Preparing to strike: Acute events in signaling by the serpentine receptor for thromboxane A2.

Over the last two decades, awareness of the (patho)physiological roles of thromboxane A2 signaling has been greatly extended. From humble beginnings as a short-lived stimulus that activates platelets and causes vasoconstriction to a dichotomous receptor system involving multiple endogenous ligands capable of modifying tissue homeostasis and disease generation in almost every tissue of the body. Thromboxane A2 receptor (TP) signal transduction is associated with the pathogenesis of cancer, atherosclerosis, heart disease, asthma, and host response to parasitic infection amongst others. The two receptors mediating these cellular responses (TPα and TPß) are derived from a single gene (TBXA2R) through alternative splicing. Recently, knowledge about the mechanism(s) of signal propagation by the two receptors has undergone a revolution in understanding. Not only have the structural relationships associated with G-protein coupling been established but the modulation of that signaling by post-translational modification to the receptor has come sharply into focus. Moreover, the signaling of the receptor unrelated to G-protein coupling has become a burgeoning field of endeavor with over 70 interacting proteins currently identified. These data are reshaping the concept of TP signaling from a mere guanine nucleotide exchange factors for Gα activation to a nexus for the convergence of diverse and poorly characterized signaling pathways. This review summarizes the advances in understanding in TP signaling, and the potential for new growth in a field that after almost 50 years is finally coming of age.

Novel Peptide Motifs Containing Asp-Glu-Gly Target P2Y12 and Thromboxane A2 Receptors to Inhibit Platelet Aggregation and Thrombus Formation.

Increasing evidence has shown that collagen peptides have multiple biological activities. Our previous study has separated and identified antiplatelet aggregation peptides Asp-Glu-Gly-Pro (DEGP) from Salmo salar skin. This study is to investigate the cellular target of DEGP on platelets and its underlying mechanism. DEGP inhibited platelet aggregation in a dose-dependent manner induced by 2MeS-ADP and U46619 and significantly attenuated tail thrombosis formation by 30% in mice at the dose of 50 mg/kg body weight. Mechanically, DEGP displayed apparent antagonism effects on TP and P2Y12 receptors by the drug affinity responsive target stability (DARTS) technique to regulate the phosphorylation of RhoAS188, PLCß3S537, as well as VASPS157. The molecular docking results revealed a stronger binding energy with the target protein of modified peptides DEGI and DDEGL. Practically, DEGI exhibited the highest inhibition activity against 2MeS-ADP- and U46619-induced platelet aggregation in vitro with IC50 values of 0.88 ± 0.10 and 0.85 ± 0.10 mM, respectively, and comparable antithrombosis activity with aspirin at the dose of 25 mg/kg body weight in vivo. These results indicated the possibility that the peptide motifs containing Asp-Glu-Gly could potentially be developed as a novel therapeutic agent in the prevention and treatment of thrombotic diseases.

Antiplatelet properties of Pim kinase inhibition are mediated through disruption of thromboxane A2 receptor signaling.

Pim kinases are upregulated in several forms of cancer, contributing to cell survival and tumour development, but their role in platelet function and thrombotic disease has not been explored. We report for the first time that Pim-1 is expressed in human and mouse platelets. Genetic deletion or pharmacological inhibition of Pim kinase results in reduced thrombus formation but is not associated with impaired haemostasis. Attenuation of thrombus formation was found to be due to inhibition of the thromboxane A2 receptor as effects on platelet function was non-additive to inhibition caused by the cyclooxygenase inhibitor indomethacin or thromboxane A2 receptor antagonist GR32191. Treatment with Pim kinase inhibitors caused reduced surface expression of the thromboxane A2 receptor and resulted in reduced responses to thromboxane A2 receptor agonists, indicating a role for Pim kinase in the regulation of thromboxane A2 receptor function. Our research identifies a novel, Pim kinase dependent regulatory mechanism for the thromboxane A2 receptor and represents a new targeting strategy that is independent of COX-1 inhibition or direct antagonism of the thromboxane A2 receptor that whilst attenuating thrombosis does not increase bleeding.

Differential expression of the TPα and TPß isoforms of the human T Prostanoid receptor during chronic inflammation of the prostate: Role for FOXP1 in the transcriptional regulation of TPß during monocyte-macrophage differentiation.

Inflammation is linked to prostate cancer (PCa) and to other diseases of the prostate. The prostanoid thromboxane (TX)A2 is a pro-inflammatory mediator implicated in several prostatic diseases, including PCa. TXA2 signals through the TPα and TPß isoforms of the T Prostanoid receptor (TP) which exhibit several functional differences and transcriptionally regulated by distinct promoters Prm1 and Prm3, respectively, within the TBXA2R gene. This study examined the expression of TPα and TPß in inflammatory infiltrates within human prostate tissue. Strikingly, TPß expression was detected in 94% of infiltrates, including in B- and T-lymphocytes and macrophages. In contrast, TPα was more variably expressed and, where present, expression was mainly confined to macrophages. To gain molecular insight into these findings, expression of TPα and TPß was evaluated as a function of monocyte-to-macrophage differentiation in THP-1 cells. Expression of both TPα and TPß was upregulated following phorbol-12-myristate-13-acetate (PMA)-induced differentiation of monocytic THP-1 to their macrophage lineage. Furthermore, FOXP1, an essential transcriptional regulator down-regulated during monocyte-to-macrophage differentiation, was identified as a key trans-acting factor regulating TPß expression through Prm3 in THP-1 cells. Knockdown of FOXP1 increased TPß, but not TPα, expression in THP-1 cells, while genetic reporter and chromatin immunoprecipitation (ChIP) analyses established that FOXP1 exerts its repressive effect on TPß through binding to four cis-elements within Prm3. Collectively, FOXP1 functions as a transcriptional repressor of TPß in monocytes. This repression is lifted in differentiated macrophages, allowing for upregulation of TPß expression and possibly accounting for the prominent expression of TPß in prostate tissue-resident macrophages.

Multiplate® evaluation of acetylsalicylic acid efficacy in carotid surgery: routine and genetic influencing factors.

Essentials Acetylsalicylic acid (ASA) is prescribed to patients scheduled for carotid endarterectomy (CEA). We measured ASA efficacy during CEA by Multiplate® and searched for influencing factors. Most patients scheduled for CEA and treated by ASA are sensitive to this therapy. Influencing genomic factors are involved in ASA metabolism and in platelet function modulations. SUMMARY: Background Acetylsalicylic acid (ASA) is recommended before, during and after carotid endarterectomy (CEA). The efficacy of ASA is influenced by numerous biological and genotypic factors. Objectives To determine the biological efficacy of ASA by using the Multiplate® method, and to explore the biological parameters and genomic factors influencing this efficacy. Methods This descriptive cross-sectional study included all patients scheduled for CEA between January 2012 and April 2013. Multiplate® tests were performed at day 0 and day 30. A set of 66 single-nucleotide polymorphisms (SNPs) from 38 genes or DNA regions were selected and studied along with phenotypic parameters by the use of hierarchical clustering (HC) for multidimensional data management. Results Fifty-five patients receiving ASA were analyzed. Of the patients, 95% were found to be sensitive to ASA, with values under the threshold of normality (400 AU min-1 ). However, there were notable differences in residual aggregation among subjects over a wide range. HC revealed four subclusters comprising three categories of parameters: (i) routine and functional parameters - in ASA-treated patients, the ASPItest was highly linked to the ADPtest, to platelet count, and, to a lesser extent, to fibrinogen and hematocrit; (ii) polymorphisms in genes involved in ASA absorption and in the arachidonic acid pathway (ABCB1 and COX-1); and (iii) polymorphisms in genes modulating basal platelet function, i.e. TBXA2R, ADRA2A, PEAR1, ITGA2 and ITGB1. Conclusion Most patients treated with ASA before CEA were sensitive to it, according to Multiplate® ASPItest results. Genomic factors influencing this efficacy are SNPs involved in ASA absorption and metabolic pathway, and in modulations in basal platelet function.

Regulated expression of the TPß isoform of the human T prostanoid receptor by the tumour suppressors FOXP1 and NKX3.1: Implications for the role of thromboxane in prostate cancer.

The prostanoid thromboxane (TX)A2 signals through the TPα and TPß isoforms of T Prostanoid receptor (TP) that are transcriptionally regulated by distinct promoters termed Prm1 and Prm3, respectively, within the TBXA2R gene. We recently demonstrated that expression of TPα and TPß is increased in PCa, differentially correlating with Gleason grade and with altered CpG methylation of the individual Prm1/Prm3 regions within the TBXA2R. The current study sought to localise the sites of CpG methylation within Prm1 and Prm3, and to identify the main transcription factors regulating TPß expression through Prm3 in the prostate adenocarcinoma PC-3 and LNCaP cell lines. Bisulfite sequencing revealed extensive differences in the pattern and status of CpG methylation of the individual Prm1 and Prm3 regions that regulate TPα and TPß expression, respectively, within the TBXA2R. More specifically, Prm1 is predominantly hypomethylated while Prm3 is hypermethylated across its entire sequence in PC-3 and LNCaP cells. Furthermore, the tumour suppressors FOXP1 and NKX3.1, strongly implicated in PCa development, were identified as key transcription factors regulating TPß expression through Prm3 in both PCa cell lines. Specific siRNA-disruption of FOXP1 and NKX3.1 each coincided with up-regulated TPß protein and mRNA expression, while genetic-reporter and chromatin immunoprecipitation (ChIP) analyses confirmed that both FOXP1 and NKX3.1 bind to cis­elements within Prm3 to transcriptionally repress TPß in the PCa lines. Collectively these data identify Prm3/TPß as a bona fide target of FOXP1 and NKX3.1 regulation, providing a mechanistic basis, at least in part, for the highly significant upregulation of TPß expression in PCa.

Aluminum exposure at human dietary levels promotes vascular dysfunction and increases blood pressure in rats: A concerted action of NAD(P)H oxidase and COX-2.

Aluminum (Al) is a non-essential metal and a significant environmental contaminant and is associated with a number of human diseases including cardiovascular disease. We investigated the effects of Al exposure at doses similar to human dietary levels on the cardiovascular system over a 60day period. Wistar male rats were divided into two major groups and received orally: 1) Low aluminum level - rats were subdivided and treated for 60days as follows: a) Untreated - ultrapure water; b) AlCl3 at a dose of 8.3mg/kg bw for 60days, representing human Al exposure by diet; and 2) High aluminum level - rats were subdivided and treated for 42days as follows: C) Untreated - ultrapure water; d) AlCl3 at 100mg/kg bw for 42days, representing a high level of human exposure to Al. Effects on systolic blood pressure (SBP) and vascular function of aortic and mesenteric resistance arteries (MRA) were studied. Endothelium and smooth muscle integrity were evaluated by concentration-response curves to acetylcholine (ACh) and sodium nitroprusside. Vasoconstrictor responses to phenylephrine (Phe) in the presence and absence of endothelium and in the presence of the NOS inhibitor L-NAME, the potassium channels blocker TEA, the NAD(P)H oxidase inhibitor apocynin, superoxide dismutase (SOD), the non-selective COX inhibitor indomethacin and the selective COX-2 inhibitor NS 398 were analyzed. Vascular reactive oxygen species (ROS), lipid peroxidation and total antioxidant capacity, were measured. The mRNA expressions of eNOS, NAD(P)H oxidase 1 and 2, SOD1, COX-2 and thromboxane A2 receptor (TXA-2 R) were also investigated. Al exposure at human dietary levels impaired the cardiovascular system and these effects were almost the same as Al exposure at much higher levels. Al increased SBP, decreased ACh-induced relaxation, increased response to Phe, decreased endothelial modulation of vasoconstrictor responses, the bioavailability of nitric oxide (NO), the involvement of potassium channels on vascular responses, as well as increased ROS production from NAD(P)H oxidase and contractile prostanoids mainly from COX-2 in both aorta and mesenteric arteries. Al exposure increased vascular ROS production and lipid peroxidation as well as altered the antioxidant status in aorta and MRA. Al decreased vascular eNOS and SOD1 mRNA levels and increased the NAD(P)H oxidase 1, COX-2 and TXA-2 R mRNA levels. Our results point to an excess of ROS mainly from NAD(P)H oxidase after Al exposure and the increased vascular prostanoids from COX-2 acting in concert to decrease NO bioavailability, thus inducing vascular dysfunction and increasing blood pressure. Therefore, 60-day chronic exposure to Al, which reflects common human dietary Al intake, appears to pose a risk for the cardiovascular system.

Regulation of protein kinase C-related kinase (PRK) signalling by the TPα and TPß isoforms of the human thromboxane A2 receptor: Implications for thromboxane- and androgen- dependent neoplastic and epigenetic responses in prostate cancer.

The prostanoid thromboxane (TX) A2 and its T Prostanoid receptor (the TP) are increasingly implicated in prostate cancer (PCa). Mechanistically, we recently discovered that both TPα and TPß form functional signalling complexes with members of the protein kinase C-related kinase (PRK) family, AGC- kinases essential for the epigenetic regulation of androgen receptor (AR)-dependent transcription and promising therapeutic targets for treatment of castrate-resistant prostate cancer (CRPC). Critically, similar to androgens, activation of the PRKs through the TXA2/TP signalling axis induces phosphorylation of histone H3 at Thr11 (H3Thr11), a marker of androgen-induced chromatin remodelling and transcriptional activation, raising the possibility that TXA2-TP signalling can mimic and/or enhance AR-induced cellular changes even in the absence of circulating androgens such as in CRPC. Hence the aim of the current study was to investigate whether TXA2/TP-induced PRK activation can mimic and/or enhance AR-mediated cellular responses in the model androgen-responsive prostate adenocarcinoma LNCaP cell line. We reveal that TXA2/TP signalling can act as a neoplastic- and epigenetic-regulator, promoting and enhancing both AR-associated chromatin remodelling (H3Thr11 phosphorylation, WDR5 recruitment and acetylation of histone H4 at lysine 16) and AR-mediated transcriptional activation (e.g of the KLK3/prostate-specific antigen and TMPRSS2 genes) through mechanisms involving TPα/TPß mediated-PRK1 and PRK2, but not PRK3, signalling complexes. Overall, these data demonstrate that TPα/TPß can act as neoplastic and epigenetic regulators by mimicking and/or enhancing the actions of androgens within the prostate and provides further mechanistic insights into the role of the TXA2/TP signalling axis in PCa, including potentially in CRPC.

Glycated albumin modifies platelet adhesion and aggregation responses.

A diabetic vasculature is detrimental to cardiovascular health through the actions of advanced glycation end products (AGEs) on endothelial cells and platelets. Platelets activated by AGEs agonize endothelial responses promoting cardiovascular disease (CVD) development. While it has been established that AGEs can alter platelet functions, little is known about the specific platelet pathways that AGEs modify. Therefore, we evaluated the effects of AGEs on specific salient platelet pathways related to CVDs and whether the effects that AGEs elicit are dependent on glycation extent. To accomplish our objective, platelets were incubated with reversibly or irreversibly glycated albumin. A time course for adhesion and aggregation agonist receptor expression was assessed. Optical platelet aggregometry was used to confirm the functional activity of platelets after AGE exposure. In general, platelets subjected to glycated albumin had a significantly enhanced adhesion and aggregation potential. Furthermore, we observed an enhancement in dense body secretion and intracellular calcium concentration. This was especially prevalent for platelets exposed to irreversibly glycated albumin. Additionally, functional aggregation correlated well with receptor expression, suggesting that AGE-induced altered receptor sensitivity translated to altered platelet functions. Our findings indicate that under diabetic vascular conditions platelets become more susceptible to activation and aggregation due to an overall enhanced receptor expression, which may act to promote CVD development.

Expression of the TPα and TPß isoforms of the thromboxane prostanoid receptor (TP) in prostate cancer: clinical significance and diagnostic potential.

The prostanoid thromboxane (TX)A2 plays a central role in haemostasis and is increasingly implicated in cancer progression. TXA2 signals through two T Prostanoid receptor (TP) isoforms termed TPα and TPß, with both encoded by the TBXA2R gene. Despite exhibiting several functional and regulatory differences, the role of the individual TP isoforms in neoplastic diseases is largely unknown.This study evaluated expression of the TPα and TPß isoforms in tumour microarrays of the benign prostate and different pathological (Gleason) grades of prostate cancer (PCa). Expression of TPß was significantly increased in PCa relative to benign tissue and strongly correlated with increasing Gleason grade. Furthermore, higher TPß expression was associated with increased risk of biochemical recurrence (BCR) and significantly shorter disease-free survival time in patients post-surgery. While TPα was more variably expressed than TPß in PCa, increased/high TPα expression within the tumour also trended toward increased BCR and shorter disease-free survival time. Comparative genomic CpG DNA methylation analysis revealed substantial differences in the extent of methylation of the promoter regions of the TBXA2R that specifically regulate expression of TPα and TPß, respectively, both in benign prostate and in clinically-derived tissue representative of precursor lesions and progressive stages of PCa. Collectively, TPα and TPß expression is differentially regulated both in the benign and tumourigenic prostate, and coincides with clinical pathology and altered CpG methylation of the TBXA2R gene. Analysis of TPß, or a combination of TPα/TPß, expression levels may have significant clinical potential as a diagnostic biomarker and predictor of PCa disease recurrence.

Associations of MDR1, TBXA2R, PLA2G7, and PEAR1 genetic polymorphisms with the platelet activity in Chinese ischemic stroke patients receiving aspirin therapy.

AIM: Aspirin resistance has an incidence of 5%-65% in patients with ischemic stroke, who receive the standard dose of aspirin, but the platelet function is inadequately inhibited, thereby leading to thrombotic events. Numerous evidence shows that thromboxane A2 receptor (TXA2 receptor, encoded by TBXA2R), lipoprotein-associated phospholipase A2 (Lp-PLA2, encoded by PLA2G7) and platelet endothelial aggregation receptor-1 (PEAR1, encoded by PEAR1) are crucial in regulating platelet activation, and P-glycoprotein (P-gp, encoded by MDR1) influences the absorption of aspirin in the intestine. In this study we examined the correlation between MDR1, TBXA2R, PLA2G7, PEAR1 genetic polymorphisms and platelet activity in Chinese ischemic stroke patients receiving aspirin therapy. METHODS: A total of 283 ischemic stroke patients receiving 100 mg aspirin for 7 d were genotyped for polymorphisms in MDR1 C3435T, TBXA2R (rs1131882), PLA2G7 (rs1051931, rs7756935), and PEAR1 (rs12566888, rs12041331). The platelet aggregation response was measured using an automatic platelet aggregation analyzer and a commercially available TXB2 ELISA kit. RESULTS: Thirty-three patients (11.66%) were insensitive to aspirin treatment. MDR1 3435TT genotype carriers, whose arachidonic acid (AA) or adenosine diphosphate (ADP)-induced platelet aggregation was lower than that of CC+CT genotype carriers, were less likely to suffer from aspirin resistance (odds ratio=0.421, 95% CI: 0.233-0.759). The TBXA2R rs1131882 CC genotype, which was found more frequently in the aspirin-insensitive group (81.8% vs 62.4%) than in the sensitive group, was identified as a risk factor for aspirin resistance (odds ratio=2.712, 95% CI: 1.080-6.810) with a higher level of AA-induced platelet aggregation. Due to the combined effects of PLA2G7 rs1051931 and rs7756935, carriers of the AA-CC haplotype had a higher level of ADP-induced platelet aggregation, and were at considerably higher risk of aspirin resistance than noncarriers (odds ratio=8.233, 95% CI: 1.590-42.638). CONCLUSION: A considerable portion (11.66%) of Chinese ischemic stroke patients are insensitive to aspirin treatment, which may be correlated with the MDR1 C3435T, TBXA2R (rs1131882), and PLA2G7 (rs1051931-rs7756935) polymorphisms.

Thromboxane A2 receptor (TBXA2R) is a potent survival factor for triple negative breast cancers (TNBCs).

Triple Negative Breast Cancer (TNBC) is defined by the lack of ERα, PR expression and HER2 overexpression and is the breast cancer subtype with the poorest clinical outcomes. Our aim was to identify genes driving TNBC proliferation and/or survival which could represent novel therapeutic targets.We performed microarray profiling of primary TNBCs and generated differential genelists based on clinical outcomes following the chemotherapy regimen FEC (5-Fluorouracil/Epirubicin/Cyclophosphamide -'good' outcome no relapse > 3 years; 'poor' outcome relapse < 3 years). Elevated expression of thromboxane A2 receptor (TBXA2R) was observed in 'good' outcome TNBCs. TBXA2R expression was higher specifically in TNBC cell lines and TBXA2R knockdowns consistently showed dramatic cell killing in TNBC cells. TBXA2R mRNA and promoter activities were up-regulated following BRCA1 knockdown, with c-Myc being required for BRCA1-mediated transcriptional repression.We demonstrated that TBXA2R enhanced TNBC cell migration, invasion and activated Rho signalling, phenotypes which could be reversed using Rho-associated Kinase (ROCK) inhibitors. TBXA2R also protected TNBC cells from DNA damage by negatively regulating reactive oxygen species levels. In summary, TBXA2R is a novel breast cancer-associated gene required for the survival and migratory behaviour of a subset of TNBCs and could provide opportunities to develop novel, more effective treatments.

Role for the thromboxane A2 receptor ß-isoform in the pathogenesis of intrauterine growth restriction.

Intrauterine growth restriction (IUGR) is a pathology of pregnancy that results in failure of the fetus to reach its genetically determined growth potential. In developed nations the most common cause of IUGR is impaired placentation resulting from poor trophoblast function, which reduces blood flow to the fetoplacental unit, promotes hypoxia and enhances production of bioactive lipids (TXA2 and isoprostanes) which act through the thromboxane receptor (TP). TP activation has been implicated as a pathogenic factor in pregnancy complications, including IUGR; however, the role of TP isoforms during pregnancy is poorly defined. We have determined that expression of the human-specific isoform of TP (TPß) is increased in placentae from IUGR pregnancies, compared to healthy pregnancies. Overexpression of TPα enhanced trophoblast proliferation and syncytialisation. Conversely, TPß attenuated these functions and inhibited migration. Expression of the TPß transgene in mice resulted in growth restricted pups and placentae with poor syncytialisation and diminished growth characteristics. Together our data indicate that expression of TPα mediates normal placentation; however, TPß impairs placentation, and promotes the development of IUGR, and represents an underappreciated pathogenic factor in humans.

Pravastatin and C reactive protein modulate protease- activated receptor-1 expression in vitro blood platelets.

Protease-activated receptor-1 (PAR-1) plays an important role in mediating activation of human platelets by thrombin. However, mechanism of statin in ADP-induced platelet PAR-1 expression is also unknown. Aggregometry, flow cytometry, immunoblotting and ELISA were used to determine role of pravastatin participating in ADP-induced platelet activation and PAR-1 expression. ADP stimulation significantly increased PAR-1 expression on platelets. PAR-1 antagonist SCH-79797 inhibited platelet aggregation as well as decreased platelet P-selectin expression induced by ADP. CRP inhibited PAR-1 expression induced by ADP in a concentration-dependent manner. Pravastatin treatment reduced PAR-1 expression in a concentration-dependent manner. Combination treatment of CRP and Pravastatin significantly reduced platelet PAR-1 expression induced by ADP. By western-blot analysis, pravastatin treatment did not influence total PAR-1 after ADP treatment. CRP decreased platelet total PAR-1 expression induced by ADP. Pravastatin and CRP reduced TXB2 formation by ADP significantly. CRP decreased thrombin fragment F1+2 level with ADP treatment. Pravastatin, in contrast, did not influence F1+2 level. Upon treatment with Pravastatin reduced platelet LOX-1 expression induced by ADP. In conclusion, PAR-1 served as a critical mechanism to relay platelet activation process induced by ADP. CRP and pravastatin reduce PAR-1 expression in platelet by ADP pathway.

Thromboxane A2 Receptor Inhibition Suppresses Multiple Myeloma Cell Proliferation by Inducing p38/c-Jun N-terminal Kinase (JNK) Mitogen-activated Protein Kinase (MAPK)-mediated G2/M Progression Delay and Cell Apoptosis.

Multiple myeloma (MM) is a plasma cell malignancy without effective therapeutics. Thromboxane A2 (TxA2)/TxA2 receptor (T prostanoid receptor (TP)) modulates the progression of some carcinomas; however, its effects on MM cell proliferation remain unclear. In this study, we evaluated cyclooxygenase (COX) enzymes and downstream prostaglandin profiles in human myeloma cell lines RPMI-8226 and U-266 and analyzed the effects of COX-1/-2 inhibitors SC-560 and NS-398 on MM cell proliferation. Our observations implicate COX-2 as being involved in modulating cell proliferation. We further incubated MM cells with prostaglandin receptor antagonists or agonists and found that only the TP antagonist, SQ29548, suppressed MM cell proliferation. TP silencing and the TP agonist, U46619, further confirmed this finding. Moreover, SQ29548 and TP silencing promoted MM cell G2/M phase delay accompanied by reducing cyclin B1/cyclin-dependent kinase-1 (CDK1) mRNA and protein expression. Notably, cyclin B1 overexpression rescued MM cells from G2/M arrest. We also found that the TP agonist activated JNK and p38 MAPK phosphorylation, and inhibitors of JNK and p38 MAPK depressed U46619-induced proliferation and cyclin B1/CDK1 protein expression. In addition, SQ29548 and TP silencing led to the MM cell apoptotic rate increasing with improving caspase 3 activity. The knockdown of caspase 3 reversed the apoptotic rate. Taken together, our results suggest that TxA2/TP promotes MM cell proliferation by reducing cell delay at G2/M phase via elevating p38 MAPK/JNK-mediated cyclin B1/CDK1 expression and hindering cell apoptosis. The TP inhibitor has potential as a novel agent to target kinase cascades for MM therapy.

Abnormal megakaryopoiesis and platelet function in cyclooxygenase-2-deficient mice.

Previous studies suggest that cyclooxygenase-2 (COX-2) might influence megakaryocyte (MK) maturation and platelet production in vitro. Using a gene deletion model, we analysed the effect of COX-2 deficiency on megakaryopoiesis and platelet function. COX-2-/- mice (10-12 weeks old) have hyper-responsive platelets as suggested by their enhanced aggregation, TXA2 biosynthesis, CD62P and CD41/CD61 expression, platelet-fibrinogen binding, and increased thromboembolic death after collagen/epinephrine injection compared to wild-type (WT). Moreover, increased platelet COX-1 expression and reticulated platelet fraction were observed in COX-2-/- mice while platelet count was similar to WT. MKs were significantly reduced in COX-2-/- bone marrows (BMs), with high nuclear/cytoplasmic ratios, low ploidy and poor expression of lineage markers of maturation (CD42d, CD49b). However, MKs were significantly increased in COX-2-/- spleens, with features of MK maturation markers which were not observed in MKs of WT spleens. Interestingly, the expression of COX-1, prostacyclin and PGE2 synthases and prostanoid pattern were modified in BMs and spleens of COX-2-/- mice. Moreover, COX-2 ablation reduced the percentage of CD49b+ cells, the platelet formation and the haematopoietic stem cells in bone marrow and increased their accumulation in the spleen. Splenectomy decreased peripheral platelet number, reverted their hyper-responsive phenotype and protected COX-2-/- mice from thromboembolism. Interestingly, fibrosis was observed in spleens of old COX-2-/- mice (28 weeks old). In conclusion, COX-2 deletion delays BM megakaryopoiesis promoting a compensatory splenic MK hyperplasia, with a release of hyper-responsive platelets and increased thrombogenicity in vivo. COX-2 seems to contribute to physiological MK maturation and pro-platelet formation.

Protein kinase C-related kinase 1 and 2 play an essential role in thromboxane-mediated neoplastic responses in prostate cancer.

The prostanoid thromboxane (TX) A2 is increasingly implicated in neoplastic progression, including prostate cancer (PCa). Mechanistically, we recently identified protein kinase C-related kinase (PRK) 1 as a functional interactant of both the TPα and TPß isoforms of the human T prostanoid receptor (TP). The interaction with PRK1 was not only essential for TPα/TPß-induced PCa cell migration but also enabled the TXA2-TP axis to induce phosphorylation of histone H3 at Thr11 (H3Thr11), an epigenetic marker both essential for and previously exclusively associated with androgen-induced chromatin remodelling and transcriptional activation. PRK1 is a member of a subfamily of three structurally related kinases comprising PRK1/PKNα, PRK2/PKNγ and PRK3/PKNß that are widely yet differentially implicated in various cancers. Hence, focusing on the setting of prostate cancer, this study investigated whether TPα and/or TPß might also complex with PRK2 and PRK3 to regulate their activity and neoplastic responses. While TPα and TPß were found in immune complexes with PRK1, PRK2 and PRK3 to regulate their activation and signalling, they do so differentially and in a TP agonist-regulated manner dependent on the T-loop activation status of the PRKs but independent of their kinase activity. Furthermore, TXA2-mediated neoplastic responses in prostate adenocarcinoma PC-3 cells, including histone H3Thr11 phosphorylation, was found to occur through a PRK1- and PRK2-, but not PRK3-, dependent mechanism. Collectively, these data suggest that TXA2 acts as both a neoplastic and epigenetic regulator and provides a mechanistic explanation, at least in part, for the prophylactic benefits of Aspirin in reducing the risk of certain cancers.

Adhesion of platelets through thromboxane A2 receptor signaling facilitates liver repair during acute chemical-induced hepatotoxicity.

AIMS: Platelets have been suggested to play an important role in liver regeneration and repair after hepatic resection and acute liver injury. However, the underlying mechanisms of liver repair remain elusive. Signaling through thromboxane prostanoid (TP) receptor participates in inflammation and tissue injury through platelet aggregation. On the other hand, TP receptor signaling also is involved in tissue repair and tumor growth through angiogenesis. The present study was examined whether or not TP receptor signaling contributes to liver repair and sinusoidal restoration from acute liver injury through platelet adhesion to the hepatic sinusoids. MAIN METHODS: Carbon tetrachrolide (CCl4) was used to induce acute liver injury in TP receptor knockout mice (TP(-/-) mice) and their wild-type littermates (WT mice). KEY FINDINGS: Compared with WT mice, TP(-/-) mice exhibited delayed in liver repair and sinusoidal restoration after CCl4 treatment, which were associated with attenuated hepatic expression of pro-angiogenic factors. Intravital microscopic observation revealed that adhering platelets to the sinusoids was increased in WT livers during the repair phase as compared with TP(-/-) livers, and platelet adhesion was dependent on TP receptor signaling. The levels of hepatocyte growth factor (HGF) in platelets from WT mice treated with CCl4 for 48h were greater than those form TP(-/-) mice, and HGF enhanced the expression of angiogenic factors in cultured human umbilical vein endothelial cells (HUVECs). SIGNIFICANCE: These results suggested that TP receptor signaling facilitates liver repair and sinusoidal restoration from acute liver injury through HGF release from platelets adhering to the sinusoids.

Thromboxane synthase deficiency improves insulin action and attenuates adipose tissue fibrosis.

Thromboxane A2, an arachidonic acid-derived eicosanoid generated by thromboxane synthase (TBXAS), plays critical roles in hemostasis and inflammation. However, the contribution of thromboxane A2 to obesity-linked metabolic dysfunction remains incompletely understood. Here, we used in vitro and mouse models to better define the role of TBXAS in metabolic homeostasis. We found that adipose expression of Tbxas and thromboxane A2 receptor (Tbxa2r) was significantly upregulated in genetic and dietary mouse models of obesity and diabetes. Expression of Tbxas and Tbxa2r was detected in adipose stromal cells, including macrophages. Furthermore, stimulation of macrophages with interferon-γ or resistin factors known to be upregulated in obesity induced Tbxas and Tbxa2r expression. Mice lacking Tbxas had similar weight gain, food intake, and energy expenditure. However, loss of Tbxas markedly enhanced insulin sensitivity in mice fed a low-fat diet. Improvement in glucose homeostasis was correlated with the upregulated expression of multiple secreted metabolic regulators (Ctrp3, Ctrp9, and Ctrp12) in the visceral fat depot. Following a challenge with a high-fat diet, Tbxas deficiency led to attenuated adipose tissue fibrosis and reduced circulating IL-6 levels without adipose tissue macrophages being affected; however, these changes were not sufficient to improve whole body insulin action. Together, our results highlight a novel, diet-dependent role for thromboxane A2 in modulating peripheral tissue insulin sensitivity and adipose tissue fibrosis.

Regulatory SNPs and transcriptional factor binding sites in ADRBK1, AKT3, ATF3, DIO2, TBXA2R and VEGFA.

Abstract Regulatory single nucleotide polymorphisms (rSNPs) which change the transcriptional factor binding sites (TFBS) for transcriptional factors (TFs) to bind DNA were reviewed for the ADRBK1 (GRK2), AKT3, ATF3, DIO2, TBXA2R and VEGFA genes. Changes in the TFBS where TFs attach to regulate these genes may result in human sickness and disease. The highlights of this previous work were reviewed for these genes.