Droplet Digital PCR Is a Novel Screening Method Identifying Potential Cardiac G-Protein-Coupled Receptors as Candidate Pharmacological Targets in a Rat Model of Pressure-Overload-Induced Cardiac Dysfunction.

The identification of novel drug targets is needed to improve the outcomes of heart failure (HF). G-protein-coupled receptors (GPCRs) represent the largest family of targets for already approved drugs, thus providing an opportunity for drug repurposing. Here, we aimed (i) to investigate the differential expressions of 288 cardiac GPCRs via droplet digital PCR (ddPCR) and bulk RNA sequencing (RNAseq) in a rat model of left ventricular pressure-overload; (ii) to compare RNAseq findings with those of ddPCR; and (iii) to screen and test for novel, translatable GPCR drug targets in HF. Male Wistar rats subjected to transverse aortic constriction (TAC, n = 5) showed significant systolic dysfunction vs. sham operated animals (SHAM, n = 5) via echocardiography. In TAC vs. SHAM hearts, RNAseq identified 69, and ddPCR identified 27 significantly differentially expressed GPCR mRNAs, 8 of which were identified using both methods, thus showing a correlation between the two methods. Of these, Prostaglandin-F2α-receptor (Ptgfr) was further investigated and localized on cardiomyocytes and fibroblasts in murine hearts via RNA-Scope. Antagonizing Ptgfr via AL-8810 reverted angiotensin-II-induced cardiomyocyte hypertrophy in vitro. In conclusion, using ddPCR as a novel screening method, we were able to identify GPCR targets in HF. We also show that the antagonism of Ptgfr could be a novel target in HF by alleviating cardiomyocyte hypertrophy.

Expression of PTGS2, PGFS and PTGFR during downregulation and restart of spermatogenesis following GnRH agonist treatment in the dog.

Prostaglandins (PGs) and prostaglandin endoperoxide synthase (PTGS) are considered to be relevant for spermatogenesis and steroidogenesis. PTGS2, prostaglandin F synthase (PGFS) and PGF receptor (PTGFR) are investigated in the adult male dog using the model of the GnRH-agonist implant downregulated canine testis and its subsequent restart of spermatogenesis following abolition of treatment (3, 6, 9, 12 weeks after implant removal). On the mRNA level (ratio), PTGS2, PGFS and PTGFR expression did not differ between downregulation, different stages of recovery of spermatogenesis and untreated adult controls (CG). On the protein level, Sertoli and Leydig cells in all samples and some peritubular cells stained immunopositive for PTGS2. In the tubular compartment, the percentage of the PTGS2-immunopositive area (PIA) and the mean PTGS2-staining intensity (gray scale, GS) did not differ between groups but in the interstitial compartment, the PIA (p = 0.0494) and the GS (p < 0.0001) were significantly upregulated during early recrudescence (week 3/6). Comparing downregulation by two GnRH-agonist implants with juvenile controls (JG) and CG, the mRNA expression (ratio) did not differ. In the tubular compartment, the GS (p = 0.0321) was significantly higher at downregulation compared to CG and in the interstitial compartment, the PIA (p = 0.0073) and the GS (p = 0.0097) were significantly higher in JG compared to downregulation/CG. PTGS2, PGFS and PTGFR mRNA and PTGS2 protein are regularly expressed in the adult, juvenile and downregulated canine testis and downregulation and subsequent recrudescence affect PTGS2 protein expression mainly in Leydig cells. PTGS2 expression in the downregulated testis resembles the one in seasonal Syrian hamster but not juvenile canine testis.

The prostaglandin E2 receptor PTGER2 and prostaglandin F2α receptor PTGFR mediate oviductal glycoprotein 1 expression in bovine oviductal epithelial cells.

Oviductal glycoprotein 1 (OVGP1), an oviductin, is involved in the maintenance of sperm viability and motility and contributes to sperm capacitation in the oviduct. In this study, the regulatory effects exerted by prostaglandin E2 (PGE2) and F2α (PGF2α) on OVGP1 expression via their corresponding receptors in bovine oviductal epithelial cells (BOECs) were investigated. BOECs were cultured in vitro, and their expression of receptors of PGE2 (PTGER1, PTGER2, PTGER3, and PTGER4) and PGF2α (PTGFR) was measured using RT-qPCR. Ca2+ concentration was determined with a fluorescence-based method and cAMP was quantified by enzyme-linked immunosorbent assays to verify activation of PTGER2 and PTGFR by their corresponding agonists in these cells. OVGP1 mRNA and protein expression was measured using RT-qPCR and western blotting, respectively, following PTGER2 and PTGFR agonist-induced activation. PTGER1, PTGER2, PTGER4, and PTGFR were found to be present in BOECs; however, PTGER3 expression was not detected. OVGP1 expression was significantly promoted by 10-6 M butaprost (a PTGER2 agonist) and decreased by 10-6 M fluprostenol (a PTGFR agonist). In addition, 3 µM H-89 (a PKA inhibitor) and 3 µM U0126 (an ERK inhibitor) effectively inhibited PGE2-induced upregulation of OVGP1, and 5 µM chelerythrine chloride (a PKC inhibitor) and 3 µM U0126 negated OVGP1 downregulation by PGF2α. In conclusion, this study demonstrates that OVGP1 expression in BOECs is enhanced by PGE2 via PTGER2-cAMP-PKA signaling, and reduced by PGF2α through the PTGFR-Ca2+-PKC pathway.

Correlations of AFAP1, GMDS and PTGFR gene polymorphisms with intra-ocular pressure response to latanoprost in patients with primary open-angle glaucoma.

WHAT IS KNOWN AND OBJECTIVE: Genomewide association studies have identified a number of genetic variants that are associated with the development of primary open-angle glaucoma (POAG). This study aimed to explore possible correlations of common single nucleotide polymorphisms (SNPs) in AFAP1, GMDS and PTGFR genes with intra-ocular pressure (IOP) response to latanoprost in POAG patients. METHODS: From January 2012 to December 2014, 135 patients with POAG were enrolled into our study. Direct sequencing of polymerase chain reaction (PCR) products was used to analyse the distribution of allelic and genotypic frequencies of AFAP1 rs11723068 G>A and rs757253 T>C, GMDS rs9503012 C>T and rs17134549 T>A, and PTGFR rs3753380 C>T and rs3766355 A>C. RESULTS AND DISCUSSION: After 1, 2 and 4 weeks of latanoprost treatment, the IOP of POAG patients decreased significantly (all P < 0·05). The genotype frequencies of six SNPs in AFAP1, GMDS and PTGFR genes were conformed to Hardy-Weinberg equilibrium (HWE). Before latanoprost treatment, the baseline IOP levels of POAG patients carrying CC+AC genotypes of PTGFR rs3766355 A>C were higher than those carrying AA genotype (P < 0·05). After 1, 2 and 4 weeks of latanoprost treatment, POAG patients carrying TT genotype of GMDS rs9503012 C>T showed better response to latanoprost than those carrying CC+CT genotype (P < 0·05). Similarly, POAG patients carrying AA genotype of PTGFR rs3766355 A>C showed better response to latanoprost than those carrying CC+AC genotypes (P < 0·05). Logistic regression analysis showed that age, CC+CT genotypes of GMDS rs9503012 C>T and CC+AC genotypes of PTGFR rs3766355 A>C were independent risk factors for poor response to latanoprost in POAG patients. WHAT IS NEW AND CONCLUSIONS: GMDS rs9503012 C>T and PTGFR rs3766355 A>C may be associated with IOP response to latanoprost in POAG patients.

Prostaglandin F2α-PTGFR signaling promotes proliferation of endometrial epithelial cells of cattle through cell cycle regulation.

There is production of prostaglandin F2α (PGF2α) and there is PGF2α receptor (PTGFR) mRNA transcript in endometrial epithelial cells of cattle. The aims of the present study were to (1) determine whether PGF2α-PTGFR signaling modulates the proliferation of endometrial epithelial cells and (2) increase knowledge of PGF2α-PTGFR signaling on the physiological and pharmacological processes in the endometrium of cattle. Amount of cellular proliferation was determined using real-time cell analysis and cell proliferation reagent WST-1 procedures. Abundance of cyclins, cyclin-dependent kinases (CDKs), cyclin-kinase inhibitors, proliferating cell nuclear antigen (PCNA), cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), PTGFR, epidermal growth factor (EGF) mRNA and protein abundances were evaluated using real-time RT-PCR and western blot analyses. The PGF2α-PTGFR signaling promoted the proliferation of endometrial epithelial cells by inducing changes in abundance of mRNA transcript and protein that resulted in an increase in the abundance for the cyclins (A, B1, D1, D3), CDKs (1, 2, 4, 6), and PCNA; decrease in abundance for p21; and increase in abundance for EGF, COX-1, COX-2, and PTGFR. There was a direct molecular association between PGF2α-PTGFR signaling and cell cycle regulation in endometrial epithelial cells of cattle. In addition, findings improve the understanding of PGF2α-PTGFR signaling in the physiological and pharmacological processes of the endometrium of cattle.

PTGFR activation promotes the expression of PTGS-2 and growth factors via activation of the PKC signaling pathway in bovine endometrial epithelial cells.

The endometrium of domestic animals has a remarkable capacity to self-repair. Prostaglandin F2α (PGF2α) is one of the major prostaglandins secreted from the endometrium. The role of PGF2α in endometrial repair, however, is still unknown. In the present study, it was investigated whether prostaglandin F2α receptor (PTGFR) activation could induce expression of prostaglandin-endoperoxide synthase 2 (PTGS-2) and growth factors associated with endometrial repair via activation of protein kinase C (PKC) signaling in endometrial epithelial cells (bEECs) of cattle. Results of the present study indicated that the treatment with the PTGFR agonist, fluprostenol, resulted in an increase in abundance of proteins for PTGS-2, vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF), transforming growth factor beta 1 (TGF-ß1), and interleukin-8 (IL-8). The increased abundances of these proteins were suppressed by the treatment with the PTGFR antagonist, AL8810.Furthermore, fluprostenol treatment also induced PKC phosphorylation. Subsequently, treatment with AL8810 inhibited the fluprostenol-induced PKC phosphorylation. Additionally, treatment with the PKC inhibitor, chelerythrine, reduced the fluprostenol-induced increase in the relative abundance of VEGF, CTGF, TGF-ß1, and IL-8 mRNA in bEECs. Taken together, these results suggest that PTGFR activation may induce endometrial repair by upregulating PTGS-2 gene expression and stimulating VEGF, CTGF, TGF-ß1, and IL-8 gene expression via activation of the PKC signaling pathway.

The impact of flutamide on prostaglandin F2α synthase and prostaglandin F2α receptor expression, and prostaglandin F2α concentration in the porcine corpus luteum of pregnancy.

Recently, we have indicated that flutamide-induced androgen deficiency diminished progesterone production in the porcine corpus luteum (CL) during late pregnancy and before parturition, as a sign of functional luteolysis. In pigs, the main luteolytic factor is prostaglandin F2α (PGF2α), which acts via specific receptors (PTGFRs), and its biosynthesis is catalyzed by prostaglandin F2α synthase (PGFS). The present study investigated the impact of flutamide on luteal PGFS and PTGFR expression, as well as intraluteal PGF2α content during pregnancy in pigs. Flutamide (50 mg/kg BW per day, for 7 d) or corn oil (control groups) were administered subcutaneously into pregnant gilts (n = 3 per group) between 83 and 89 (GD90) or 101-107 (GD108) days of gestation (GD). On GD90 and GD108 ovaries were collected and CLs were obtained. Real-time PCR and Western blot analyses were conducted to quantify PGFS and PTGFR mRNA and protein expression, respectively. In addition, immunohistochemical localization of both proteins was performed and the concentration of PGF2α was analyzed by enzyme immunoassay method. Flutamide caused upregulation of PGFS mRNA and protein in GD90F (P = 0.008; P = 0.008, respectively) and GD108F (P = 0.041; P = 0.009, respectively) groups. The level of PTGFR mRNA increased only in the GD90F (P = 0.007) group, whereas PTGFR protein expression was greater in both gestational periods (P = 0.035; P = 0.038, respectively). On GD90 PGFS was immunolocalized in the cytoplasm of large luteal cells only, whereas on GD108, sparse small luteal cells also displayed positive staining. PTGFR showed membranous localization within large luteal cells on both days of pregnancy. In luteal tissue, PGF2α concentration was greater after flutamide exposure on both days (P = 0.041; P = 0.038, respectively), when compared with control groups. Overall, the enhanced luteal PGF2α content due to increased PGFS expression after flutamide administration might contribute to premature CL regression. Moreover, higher PTGFR protein levels indicate enhanced sensitivity of luteal cells to PGF2α under androgen deficiency.

Cloprostenol administration in the first week postpartum reduces expression of oxytocin receptors in the endometrium in Holstein-Zebu cows / Administração de cloprostenol na primeira semana pós-parto pode reduzir a expressão de receptores de ocitocina no endométrio de vacas mestiças Holandês-Zebu

The present study investigated the hormonal profile and expression of prostaglandin F2α (PGF2α), oxytocin and estrogen receptors in uterine tissues of postpartum cows treated with cloprostenol. Twenty Holstein-Zebu crossbred cows were treated with saline solution (treatment CONT) or cloprostenol (treatment CLO), both administered two and five days postpartum. Blood samples were collected on days two, seven, 14, 21 and 28 postpartum for progesterone, PGF2α metabolite (PGFM) and estradiol determination, and endometrial biopsy was performed in order to quantify the expression of oxytocin receptor (OXTR), prostaglandin F receptor (PTGFR) and estrogen receptor 1 (ERS1) genes. In the CLO treatment, expression of OXTR was reduced (P<0.05) but no difference (P>0.05) between treatments was found for PTGFR and ERS1 expression. Estrogen concentrations increased progressively until day 14 (P<0.05) and the highest OXTR expression and lowest PTGFR expression were observed on day 14 (P<0.05) in both treatments. Serum PGFM concentrations were high throughout the experiment. In conclusion, cloprostenol administration at days two and five of postpartum seems to reduce OXTR expression in the endometrium in crossbred cows.(AU)

O presente estudo avaliou o perfil hormonal e a expressão gênica de receptores de prostaglandina F2α (PGF2α), ocitocina e estrógeno no endométrio de vacas pós-parto tratadas com cloprostenol. Vinte vacas mestiças Holandês-Zebu foram tratadas com solução salina (tratamento CONT, n = 10) ou cloprostenol (tratamento CLO, n = 10), ambos administrados dois e cinco dias após o parto. Amostras de sangue foram coletadas nos dias dois, sete, 14, 21 e 28 pós-parto para mensuração de progesterona, de metabólito de PGF2α (PGFM) e de estradiol, e foram obtidas biópsias endometriais para quantificar a expressão de PTGFR, OXTR e ESR1. No tratamento CLO, a expressão gênica de receptores de ocitocina foi menor (P<0,05). As concentrações de estrógeno aumentaram progressivamente até o dia 14 (P<0,05). A maior expressão de OXTR foi observada no dia 14 (P<0,05). A expressão de ESR1 foi semelhante entre os tratamentos (P>0,05). Os níveis de PGFM foram altos durante todo o estudo. Conclui-se que a administração de cloprostenol nos dias dois e cinco pós-parto parece diminuir a expressão de OXTR no endométrio em vacas mestiças.(AU)