Now showing 1 - 10 of 27
  • Publication
    The Wilms’ tumor suppressor protein WT1 acts as a key transcriptional repressor of the human thromboxane A2 receptor gene in megakaryocytes
    (Wiley-Blackwell, 2009-11) ;
    In humans, TPα and TPβ isoforms of the thromboxane A2 receptor are transcriptionally regulated by distinct promoters, designated Prm1 and Prm3. Previous investigations identified two upstream repressor regions (URR) 1 and URR2 within Prm1. Herein, it was sought to characterize Prm1, identifying the factor(s) regulating URR1 and URR2 in human erythroleukemia (HEL) 92.1.7 cells. Genetic reporter assays and 5’ deletions confirmed the presence of URR1 and URR2 but also identified a third repressor, designated RR3, within the proximal “core” promoter. Bioinformatic analysis revealed several GC elements representing putative sites for Egr1/Sp1/Wilms tumor (WT)1 within URR1, URR2 and RR3. While mutation of three GC elements within URR1 and of an adjacent GC element suggested that repressor binding occurs through a cooperative mechanism, repressors binding to the single GC elements within URR2 and RR3 act independently to regulate Prm1. While EMSAs and supershift assays demonstrated that each of the GC elements can bind Egr1 and WT1 in vitro, chromatin immunoprecipitations established that WT1 is the factor predominantly bound to each of the repressor regions in vivo. Additionally, ectopic expression of -KTS isoforms of WT1 decreased Prm1-directed gene expression and TPα mRNA expression. Collectively, these data establish WT1 as a critical repressor of Prm1, suppressing TPα expression in the platelet progenitor megakaryoblastic HEL cells.
      225Scopus© Citations 8
  • Publication
    Regulation of the Human Prostacyclin Receptor Gene by the Cholesterol-responsive Sterol Response Element Binding Protein (SREBP) 1.
    (American Society for Biochemistry and Molecular Biology, Inc., 2012-09-11) ;
    Prostacyclin and its prostacyclin receptor, the IP, play essential roles in regulating haemostasis and vascular tone and have also been implicated in a range cardio-protective effects, but through largely unknown mechanisms. In this study, the influence of cholesterol on human (h)IP gene expression was investigated in cultured vascular endothelial and platelet-progenitor megakaryocytic cells. Cholesterol-depletion increased hIP mRNA, hIP promoter-directed reporter gene expression and hIP-induced cAMP generation in all cell types. Furthermore, the constitutively active SREBP1a, but not SREBP2, increased hIP mRNA and promoter-directed gene expression while 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, while immunofluorescence microscopy corroborated that cholesterol-depletion significantly increases hIP expression levels. In conclusion, the hIP gene is directly regulated by cholesterol-depletion that 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 prostacyclin receptor (IP) expression within the human vasculature.
      181Scopus© Citations 7
  • 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.
      449Scopus© Citations 2
  • Publication
    Identification of a novel endoplasmic reticulum export motif within the eighth alpha-helical domain (alpha-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 Val299 – Val307 within the eighth helical domain (alpha-H8) adjacent to the palmitoylated residues at Cys308 – Cys311. 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 Lys297, Arg302 and Lys304 located within alpha-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 alpha-H8 of the hIP. This ER export motif, defined by ‘K/R(X)4K/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.
  • Publication
    The role of N-linked glycosylation in determining the surface expression, G protein interaction and effector coupling of the alpha isoform of the human thromboxane A2 receptor
    In humans, thromboxane (TX) A2 signals through two TXA2 receptor (TP) isoforms, termed TPalpha and TPbeta, that diverge exclusively within their carboxyl terminal cytoplasmic domains. The amino terminal extracellular region of the TPs contains two highly conserved Asn (N)-linked glycosylation sites at Asn4 and Asn16. Whilst it has been established that impairment of N-glycosylation of TPalpha significantly affects ligand binding/intracellular signalling, previous studies did not ascertain whether N-linked glycosylation was critical for ligand binding per se or whether it was required for the intracellular trafficking and the functional expression of TPalpha on the plasma membrane (PM). In the current study, we investigated the role of N-linked glycosylation in determining the functional expression of TPalpha, by assessment of its ligand binding, G-protein coupling and intracellular signalling properties, correlating it with the level of antigenic TPalpha protein expressed on the PM and/or retained intracellularly. From our data, we conclude that N-glycosylation of either Asn4 or Asn16 is required and sufficient for expression of functionally active TPalpha on the PM while the fully non-glycosylated TPalphaN4,N16-Q4,Q16 is almost completely retained within the endoplasmic reticulum and remains functionally inactive, failing to associate with its coupling G protein Galphaq and, in turn, failing to mediate phospholipase Cbeta activation.
      444Scopus© Citations 23
  • Publication
    Homologous desensitization of signalling by the beta isoform of the human thromboxane A2 receptor
    Thromboxane (TX) A2 is a potent stimulator of platelet activation/aggregation and smooth muscle contraction and contributes to a variety of pathologies within the vasculature. In this study, we investigated the mechanism whereby the cellular responses to TXA2 mediated through the TPbeta isoform of the human TXA2 receptor (TP) are dynamically regulated by examining the mechanism of agonist-induced desensitization of intracellular signalling and second messenger generation by TPbeta. It was established that TPbeta is subject to profound agonist-induced homologous desensitization of signalling (intracellular calcium mobilization and inositol 1,3,5 trisphosphate generation) in response to stimulation with the TXA2 mimetic U46619 and this occurs through two key mechanisms: TPbeta undergoes partial agonist-induced desensitization that occurs through a GF 109203X-sensitive, protein kinase (PK)C mechanism whereby Ser145 within intracellular domain (IC)2 has been identified as the key phospho-target. In addition, TPbeta also undergoes more profound and sustained agonist-induced desensitization involving G protein-coupled receptor kinase (GRK)2/3-phosphorylation of both Ser239 and Ser357 within its IC3 and carboxyl-terminal C-tail domains, respectively. Inhibition of phosphorylation of either Ser239 or Ser357, through site directed mutagenesis, impaired desensitization while mutation of both Ser239 and Ser357 almost completely abolished desensitization of signalling, GRK phosphorylation and beta-arrestin association, thereby blocking TPbeta internalization. These data suggest a model whereby agonist-induced PKC phosphorylation of Ser145 partially impairs TPbeta signalling while GRK2/3 phosphorylation at both Ser239 and Ser357 within its IC3 and C-tail domains, respectively, sterically inhibits G-protein coupling, profoundly desensitizing signalling, and promotes beta-arrestin association and, in turn, facilitates TPbeta internalization.
      151Scopus© Citations 26
  • Publication
    Thromboxane A2 receptor mediated activation of the mitogen activated protein kinase cascades in human uterine smooth muscle cells
    Both thromboxane (TX) A2 and 8-epi prostaglandin (PG) F2alpha have been reported to stimulate mitogenesis of vascular smooth muscle (SM) in a number of species. However, TXA2 and 8-epiPGF2alpha mediated mitogenic signalling have not been studied in detail in human vascular SM. Thus, using the human uterine ULTR cell line as a model, we investigated TXA2 receptor (TP) mediated mitogenic signalling in cultured human vascular SM cells. Both the TP agonist U46619 and 8-epiPGF2alpha elicited time and concentration dependent activation of the extracellular signal regulated kinase (ERK)s and c-Jun N-terminal kinase (JNK)s in ULTR cells. Whereas the TP antagonist SQ29,548 abolished U46619-mediated signalling, it only partially inhibited 8-epiPGF2alpha mediated ERK and JNK activation in ULTR cells. Both U46619 and 8-epiPGF2alpha induced ERK activations were inhibited by the protein kinase (PK) C, PKA and phosphoinositide 3-kinase inhibitors GF 109203X, H-89 and wortmannin, respectively, but were unaffected by pertussis toxin. In addition, U46619 mediated ERK activation in ULTR cells involves transactivation of the EGF receptor. In humans, TXA2 signals through two distinct TP isoforms. In investigating the involvement of the TP isoforms in mitogenic signalling, both TPalpha and TPbeta, independently directed U46619 and 8-epiPGF2alpha mediated ERK and JNK activation in human embryonic kidney (HEK) 293 cells over-expressing the individual TP isoforms. However, in contrast to that which occurred in ULTR cells, SQ29,548 abolished 8-epiPGF2alpha mediated ERK and JNK activation through both TPalpha and TPbeta in HEK 293 cells providing further evidence that 8-epiPGF2alpha may signal through alternative receptors, in addition to the TPs, in human uterine ULTR cells.
      315Scopus© Citations 68
  • 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.
      239Scopus© Citations 3
  • Publication
    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.
      407Scopus© Citations 6
  • 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.
      197Scopus© Citations 23