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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.
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Background: Pulmonary 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. Methods: The 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). Results: In 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. Conclusion: This 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.
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NTP42 is a novel antagonist of the thromboxane A2 receptor (TP) in development for the treatment of pulmonary arterial hypertension (PAH). Recent studies demonstrated that NTP42 and TP antagonism have a role in alleviating PAH pathophysiology. However, the efficacy of NTP42 when used in combination with existing PAH therapies has not yet been investigated. Herein, the Sugen 5416/hypoxia (SuHx)-induced PAH model was employed to evaluate the efficacy of NTP42 when used alone or in dual-therapy with Sildenafil, a PAH standard-of-care. PAH was induced in rats by injection of Sugen 5416 and exposure to hypoxia for 21 days. Thereafter, animals were treated orally twice-daily for 28 days with either vehicle, NTP42 (0.05 mg/kg), Sildenafil (50 mg/kg), or NTP42+Sildenafil (0.05 mg/kg + 50 mg/kg, respectively). While Sildenafil or NTP42 mono-therapy led to non-significant reductions in the SuHx-induced rises in mean pulmonary arterial pressure (mPAP) or right ventricular systolic pressure (RSVP), combined use of NTP42+Sildenafil significantly reduced these increases in mPAP and RVSP. Detailed histologic analyses of pulmonary vessel remodelling, right ventricular hypertrophy and fibrosis demonstrated that while NTP42 and Sildenafil in mono-therapy resulted in significant benefits, NTP42+Sildenafil in dual-therapy showed an even greater benefit over either drug used alone. In summary, combined use of NTP42+Sildenafil in dual-therapy confers an even greater benefit in treating or offsetting key aetiologies underlying PAH. These findings corroborate earlier preclinical findings suggesting that, through antagonism of TP signalling, NTP42 attenuates PAH pathophysiology, positioning it as a novel therapeutic for use alone or in combination therapy regimens.
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Hipoxia/tratamiento farmacológico , Indoles/toxicidad , Hipertensión Arterial Pulmonar/tratamiento farmacológico , Pirroles/toxicidad , Receptores de Tromboxano A2 y Prostaglandina H2/antagonistas & inhibidores , Citrato de Sildenafil/administración & dosificación , Inhibidores de la Angiogénesis/toxicidad , Animales , Quimioterapia Combinada , Hipoxia/inducido químicamente , Hipoxia/metabolismo , Masculino , Hipertensión Arterial Pulmonar/inducido químicamente , Hipertensión Arterial Pulmonar/metabolismo , Ratas , Ratas Wistar , Receptores de Tromboxano A2 y Prostaglandina H2/metabolismo , Resultado del Tratamiento , Vasodilatadores/administración & dosificaciónRESUMEN
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.
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Antihipertensivos/farmacología , Hipertensión Arterial Pulmonar/tratamiento farmacológico , Arteria Pulmonar/fisiopatología , Receptores de Tromboxanos/antagonistas & inhibidores , Acetamidas/farmacología , Animales , Modelos Animales de Enfermedad , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/patología , Hemodinámica/efectos de los fármacos , Humanos , Hipertrofia Ventricular Derecha/inducido químicamente , Hipertrofia Ventricular Derecha/tratamiento farmacológico , Hipertrofia Ventricular Derecha/metabolismo , Hipertrofia Ventricular Derecha/patología , Masculino , Monocrotalina , Hipertensión Arterial Pulmonar/inducido químicamente , Hipertensión Arterial Pulmonar/metabolismo , Hipertensión Arterial Pulmonar/patología , Pirazinas/farmacología , Ratas , Ratas Endogámicas WKY , Citrato de Sildenafil/farmacología , Remodelación Vascular/efectos de los fármacosRESUMEN
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.
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Diferenciación Celular/genética , Perfilación de la Expresión Génica , Inflamación/genética , Próstata/metabolismo , Receptores de Tromboxano A2 y Prostaglandina H2/genética , Enfermedad Crónica , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Humanos , Inflamación/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Masculino , Monocitos/citología , Monocitos/metabolismo , Prostaglandinas/metabolismo , Próstata/patología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferencia de ARN , Receptores de Tromboxano A2 y Prostaglandina H2/metabolismo , Células THP-1RESUMEN
Aims: Pulmonary arterial hypertension (PAH) is a devastating disease and treatment options are limited. Urocortin-2 (Ucn-2) has shown promising therapeutic effects in experimental and clinical left ventricular heart failure (HF). Our aim was to analyse the expression of Ucn-2 in human and experimental PAH, and to investigate the effects of human Ucn-2 (hUcn-2) administration in rats with monocrotaline (MCT)-induced pulmonary hypertension (PH). Methods and results: Tissue samples were collected from patients with and without PAH and from rats with MCT-induced PH. hUcn-2 (5 µg/kg, bi-daily, i.p., for 10 days) or vehicle was administered to male wistar rats subjected to MCT injection or to pulmonary artery banding (PAB) to induce right ventricular (RV) overload without PAH. Expression of Ucn-2 and its receptor was increased in the RV of patients and rats with PAH. hUcn-2 treatment reduced PAH in MCT rats, resulting in decreased morbidity, improved exercise capacity and attenuated pulmonary arterial and RV remodelling and dysfunction. Additionally, RV gene expression of hypertrophy and failure signalling pathways were attenuated. hUcn-2 treatment also attenuated PAB-induced RV hypertrophy. Conclusions: Ucn-2 levels are altered in human and experimental PAH. hUcn-2 treatment attenuates PAH and RV dysfunction in MCT-induced PH, has direct anti-remodelling effects on the pressure-overloaded RV, and improves pulmonary vascular function.
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Antihipertensivos/farmacología , Presión Arterial/efectos de los fármacos , Hormona Liberadora de Corticotropina/farmacología , Ventrículos Cardíacos/efectos de los fármacos , Hipertensión Pulmonar/prevención & control , Hipertrofia Ventricular Derecha/prevención & control , Arteria Pulmonar/efectos de los fármacos , Urocortinas/farmacología , Disfunción Ventricular Derecha/prevención & control , Función Ventricular Derecha/efectos de los fármacos , Animales , Estudios de Casos y Controles , Hormona Liberadora de Corticotropina/metabolismo , Modelos Animales de Enfermedad , Tolerancia al Ejercicio/efectos de los fármacos , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/fisiopatología , Humanos , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/fisiopatología , Hipertrofia Ventricular Derecha/metabolismo , Hipertrofia Ventricular Derecha/fisiopatología , Masculino , Arteria Pulmonar/fisiopatología , Ratas Wistar , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Transducción de Señal/efectos de los fármacos , Urocortinas/metabolismo , Remodelación Vascular/efectos de los fármacos , Vasodilatación/efectos de los fármacos , Disfunción Ventricular Derecha/metabolismo , Disfunción Ventricular Derecha/fisiopatología , Remodelación Ventricular/efectos de los fármacosRESUMEN
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.
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Factores de Transcripción Forkhead/metabolismo , Proteínas de Homeodominio/metabolismo , Neoplasias de la Próstata/metabolismo , Receptores de Tromboxano A2 y Prostaglandina H2/metabolismo , Proteínas Represoras/metabolismo , Tromboxano A2/metabolismo , Factores de Transcripción/metabolismo , Línea Celular Tumoral , Islas de CpG , Metilación de ADN , Regulación hacia Abajo , Factores de Transcripción Forkhead/genética , Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/genética , Humanos , Masculino , Regiones Promotoras Genéticas , Neoplasias de la Próstata/genética , Protaminas/genética , Isoformas de Proteínas , Receptores de Tromboxano A2 y Prostaglandina H2/genética , Proteínas Represoras/genética , Factores de Transcripción/genética , Regulación hacia ArribaRESUMEN
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.
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Dominios y Motivos de Interacción de Proteínas/genética , Receptores de Epoprostenol/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Secuencia de Aminoácidos/genética , Células HeLa , Humanos , Unión Proteica , Conformación Proteica en Hélice alfa/genética , Transporte de Proteínas/genética , Receptores de Epoprostenol/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Unión al GTP rab/química , Proteínas de Unión al GTP rab/genéticaRESUMEN
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.
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Andrógenos/farmacología , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas de Neoplasias/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Tromboxano A2 y Prostaglandina H2/metabolismo , Transducción de Señal , Tromboxano A2/farmacología , Acetilación/efectos de los fármacos , Andrógenos/metabolismo , Línea Celular Tumoral , Humanos , Masculino , Proteínas de Neoplasias/genética , Fosforilación/efectos de los fármacos , Fosforilación/genética , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/patología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Receptores de Tromboxano A2 y Prostaglandina H2/genética , Tromboxano A2/metabolismoRESUMEN
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.
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Regulación Neoplásica de la Expresión Génica , Neoplasias de la Próstata/genética , Receptores de Tromboxano A2 y Prostaglandina H2/genética , Metilación de ADN , Progresión de la Enfermedad , Humanos , Masculino , Clasificación del Tumor , Pronóstico , Regiones Promotoras Genéticas , Próstata/metabolismo , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/mortalidad , Isoformas de Proteínas , Receptores de Tromboxano A2 y Prostaglandina H2/química , Transcripción GenéticaRESUMEN
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.
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Adenocarcinoma/enzimología , Neoplasias de la Próstata/enzimología , Proteína Quinasa C/metabolismo , Receptores de Tromboxano A2 y Prostaglandina H2/agonistas , Tromboxano A2/farmacología , Adenocarcinoma/genética , Adenocarcinoma/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Activación Enzimática , Epigénesis Genética/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Histonas/metabolismo , Humanos , Masculino , Fosforilación , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Unión Proteica , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/genética , Inhibidores de Proteínas Quinasas/farmacología , Receptores de Tromboxano A2 y Prostaglandina H2/genética , Receptores de Tromboxano A2 y Prostaglandina H2/metabolismo , Transducción de Señal/efectos de los fármacos , Treonina/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
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.
Asunto(s)
Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica , Factores Reguladores Miogénicos/metabolismo , Neoplasias de la Próstata/genética , Receptores de Tromboxano A2 y Prostaglandina H2/genética , Proteínas WT1/metabolismo , Western Blotting , Neoplasias de la Mama/metabolismo , Movimiento Celular , Proliferación Celular , Inmunoprecipitación de Cromatina , Femenino , Humanos , Técnicas para Inmunoenzimas , Luciferasas/metabolismo , Masculino , Mutagénesis Sitio-Dirigida , Factores Reguladores Miogénicos/genética , Regiones Promotoras Genéticas/genética , Neoplasias de la Próstata/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Tromboxano A2 y Prostaglandina H2/metabolismo , Elementos Reguladores de la Transcripción , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transactivadores , Transcripción Genética , Células Tumorales Cultivadas , Proteínas WT1/genéticaRESUMEN
The prostanoid prostacyclin, or prostaglandin I2, plays an essential role in many aspects of cardiovascular disease. The actions of prostacyclin are mainly mediated through its activation of the prostacyclin receptor or, in short, the IP. In recent studies, the cytoplasmic carboxy-terminal domain of the IP was shown to bind several PDZ domains of the multi-PDZ adaptor PDZK1. The interaction between the two proteins was found to enhance cell surface expression of the IP and to be functionally important in promoting prostacyclin-induced endothelial cell migration and angiogenesis. To investigate the interaction of the IP with the first PDZ domain (PDZ1) of PDZK1, we generated a nine residue peptide (KK(411)IAACSLC(417)) containing the seven carboxy-terminal amino acids of the IP and measured its binding affinity to a recombinant protein corresponding to PDZ1 by isothermal titration calorimetry. We determined that the IP interacts with PDZ1 with a binding affinity of 8.2 µM. Using the same technique, we also determined that the farnesylated form of carboxy-terminus of the IP does not bind to PDZ1. To understand the molecular basis of these findings, we solved the high resolution crystal structure of PDZ1 bound to a 7-residue peptide derived from the carboxy-terminus of the non-farnesylated form of IP ((411)IAACSLC(417)). Analysis of the structure demonstrates a critical role for the three carboxy-terminal amino acids in establishing a strong interaction with PDZ1 and explains the inability of the farnesylated form of IP to interact with the PDZ1 domain of PDZK1 at least in vitro.
Asunto(s)
Proteínas Portadoras/metabolismo , Dominios PDZ , Receptores de Epoprostenol/metabolismo , Secuencia de Aminoácidos , Proteínas Portadoras/química , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Mapeo de Interacción de Proteínas , Receptores de Epoprostenol/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Neoplasias/metabolismo , Fosfoproteínas/metabolismo , Receptores de Prostaglandina/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Moléculas de Adhesión Celular , Movimiento Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Riñón/citología , Ligandos , Modelos Biológicos , Proteínas de Neoplasias/química , Neovascularización Fisiológica , Fosfoproteínas/química , Unión Proteica , Proteína Quinasa C/metabolismo , Estructura Terciaria de Proteína , Receptores de Epoprostenol , Receptores de Prostaglandina/agonistas , Receptores de Prostaglandina/química , Transducción de Señal , Intercambiadores de Sodio-Hidrógeno/químicaRESUMEN
Prostacyclin is increasingly implicated in re-endothelialization and angiogenesis but through largely unknown mechanisms. Herein the high-density lipoprotein (HDL) scavenger receptor class B, type 1 (SR-B1) adapter protein PDZ domain-containing protein 1 (PDZK1) was identified as an interactant of the human prostacyclin receptor (hIP) involving a Class I PDZ ligand at its carboxyl terminus and PDZ domains 1, 3, and 4 of PDZK1. Although the interaction is constitutive, it may be dynamically regulated following cicaprost activation of the hIP through a mechanism involving cAMP-dependent protein kinase (PK)A-phosphorylation of PDZK1 at Ser-505. Although PDZK1 did not increase overall levels of the hIP, it increased its functional expression at the cell surface, enhancing ligand binding and cicaprost-induced cAMP generation. Consistent with its role in re-endothelialization and angiogenesis, cicaprost activation of the hIP increased endothelial cell migration and tube formation/in vitro angiogenesis, effects completely abrogated by the specific IP antagonist RO1138452. Furthermore, similar to HDL/SR-B1, small interfering RNA (siRNA)-targeted disruption of PDZK1 abolished cicaprost-mediated endothelial responses but did not affect VEGF responses. Considering the essential role played by prostacyclin throughout the cardiovascular system, identification of PDZK1 as a functional interactant of the hIP sheds significant mechanistic insights into the protective roles of these key players, and potentially HDL/SR-B1, within the vascular endothelium.
Asunto(s)
Proteínas Portadoras/metabolismo , AMP Cíclico/metabolismo , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Lipoproteínas HDL/metabolismo , Receptores de Prostaglandina/metabolismo , Transducción de Señal , Animales , Antineoplásicos/farmacología , Compuestos de Bencilo/farmacología , Proteínas Portadoras/genética , Membrana Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células Endoteliales/citología , Endotelio Vascular/citología , Epoprostenol/análogos & derivados , Epoprostenol/farmacología , Regulación de la Expresión Génica , Células HEK293 , Humanos , Imidazoles/farmacología , Proteínas de la Membrana , Ratones , Ratones Noqueados , Neovascularización Fisiológica/efectos de los fármacos , Dominios PDZ , Fosforilación , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Receptores de Epoprostenol , Receptores de Prostaglandina/antagonistas & inhibidores , Receptores de Prostaglandina/genéticaRESUMEN
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.
Asunto(s)
Neoplasias de la Próstata/patología , Proteína Quinasa C/metabolismo , Receptores de Tromboxano A2 y Prostaglandina H2/metabolismo , Línea Celular Tumoral , Movimiento Celular , Histonas/metabolismo , Humanos , Masculino , Fosforilación , Neoplasias de la Próstata/metabolismo , Unión Proteica , Isoformas de Proteínas/metabolismoRESUMEN
In humans, thromboxane (TX) A2 signals through the TPα and TPß isoforms of its G-protein coupled TXA2 receptor (TP) to mediate a host of (patho)physiologic responses. Herein, angio-associated migratory cell protein (AAMP) was identified as a novel interacting partner of both TPα and TPß through an interaction dependent on common (residues 312-328) and unique (residues 366-392 of TPß) sequences within their carboxyl-terminal (C)-tail domains. While the interaction was constitutive in mammalian cells, agonist-stimulation of TPα/TPß led to a transient dissociation of AAMP from immune complexes which coincided with a transient redistribution of AAMP from its localization in an intracellular fibrous network. Although the GTPase RhoA is a downstream effector of both AAMP and the TPs, AAMP did not influence TP-mediated RhoA or vice versa. Small interfering RNA (siRNA)-mediated disruption of AAMP expression decreased migration of primary human coronary artery smooth muscle cells (1° hCoASMCs). Moreover, siRNA-disruption of AAMP significantly impaired 1° hCoASMC migration in the presence of the TXA2 mimetic U46619 but did not affect VEGF-mediated cell migration. Given their roles within the vasculature, the identification of a specific interaction between TPα/TPß and AAMP is likely to have substantial functional implications for vascular pathologies in which they are both implicated.
Asunto(s)
Proteínas Portadoras/metabolismo , Isoformas de Proteínas/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Tromboxano A2 y Prostaglandina H2/metabolismo , Tromboxano A2/metabolismo , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Actinina/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Portadoras/química , Proteínas Portadoras/genética , Movimiento Celular , Células Cultivadas , Técnicas de Silenciamiento del Gen , Humanos , Datos de Secuencia Molecular , Miocitos del Músculo Liso/fisiología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas/agonistas , ARN Interferente Pequeño/genética , Receptores de Antígenos de Linfocitos B/química , Receptores de Antígenos de Linfocitos B/genética , Receptores de Tromboxano A2 y Prostaglandina H2/agonistas , Proteínas Recombinantes de Fusión/agonistas , Proteínas Recombinantes de Fusión/metabolismo , Tromboxano A2/agonistas , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
The human prostacyclin receptor (hIP) undergoes agonist-induced internalization and subsequent recyclization in slowly recycling endosomes involving its direct physical interaction with Rab11a. Moreover, interaction with Rab11a localizes to a 22-residue putative Rab11 binding domain (RBD) within the carboxyl-terminal tail of the hIP, proximal to the transmembrane 7 (TM7) domain. Because the proposed RBD contains Cys(308) and Cys(311), in addition to Cys(309), that are known to undergo palmitoylation, we sought to identify the structure/function determinants of the RBD, including the influence of palmitoylation, on agonist-induced trafficking of the hIP. Through complementary approaches in yeast and mammalian cells along with computational structural studies, the RBD was localized to a 14-residue domain, between Val(299) and Leu(312), and proposed to be organized into an eighth alpha-helical domain (alpha-helix 8), comprising Val(299)-Val(307), adjacent to the palmitoylated residues at Cys(308)-Cys(311). From mutational and [(3)H]palmitate metabolic labeling studies, it is proposed that palmitoylation at Cys(311) in addition to agonist-regulated deacylation at Cys(309) > Cys(308) may dynamically position alpha-helix 8 in proximity to Rab11a, to regulate agonist-induced intracellular trafficking of the hIP. Moreover, Ala-scanning mutagenesis identified several hydrophobic residues within alpha-helix 8 as necessary for the interaction with Rab11a. Given the diverse membership of the G protein-coupled receptor superfamily, of which many members are also predicted to contain an alpha-helical 8 domain proximal to TM7 and, often, adjacent to palmitoylable cysteine(s), the identification of a functional role for alpha-helix 8, as exemplified as an RBD for the hIP, is likely to have broader significance for certain members of the superfamily.