Now showing 1 - 8 of 8
  • Publication
    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 haemostasis 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 (PDZD1) and, to a lesser extent, PDZD2 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.
      369Scopus© Citations 4
  • Publication
    Interaction of the human prostacyclin receptor with the PDZ adapter protein PDZK : role in endothelial cell migration and angiogenesis
    (American Society for Cell Biology, 2011-06-08) ; ; ;
    Prostacyclin is increasingly implicated in re-endothelialization and angiogenesis but through largely unknown mechanisms. Herein, the 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. While 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 Ser505. While 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, 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.
    Scopus© Citations 25  713
  • Publication
    Interaction of angio-associated migratory cell protein with the TPα and TPβ isoforms of the human thromboxane A2 receptor
    In humans, thromboxane (TX) A2 signals through the TPα and TPβ 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 TPβ through an interaction dependent on common (residues 312-328) and unique (residues 366-392 of TPβ) sequences within their carboxyl-terminal (C)-tail domains. While the interaction was constitutive in mammalian cells, agonist-stimulation of TPα/TPβ 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/TPβ and AAMP is likely to have substantial functional implications for vascular pathologies in which they are both implicated.
      1107Scopus© Citations 14
  • Publication
    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
    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.
      543Scopus© Citations 3
  • Publication
    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)A2 signals through the TPalpha and TPbeta isoforms of the TXA2 receptor, or TP. Herein, the RhoA effector protein kinase C-related kinase (PRK) 1 was identified as an interactant of both TPalpha and TPbeta involving common and unique sequences within their respective carboxyl-terminal (C)-tail domains and the kinase domain of PRK1 (PRK1640-942). While the interaction with PRK1 is constitutive, agonist-activation of TPalpha/TPbeta 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 signalling are increasingly implicated in certain neoplasms, particularly in androgen-associated prostate carcinomas. Agonist-activation of TPalpha/TPbeta led to phosphorylation of histone H3 at Thr11 (H3Thr11), 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 non-responsive PC-3 cells. Furthermore, PRK1 was confirmed to constitutively interact with TPalpha/TPbeta in both LNCaP and PC-3 cells and targeted disruption of PRK1 impaired TPalpha/TPbeta-mediated H3Thr11 phosphorylation in, and cell migration of, both prostate cell types. Collectively, considering the role of TXA2 as a potent mediator of RhoA signalling, the identification of PRK1 as a bone fide interactant of TPalpha/TPbeta, and leading to H3Thr11 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.
      271Scopus© Citations 24
  • Publication
    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 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.
      268Scopus© Citations 4
  • Publication
    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
    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.
      201Scopus© Citations 14
  • Publication
    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) 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.
      582Scopus© Citations 14