NTP42, a novel antagonist of the thromboxane receptor, attenuates experimentally induced pulmonary arterial hypertension.

BACKGROUND: NTP42 is a novel antagonist of the thromboxane prostanoid receptor (TP), currently in development for the treatment of pulmonary arterial hypertension (PAH). PAH is a devastating disease with multiple pathophysiological hallmarks including excessive pulmonary vasoconstriction, vascular remodelling, inflammation, fibrosis, in situ thrombosis and right ventricular hypertrophy. Signalling through the TP, thromboxane (TX) A2 is a potent vasoconstrictor and mediator of platelet aggregation. It is also a pro-mitogenic, pro-inflammatory and pro-fibrotic agent. Moreover, the TP also mediates the adverse actions of the isoprostane 8-iso-prostaglandin F2α, a free-radical-derived product of arachidonic acid produced in abundance during oxidative injury. Mechanistically, TP antagonists should treat most of the hallmarks of PAH, including inhibiting the excessive vasoconstriction and pulmonary artery remodelling, in situ thrombosis, inflammation and fibrosis. This study aimed to investigate the efficacy of NTP42 in the monocrotaline (MCT)-induced PAH rat model, alongside current standard-of-care drugs. METHODS: PAH was induced by subcutaneous injection of 60 mg/kg MCT in male Wistar-Kyoto rats. Animals were assigned into groups: 1. 'No MCT'; 2. 'MCT Only'; 3. MCT + NTP42 (0.25 mg/kg BID); 4. MCT + Sildenafil (50 mg/kg BID), and 5. MCT + Selexipag (1 mg/kg BID), where 28-day drug treatment was initiated within 24 h post-MCT. RESULTS: From haemodynamic assessments, NTP42 reduced the MCT-induced PAH, including mean pulmonary arterial pressure (mPAP) and right systolic ventricular pressure (RSVP), being at least comparable to the standard-of-care drugs Sildenafil or Selexipag in bringing about these effects. Moreover, NTP42 was superior to Sildenafil and Selexipag in significantly reducing pulmonary vascular remodelling, inflammatory mast cell infiltration and fibrosis in MCT-treated animals. CONCLUSIONS: These findings suggest that NTP42 and antagonism of the TP signalling pathway have a relevant role in alleviating the pathophysiology of PAH, representing a novel therapeutic target with marked benefits over existing standard-of-care therapies.

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.

Identification of α-helix 4 (α4) of Rab11a as a novel Rab11-binding domain (RBD): Interaction of Rab11a with the Prostacyclin Receptor.

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 α4 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.

Regulated expression of the prostacyclin receptor (IP) gene by androgens within the vasculature: Combined role for androgens and serum cholesterol.

The prostanoid prostacyclin plays a key cardioprotective role within the vasculature. There is increasing evidence that androgens may also confer cardioprotection but through unknown mechanisms. This study investigated whether the androgen dihydrotestosterone (DHT) may regulate expression of the prostacyclin/I prostanoid receptor or, in short, the IP in platelet-progenitor megakaryoblastic and vascular endothelial cells. DHT significantly increased IP mRNA and protein expression, IP-induced cAMP generation and promoter (PrmIP)-directed gene expression in all cell types examined. The androgen-responsive region was localised to a cis-acting androgen response element (ARE), which lies in close proximity to a functional sterol response element (SRE) within the core promoter. In normal serum conditions, DHT increased IP expression through classic androgen receptor (AR) binding to the functional ARE within the PrmIP. However, under conditions of low-cholesterol, DHT led to further increases in IP expression through an indirect mechanism involving AR-dependent upregulation of SCAP expression and enhanced SREBP1 processing & binding to the SRE within the PrmIP. Chromatin immunoprecipitation assays confirmed DHT-induced AR binding to the ARE in vivo in cells cultured in normal serum while, in conditions of low cholesterol, DHT led to increased AR and SREBP1 binding to the functional ARE and SRE cis-acting elements, respectively, within the core PrmIP resulting in further increases in IP expression. Collectively, these data establish that the human IP gene is under the transcriptional regulation of DHT, where this regulation is further influenced by serum-cholesterol levels. This may explain, in part, some of the protective actions of androgens within the vasculature.

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.

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.

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.

The prostanoid thromboxane (TX) A(2) plays a central role in hemostasis and is increasingly implicated in neoplastic disease, including prostate and breast cancers. In humans, TXA(2) signals through the TPα and TPß 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 TXA(2) 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.

Prostacyclin receptors: Transcriptional regulation and novel signalling mechanisms.

The prostanoid Prostacyclin plays diverse physiologic roles within the vasculature and other systems, and is widely implicated in several cardiovascular, pulmonary and renal diseases. Despite this, knowledge of the factors regulating expression of the I prostanoid receptor (the IP) remained largely unknown. This review details recent advances in understanding the key transcriptional regulators determining expression of the PTGIR gene in the human vasculature and the identification of novel interacting partners of the IP that impact on its function therein. Included in this are the trans-acting factors that regulate expression of the PTGIR under basal- and regulated-conditions, particularly those determining its up-regulation in response to cellular differentiation, estrogen and low serum-cholesterol. Moreover, the functional implications of the interactions between the IP with PDZK1, a multi PDZ-domain containing protein essential for reverse-cholesterol transport and endothelialization, and the IP with IKEPP, the intestinal and kidney enriched PDZ protein, for the role of the prostacyclin-IP axis within the vasculature are reviewed.

Regulation of the human prostacyclin receptor gene in megakaryocytes: major roles for C/EBPδ and PU.1.

The prostanoid prostacyclin plays a central role in haemostasis and vascular repair. Recent studies investigating the regulation of the human prostacyclin receptor (hIP) gene identified an upstream repressor region (URR) within its regulatory promoter, herein termed the PrmIP. This study aimed to identify the main trans-acting factors that bind within the URR to transcriptionally repress PrmIP-directed gene expression in the megakaryoblastic human erythroleukemia (HEL) 92.1.7 cell line. Of the putative cis-acting elements examined, disruption of C/EBP and PU.1 elements within the URR substantially increased PrmIP-directed gene expression. Chromatin immunoprecipitation (ChIP) confirmed that C/EBPδ and PU.1, but not C/EBPß, bind to the URR in vivo, while ectopic expression of C/EBPδ substantially reduced hIP mRNA levels and PrmIP-directed gene expression. Phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic differentiation increased hIP mRNA and PrmIP-directed reporter gene expression and hIP-mediated cAMP generation in HEL cells. Two PMA-responsive regions, termed PRR1 and PRR2, were identified within PrmIP. Disruption of C/EBPδ and PU.1 cis-elements within the overlapping PRR1/URR and of Sp1, PU.1 and Oct-1 cis-elements within the overlapping PRR2/core PrmIP, revealed that both PRR1 and PRR2 contribute to the PMA- induction of hIP mRNA and gene expression in HEL cells. Furthermore, ChIP analysis established that induction of PrmIP-directed gene expression during megakaryocytic differentiation is largely regulated by PMA-induced dissociation of C/EBPδ and enhanced binding of PU.1 to PRR1 in addition to increased binding of Sp1, PU.1 and Oct-1 to elements within the core promoter/PRR2 in vivo. Taken together, these data provide critical insights into the transcriptional regulation of the hIP gene within the vasculature, including during megakaryocytic differentiation.

Interaction of the human prostacyclin receptor and the NHERF4 family member intestinal and kidney enriched PDZ protein (IKEPP).

Prostacyclin and its I prostanoid receptor, the IP, play central roles in hemostasis and in re-endothelialization in response to vascular injury. Herein, intestinal and kidney enriched PDZ protein (IKEPP) was identified as an interactant of the human (h) IP mediated through binding of PDZ domain 1 (PDZ(D1)) and, to a lesser extent, PDZ(D2) of IKEPP to a carboxyl-terminal Class I 'PDZ ligand' within the hIP. While the interaction is constitutive, agonist-activation of the hIP leads to cAMP-dependent protein kinase (PK) A and PKC-phosphorylation of IKEPP, coinciding with its increased interaction with the hIP. Ectopic expression of IKEPP increases functional expression of the hIP, enhancing its ligand binding and agonist-induced cAMP generation. Originally thought to be restricted to renal and gastrointestinal tissues, herein, IKEPP was also found to be expressed in vascular endothelial cells where it co-localizes and complexes with the hIP. Furthermore, siRNA-disruption of IKEPP expression impaired hIP-induced endothelial cell migration and in vitro angiogenesis, revealing the functional importance of the IKEPP:IP interaction within the vascular endothelium. Identification of IKEPP as a functional interactant of the IP reveals novel mechanistic insights into the role of these proteins within the vasculature and, potentially, in other systems where they are co-expressed.

Evaluation of NTP42, a novel thromboxane receptor antagonist, in a first-in-human phase I clinical trial.

Background: The thromboxane receptor (TP) antagonist NTP42 is in clinical development for treatment of cardiopulmonary diseases, such as pulmonary arterial hypertension. In this randomized, placebo-controlled Phase I clinical trial, NTP42, administered as the oral formulation NTP42:KVA4, was evaluated for safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) in healthy males. Methods: The first-in-human trial had three Parts: A, single ascending dose (SAD) study with seven groups given 0.25-243 mg NTP42:KVA4 or placebo; B, food effect study where one SAD group (9 mg) was also given NTP42:KVA4 or placebo after a high-fat breakfast; C, multiple ascending dose study with three groups given 15-135 mg NTP42:KVA4 or placebo once-daily for 7 days. Results: Seventy-nine volunteers participated. No serious adverse events occurred, where any drug- or placebo-related adverse events were mild to moderate, with no correlation to NTP42:KVA4 dose. NTP42 was rapidly absorbed, yielding dose proportional increases in exposure after single and repeat dosing. PK confirmed that, with a clearance (T1/2) of 18.7 h, NTP42:KVA4 is suited to once-daily dosing, can be taken with or without food, and does not accumulate on repeat dosing. At doses ≥1 mg, NTP42 led to complete and sustained inhibition of thromboxane-, but not ADP-, induced platelet aggregation ex vivo, with direct correlation between NTP42 exposure and duration of PD effects. Conclusion: Orally administered NTP42:KVA4 was well tolerated, with favorable PK/PD profiles and evidence of specific TP target engagement. These findings support continued clinical development of NTP42:KVA4 for cardiopulmonary or other relevant diseases with unmet needs. Clinical Trial Registration: clinicaltrials.gov, identifier NCT04919863.

Regulation of the human prostacyclin receptor gene by the cholesterol-responsive SREBP1.

Prostacyclin and its prostacyclin receptor, the I Prostanoid (IP), play essential roles in regulating hemostasis and vascular tone and have been implicated in a range cardio-protective effects but through largely unknown mechanisms. In this study, the influence of cholesterol on human IP [(h)IP] gene expression was investigated in cultured vascular endothelial and platelet-progenitor megakaryocytic cells. Cholesterol depletion increased human prostacyclin receptor (hIP) mRNA, hIP promoter-directed reporter gene expression, and hIP-induced cAMP generation in all cell types. Furthermore, the constitutively active sterol-response element binding protein (SREBP)1a, but not SREBP2, increased hIP mRNA and promoter-directed gene expression, and deletional and mutational analysis uncovered an evolutionary conserved sterol-response element (SRE), adjacent to a known functional Sp1 element, within the core hIP promoter. Moreover, chromatin immunoprecipitation assays confirmed direct cholesterol-regulated binding of SREBP1a to this hIP promoter region in vivo, and immunofluorescence microscopy corroborated that cholesterol depletion significantly increases hIP expression levels. In conclusion, the hIP gene is directly regulated by cholesterol depletion, which occurs through binding of SREBP1a to a functional SRE within its core promoter. Mechanistically, these data establish that cholesterol can regulate hIP expression, which may, at least in part, account for the combined cardio-protective actions of low serum cholesterol through its regulation of IP expression within the human vasculature.

Identification of an interaction between the TPalpha and TPbeta isoforms of the human thromboxane A2 receptor with protein kinase C-related kinase (PRK) 1: implications for prostate cancer.

In humans, thromboxane (TX) A(2) signals through the TPα and TPß isoforms of the TXA(2) receptor or TP. Here, the RhoA effector protein kinase C-related kinase (PRK) 1 was identified as an interactant of both TPα and ΤPß involving common and unique sequences within their respective C-terminal (C)-tail domains and the kinase domain of PRK1 (PRK1(640-942)). Although the interaction with PRK1 is constitutive, agonist activation of TPα/TPß did not regulate the complex per se but enhanced PRK1 activation leading to phosphorylation of its general substrate histone H1 in vitro. Altered PRK1 and TP expression and signaling are increasingly implicated in certain neoplasms, particularly in androgen-associated prostate carcinomas. Agonist activation of TPα/TPß led to phosphorylation of histone H3 at Thr(11) (H3 Thr(11)), a previously recognized specific marker of androgen-induced chromatin remodeling, in the prostate LNCaP and PC-3 cell lines but not in primary vascular smooth muscle or endothelial cells. Moreover, this effect was augmented by dihydrotestosterone in androgen-responsive LNCaP but not in nonresponsive PC-3 cells. Furthermore, PRK1 was confirmed to constitutively interact with TPα/TPß in both LNCaP and PC-3 cells, and targeted disruption of PRK1 impaired TPα/TPß-mediated H3 Thr(11) phosphorylation in, and cell migration of, both prostate cell types. Collectively, considering the role of TXA(2) as a potent mediator of RhoA signaling, the identification of PRK1 as a bona fide interactant of TPα/TPß, and leading to H3 Thr(11) phosphorylation to regulate cell migration, has broad functional significance such as within the vasculature and in neoplasms in which both PRK1 and the TPs are increasingly implicated.

Identification of a novel endoplasmic reticulum export motif within the eighth α-helical domain (α-H8) of the human prostacyclin receptor.

The human prostacyclin receptor (hIP) undergoes agonist-dependent trafficking involving a direct interaction with Rab11a GTPase. The region of interaction was localised to a 14 residue Rab11a binding domain (RBD) within the proximal carboxyl-terminal (C)-tail domain of the hIP, consisting of Val(299)-Val(307) within the eighth helical domain (α-H8) adjacent to the palmitoylated residues at Cys(308)-Cys(311). However, the factors determining the anterograde transport of the newly synthesised hIP from the endoplasmic reticulum (ER) to the plasma membrane (PM) have not been identified. The aim of the current study was to identify the major ER export motif(s) within the hIP initially by investigating the role of Lys residues in its maturation and processing. Through site-directed and Ala-scanning mutational studies in combination with analyses of protein expression and maturation, functional analyses of ligand binding, agonist-induced intracellular signalling and confocal image analyses, it was determined that Lys(297), Arg(302) and Lys(304) located within α-H8 represent the critical determinants of a novel ER export motif of the hIP. Furthermore, while substitution of those critical residues significantly impaired maturation and processing of the hIP, replacement of the positively charged Lys with Arg residues, and vice versa, was functionally permissible. Hence, this study has identified a novel 8 residue ER export motif within the functionally important α-H8 of the hIP. This ER export motif, defined by "K/R(X)(4)K/R(X)K/R," has a strict requirement for positively charged, basic Lys/Arg residues at the 1st, 6th and 8th positions and appears to be evolutionarily conserved within IP sequences from mouse to man.