22 research outputs found

    The thromboxane receptor antagonist NTP42 promotes beneficial adaptation and preserves cardiac function in experimental models of right heart overload

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    BackgroundPulmonary arterial hypertension (PAH) is a progressive disease characterized by increased pulmonary artery pressure leading to right ventricular (RV) failure. While current PAH therapies improve patient outlook, they show limited benefit in attenuating RV dysfunction. Recent investigations demonstrated that the thromboxane (TX) A2 receptor (TP) antagonist NTP42 attenuates experimental PAH across key hemodynamic parameters in the lungs and heart. This study aimed to validate the efficacy of NTP42:KVA4, a novel oral formulation of NTP42 in clinical development, in preclinical models of PAH while also, critically, investigating its direct effects on RV dysfunction.MethodsThe effects of NTP42:KVA4 were evaluated in the monocrotaline (MCT) and pulmonary artery banding (PAB) models of PAH and RV dysfunction, respectively, and when compared with leading standard-of-care (SOC) PAH drugs. In addition, the expression of the TP, the target for NTP42, was investigated in cardiac tissue from several other related disease models, and from subjects with PAH and dilated cardiomyopathy (DCM).ResultsIn the MCT-PAH model, NTP42:KVA4 alleviated disease-induced changes in cardiopulmonary hemodynamics, pulmonary vascular remodeling, inflammation, and fibrosis, to a similar or greater extent than the PAH SOCs tested. In the PAB model, NTP42:KVA4 improved RV geometries and contractility, normalized RV stiffness, and significantly increased RV ejection fraction. In both models, NTP42:KVA4 promoted beneficial RV adaptation, decreasing cellular hypertrophy, and increasing vascularization. Notably, elevated expression of the TP target was observed both in RV tissue from these and related disease models, and in clinical RV specimens of PAH and DCM.ConclusionThis study shows that, through antagonism of TP signaling, NTP42:KVA4 attenuates experimental PAH pathophysiology, not only alleviating pulmonary pathologies but also reducing RV remodeling, promoting beneficial hypertrophy, and improving cardiac function. The findings suggest a direct cardioprotective effect for NTP42:KVA4, and its potential to be a disease-modifying therapy in PAH and other cardiac conditions

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

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    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 TPβ 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/TPβ mediated-PRK1 and PRK2, but not PRK3, signalling complexes. Overall, these data demonstrate that TPα/TPβ 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.Health Research BoardIrish Cancer SocietyMovember Foundatio

    Interaction of Rab11a with the Prostacyclin Receptor

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    The cellular trafficking of numerous G protein-coupled receptors (GPCRs) is known to be regulated by Rab proteins that involves a direct protein:protein interaction between the receptor and the GTPase. In the case of the human prostacyclin receptor (hIP), it undergoes agonist-induced internalization and subsequent Rab11a-dependent recyclization involving an interaction between a Rab11-binding domain (RBD) localized within its carboxyl-tail domain with Rab11a. However, the GPCR-interacting domain on Rab11a itself is unknown. Hence, we sought to identify the region within Rab11a that mediates its interaction with the RBD of the hIP. The α4 helix region of Rab11 was identified as a novel binding domain for the hIP, a site entirely distinct from the Switch I/Switch II -regions that act as specific binding domain for most other Rab and Ras-like GTPase interactants. Specifically, Glu138 within 4 helix of Rab11a appears to contact with key residues (e.g Lys304) within the RBD of the hIP, where such contacts differ depending on the agonist-activated versus -inactive status of the hIP. Through mutational studies, supported by in silico homology modelling of the inactive and active hIP:Rab11a complexes, a mechanism is proposed to explain both the constitutive and agonist-induced binding of Rab11a to regulate intracellular trafficking of the hIP. Collectively, these studies are not only the first to identify α4 helix of Rab11a as a protein binding domain on the GTPase but also reveal novel mechanistic insights into the intracellular trafficking of the hIP, and potentially of other members of the GPCR superfamily, involving Rab11-dependent mechanisms.Science Foundation Irelan

    Transcriptional regulation of the human thromboxane A2 receptor gene by Wilms' tumor (WT)1 and hypermethylated in cancer (HIC) 1 in prostate and breast cancers

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    The prostanoid thromboxane (TX) A2 plays a central role in hemostasis and is increasingly implicated in neoplastic disease, including prostate and breast cancers. In humans, TXA2 signals through the TPα and TPβ isoforms of the T prostanoid receptor, two structurally related receptors transcriptionally regulated by distinct promoters, Prm1 and Prm3, respectively, within the TP gene. Focusing on TPα, the current study investigated its expression and transcriptional regulation through Prm1 in prostate and breast cancers. Expression of TPα correlated with increasing prostate and breast tissue tumor grade while the TXA2 mimetic U46619 promoted both proliferation and migration of the respective prostate (PC3) and breast (MCF-7 and MDA-MD-231) derived-carcinoma cell lines. Through 5′ deletional and genetic reporter analyses, several functional upstream repressor regions (URRs) were identified within Prm1 in PC3, MCF-7 and MDA-MB-231 cells while site-directed mutagenesis identified the tumor suppressors Wilms' tumor (WT)1 and hypermethylated in cancer (HIC) 1 as the trans-acting factors regulating those repressor regions. Chromatin immunoprecipitation (ChIP) studies confirmed that WT1 binds in vivo to multiple GC-enriched WT1 cis-elements within the URRs of Prm1 in PC3, MCF-7 and MDA-MB-231 cells. Furthermore, ChIP analyses established that HIC1 binds in vivo to the HIC1(b)cis-element within Prm1 in PC3 and MCF-7 cells but not in the MDA-MB-231 carcinoma line. Collectively, these data establish that WT1 and HIC1, both tumor suppressors implicated in prostate and breast cancers, transcriptionally repress TPα expression and thereby provide a strong genetic basis for understanding the role of TXA2 in the progression of certain human cancers.Health Research BoardIrish Cancer SocietyMovember FoundationMolCellBiol Programme, Programme for Research in Third-Level Institution

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

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    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 TPβ 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 TPβ in inflammatory infiltrates within human prostate tissue. Strikingly, TPβ 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 TPβ was evaluated as a function of monocyte-to-macrophage differentiation in THP-1 cells. Expression of both TPα and TPβ 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 TPβ expression through Prm3 in THP-1 cells. Knockdown of FOXP1 increased TPβ, but not TPα, expression in THP-1 cells, while genetic reporter and chromatin immunoprecipitation (ChIP) analyses established that FOXP1 exerts its repressive effect on TPβ through binding to four cis-elements within Prm3. Collectively, FOXP1 functions as a transcriptional repressor of TPβ in monocytes. This repression is lifted in differentiated macrophages, allowing for upregulation of TPβ expression and possibly accounting for the prominent expression of TPβ in prostate tissue-resident macrophages.European Commission Horizon 2020Health Research BoardIrish Cancer SocietyMovember FoundationEmbargo shortened to 6 months due to H2020Update citation details at check date - H

    X-ray crystal structure of the PDZ1-IP target peptide chimera.

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    <p><b>A</b>: Amino acid sequence of the recombinant chimeric protein used for crystallization: N-terminal Gly-Ser dipeptide derived from the cloning vector (black), PDZ1 domain (residues 7–86 (PDZK1 numbering), green), partial interdomain segment (87–106, lies between the PDZ1 and PDZ2 domains, blue) and seven carboxy-terminal residues of IP (<sup>411</sup>IAACSLC<sup>417</sup>, IP numbering, yellow). Regions of secondary structure (β strands and α helices) and the carboxylate-binding loop (CBL) are indicated above the sequence. <b>B</b>: Asymmetric unit showing the head-to-tail arrangement of two chimeric molecules. This figure was generated using POVScript <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053819#pone.0053819-Fenn1" target="_blank">[47]</a> using the color scheme in panel A.</p

    Isothermal titration calorimetric analysis of the binding of a C-terminal peptide from IP to the PDZ1 domain of PDZK1.

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    <p>Recombinant wild-type PDZ1 domain (0.03 mM in 150 mM NaCl, 0.5 mM tris (2-carboxyethyl) phosphine, 25 mM Tris-pH 8.0) were placed in the titration cell and equilibrated at 20°C. A solution containing 1.0 mM of (A) the C-terminal nonapeptide from IP, KK<sup>411</sup>IAACSLC<sup>417</sup>, or (B) the octapeptide K<sup>407</sup>SEAIAAC<sup>414</sup> corresponding to the C-terminal sequence of IP from which the last three amino acid –SLC are absent, or (C) the farnesylated-carboxy-methylated form of the same peptide (K<sup>407</sup>SEAIAAC<sup>414)</sup> were injected in 10 µl aliquots with an interval of 4 minutes between each addition to permit re-equilibration. Titration curves were analyzed and K<sub>d</sub> values determined using ORIGIN 7.0 software.</p

    Structure determination and refinement statistics.

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    a<p>Values in parenthesis are for the highest resolution shell. R<sub>sym</sub> = Σ|I-<i>|/Σ(I), where I is the observed integrated intensity, <i> is the average integrated intensity obtained from multiple measurements, and the summation is over all observable reflections. R<sub>cryst</sub> = Σ||F<sub>obs</sub>|-<i>k</i>|F<sub>calc</sub>||/Σ|F<sub>obs</sub>|, where F<sub>obs</sub> and F<sub>calc</sub> are the observed and calculates structure factors, respectively. R<sub>free</sub> is calculated as R<sub>cryst</sub> using 5% of the reflection chosen randomly and omitted from the refinement calculations. Bond lengths and angles are root-mean-square deviations from ideal values.</i></i></p
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