice (3.5 apoptotic cells/crypt). The EP(2) receptor is expressed in mouse gastrointestinal epithelial cells and is upregulated following radiation injury. The effects of PGE(2) on both crypt epithelial apoptosis and intestinal crypt stem cell survival are mediated through the EP(2) receptor.

Animals↗
PubMed2002

Cloning and expression of the rabbit prostaglandin EP2 receptor.

BACKGROUND: Prostaglandin E2 (PGE2) has multiple physiologic roles mediated by G protein coupled receptors designated E-prostanoid, or "EP" receptors. Evidence supports an important role for the EP2 receptor in regulating fertility, vascular tone and renal function. RESULTS: The full-length rabbit EP2 receptor cDNA was cloned. The encoded polypeptide contains 361 amino acid residues with seven hydrophobic domains. COS-1 cells expressing the cloned rabbit EP2 exhibited specific [3H]PGE2 binding with a Kd of 19.1 +/- 1.7 nM. [3H]PGE2 was displaced by unlabeled ligands in the following order: PGE2>>PGD2=PGF2alpha=iloprost. Binding of [3H]PGE2 was also displaced by EP receptor subtype selective agonists with a rank order of affinity consistent with the EP2 receptor (butaprost>AH13205>misoprostol>sulprostone). Butaprost free acid produced a concentration-dependent increase in cAMP accumulation in rabbit EP2 transfected COS-1 cells with a half-maximal effective concentration of 480 nM. RNase protection assay revealed high expression in the ileum, spleen, and liver with lower expression in the kidney, lung, heart, uterus, adrenal gland and skeletal muscle. In situ hybridization localized EP2 mRNA to the uterine endometrium, but showed no distinct localization in the kidney. EP2 mRNA expression along the nephron was determined by RT-PCR and its expression was present in glomeruli, MCD, tDL and CCD. In cultured cells EP2 receptor was not detected in collecting ducts but was detected in renal interstitial cells and vascular smooth muscle cells. EP2 mRNA was also detected in arteries, veins, and preglomerular vessels of the kidney. CONCLUSION: EP2 expression pattern is consistent with the known functional roles for cAMP coupled PGE2 effects in reproductive and vascular tissues and renal interstitial cells. It remains uncertain whether it is also expressed in renal tubules.

Amino Acid Sequence↗
PubMed2002

Prostaglandin receptor EP2 mediates PGE2 stimulated hypercalcemia in mice in vivo.

Prostaglandin E2 (PGE2) can stimulate bone resorption by a cyclic AMP-dependent pathway. Two PGE2 receptors, EP2 and EP4 have been shown to play a role in PGE2 stimulation of osteoclast formation. In primary osteoblastic cell cultures from EP2 wild type (EP2 +/+) mice, PGE2 (0.1 microM) increased cyclic AMP production 3.5-fold, but PGE2 had no effect on cells from mice in which the EP2 receptor had been deleted (EP2 -/-). To examine the role of the EP2 receptor in the resorption response in vivo we injected PGE2 in EP2 -/- mice, and compared them with EP2 +/+ mice. Injection of PGE2 (3 mg/kg, four times daily for three days) in 9- to 12-month-old male mice on a 129 SvEv background increased serum calcium from 9.8 +/- 0.5 to 10.7 +/- 0.3 mg/dl (P < 0.01) in EP2 +/+ mice but not in EP2 -/- mice (10.1 +/- 0.3 vs. 10.2 +/- 0.3 mg/dl). PGE2 injection (6 mg/kg twice a day for three days) in 3-4 month old male mice on a C57 BL/6 X 129 SvEv background increased calcium from 8.2 +/- 0.1 to 9.0 +/- 0.3 mg/dl (P < 0.05) in EP2 +/+ mice but had no effect in EP2-/- mice (8.4 +/- 0.1 vs. 8.3 +/- 0.2 mg/dl). Injection of PGE2 over the calvariae of EP2 +/+ and EP2-/- mice increased the expression of receptor activator of nuclear factor kappaB ligand (RANKL) both locally and in the tibia, but RANKL responses were lower in EP2 -/- mice. We conclude that EP2 receptor plays a role in the hypercalcemic response to PGE2. This impaired response in EP2 -/- mice may be due to decreased ability to stimulate cyclic AMP and in part, to a smaller increase in the expression of RANKL mRNA.

Actins↗
PubMed2002

Neuronal oxidative damage from activated innate immunity is EP2 receptor-dependent.

Increase in prostaglandin (PG) E2 levels and oxidative damage are associated with diseases of brain that involve activation of innate immunity. We tested the hypothesis that cerebral oxidative damage resulting from activation of innate immunity with intracerebroventricular (icv) lipopolysaccharide (LPS) is dependent on PGE2-mediated signaling. We measured two quantitative in vivo biomarkers of lipid peroxidation: F2-isoprostanes (IsoPs) that derive from arachidonic acid (AA) that is uniformly distributed in all cell types in brain, and F4-neuroprostanes (NeuroPs) that derive from docosahexaenoic acid (DHA) that is highly concentrated in neuronal membranes. LPS stimulated delayed elevations in cerebral F2-IsoPs and F4-NeuroPs that were completely suppressed by indomethacin or ibuprofen pre-treatment. LPS-induced cerebral oxidative damage was abolished by disruption of subtype 2 receptor for PGE2 (EP2). In contrast, initial oxidative damage from icv kainic acid (KA) was more rapid than with LPS also was completely suppressed by indomethacin or ibuprofen pre-treatment but was independent of EP2 receptor activation. The protective effect of deleting the EP2 receptor was not associated with changes in cerebral eicosaniod production, but was partially related to reduced induction of nitric oxide synthase (NOS) activity. These results suggest the EP2 receptor as a therapeutic target to limit oxidative damage from activation of innate immunity in cerebrum.

Animals↗
PubMed2002

Contribution of prostaglandin EP(2) receptors to renal microvascular reactivity in mice.

The present studies were performed to determine the contribution of EP(2) receptors to renal hemodynamics by examining afferent arteriolar responses to PGE(2), butaprost, sulprostone, and endothelin-1 in EP(2) receptor-deficient male mice (EP(2)-/-). Afferent arteriolar diameters averaged 17.8 +/- 0.8 microm in wild-type (EP(2)+/+) mice and 16.7 +/- 0.7 microm in EP(2)-/- mice at a renal perfusion pressure of 100 mmHg. Vessels from both groups of mice responded to norepinephrine (0.5 microM) with similar 17-19% decreases in diameter. Diameters of norepinephrine-preconstricted afferent arterioles increased by 7 +/- 2 and 20 +/- 6% in EP(2)+/+ mice in response to 1 microM PGE(2) and 1 microM butaprost, respectively. In contrast, afferent arteriolar diameter of EP(2)-/- mice decreased by 13 +/- 3 and 16 +/- 6% in response to PGE(2) and butaprost. The afferent arteriolar vasoconstriction to butaprost in EP(2)-/- mice was eliminated by angiotensin-converting enzyme inhibition. Sulprostone, an EP(1) and EP(3) receptor ligand, decreased afferent arteriolar diameter in both groups; however, the vasoconstriction in the EP(2)-/- mice was greater than in the EP(2)+/+ mice. Endothelin-1-mediated afferent arteriolar diameter responses were enhanced in EP(2)-/- mice. Afferent arteriolar diameter decreased by 29 +/- 7% in EP(2)-/- and 12 +/- 7% in EP(2)+/+ mice after administration of 1 nM endothelin-1. These results demonstrate that the EP(2) receptor mediates a portion of the PGE(2) afferent arteriolar vasodilation and buffers the renal vasoconstrictor responses elicited by EP(1) and EP(3) receptor activation as well as endothelin-1.

Alprostadil↗
PubMed2002

Opposite effects of cyclooxygenase-1 and -2 activity on the pressor response to angiotensin II.

Therapeutic use of cyclooxygenase-inhibiting (COX-inhibiting) nonsteroidal antiinflammatory drugs (NSAIDs) is often complicated by renal side effects including hypertension and edema. The present studies were undertaken to elucidate the roles of COX1 and COX2 in regulating blood pressure and renal function. COX2 inhibitors or gene knockout dramatically augment the pressor effect of angiotensin II (Ang II). Unexpectedly, after a brief increase, the pressor effect of Ang II was abolished by COX1 deficiency (either inhibitor or knockout). Ang II infusion also reduced medullary blood flow in COX2-deficient but not in control or COX1-deficient animals, suggesting synthesis of COX2-dependent vasodilators in the renal medulla. Consistent with this, Ang II failed to stimulate renal medullary prostaglandin E(2) and prostaglandin I(2) production in COX2-deficient animals. Ang II infusion normally promotes natriuresis and diuresis, but COX2 deficiency blocked this effect. Thus, COX1 and COX2 exert opposite effects on systemic blood pressure and renal function. COX2 inhibitors reduce renal medullary blood flow, decrease urine flow, and enhance the pressor effect of Ang II. In contrast, the pressor effect of Ang II is blunted by COX1 inhibition. These results suggest that, rather than having similar cardiovascular effects, the activities of COX1 and COX2 are functionally antagonistic.

Angiotensin II↗
PubMedDate not supplied

Prostaglandin E2 receptor subtype 2 (EP2) null mice are protected against murine lung tumorigenesis.

BACKGROUND: Manipulating prostaglandin (PG) production modulates tumor development. Elevated PGI2 production prevents murine lung cancer, while decreasing PGE2 content protects against colon cancer. PGE2 receptor subtype 2 (EP2) -deficient mice were hypothesized to be resistant to lung tumorigenesis. MATERIALS AND METHODS: EP2 null BALB/c mice and their wild-type littermates were exposed to an initiation-promotion carcinogenesis protocol and lung tumorigenesis was examined. Chronic lung inflammation was induced to determine whether EP2 ablation influenced inflammatory cell infiltration. RESULTS: Tumor multiplicity in EP2 null mice was 34% lower than in their wild-type littermates (21.9+/-3.0 vs. 14.5+/-2.9 tumors/mouse, p<0.001). The lung tumor burden, an indicator of growth rate, also declined (57%, p<0.05). All the mice exhibited similar inflammatory cell infiltration. CONCLUSION: PGE2, acting through EP2, enhanced lung tumorigenesis through a mechanism that may be distinct from its proinflammatory activity. Thus, EP2 is a potential target for novel chemoprevention strategies.

Animals↗