Now showing 1 - 10 of 27
  • 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.
    Scopus© Citations 14  578
  • Publication
    Synthetic peroxisome proliferator-activated receptor gamma agonists rosiglitazone and troglitazone suppress transcription by promoter 3 of the human thromboxane A2 receptor gene in human erythroleukemia cells
    The human thromboxane (TX)A2 receptor (TP) gene encodes two TP isoforms, TPalpha and TP beta that are regulated by distinct promoters designated promoter (Prm) 1 and Prm3, respectively. Previous studies established that 15d-delta 12,14-prostaglandin J2 (15d-PGJ2) selectively inhibits Prm3 activity and TP beta expression through a peroxisome proliferator-activated receptor (PPAR)gamma mechanism without affecting Prm1 activity or TPalpha expression in human megakaryocytic erythroleukemia (HEL) 92.1.7 cells. Herein, we investigated the effect of synthetic thiazolidinedione (TZD) PPARgamma ligands rosiglitazone and troglitazone on TP gene expression in HEL cells. Like 15d-PGJ2, both TZDs suppressed Prm3 activity, TPbeta mRNA expression and TP-mediated calcium mobilization without affecting Prm1 or TPalpha mRNA expression. However, unlike 15d-PGJ2, both TZDs mediated their PPARgamma-dependent effects through trans-repression of an activator protein-1 (AP-1) element, a site previously found to be critical for basal Prm3 activity. These data provide further evidence for the role of PPARgamma in regulating the human TP gene; they highlight further differences in TPalpha and TPbeta expression/regulation and point to essential differences between natural and synthetic PPARgamma agonists in mediating those effects.
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  • 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.
      368Scopus© Citations 4
  • Publication
    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.
      650Scopus© Citations 15
  • 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.
    Scopus© Citations 14  1104
  • 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.
    Scopus© Citations 69  367
  • Publication
    Differential regulation of RhoA-mediated signaling by the TPalpha and TPbeta isoforms of the human thromboxane A2 receptor : independent modulation of TPalpha signaling by prostacyclin and nitric oxide
    In humans, thromboxane (TX) A2 signals through the TPalpha and TPbeta isoforms of the TXA2 receptor that exhibit common and distinct roles. For example, Gq/phospholipase (PL)Cbeta signaling by TPalpha is directly inhibited by the vasodilators prostacyclin and nitric oxide (NO) whereas that signaling by TPbeta is unaffected. Herein, we investigated whether TPalpha and/or TPbeta regulate G12/Rho activation and whether that signaling might be differentially regulated by prostacyclin and/or NO. Both TPalpha and TPbeta independently regulated RhoA activation and signaling in clonal cells over-expressing TPalpha or TPbeta and in primary human aortic smooth muscle cells (1o AoSMCs). While RhoA- signaling by TPalpha was directly impaired by prostacyclin and NO through protein kinase (PK)A- and PKG-dependent phosphorylation, respectively, signaling by TPbeta was not directly affected by either agent. Collectively, while TPalpha and TPbeta contribute to RhoA activation, our findings support the hypothesis that TPalpha is involved in the dynamic regulation of haemostasis and vascular tone, such as in response to prostacyclin and NO. Conversely, the role of TPbeta in such processes remains unsolved. Data herein provide essential new insights into the physiologic roles of TPalpha and TPbeta and, through studies in AoSMCs, reveal an additional mode of regulation of VSM contractile responses by TXA2.
    Scopus© Citations 35  612
  • Publication
    Transcriptional regulation of the human prostacyclin receptor gene is dependent on Sp1, PU.1 and Oct-1 in megakaryocytes and endothelial cells
    Prostacyclin plays a central role in haemostasis, inflammation and nociception. However, the factors regulating expression of the prostacyclin receptor (IP) gene in humans, or in other species, have not been identified. Herein it was sought to identify the key trans-acting factors and cis-acting elements regulating IP expression in the megakaryoblastic human erythroleukemia (HEL) 92.1.7 and the vascular endothelial EA.hy 926 cell lines. Using deletion and genetic reporter analyses, the essential core promoter, termed PrmIP, was localized to -1022 to -895 proximal to the transcription initiation site, while an upstream repressor region, localized to -1502 to -1271, was also identified. Bioinformatic analysis revealed evolutionary conserved Sp1, PU.1 and Oct-1 sites within the core PrmIP and disruption of those elements each led to substantial reductions in PrmIP-directed gene expression in both HEL and EA.hy 926 cells. Electrophoretic mobility shift assays (EMSAs) and supershift assays established that Sp1, PU.1 and Oct-1 can bind to elements within the core promoter in vitro while chromatin immunoprecipitiation (ChIP) assays confirmed their specific binding to chromatin in vivo. Furthermore, combination mutations of the Sp1, PU.1 and Oct-1 elements revealed that they act independently to co-regulate basal transcription of the IP gene while ectopic expression of each of the trans-acting factors led to substantial increases in PrmIP-directed gene expression and IP mRNA expression in both HEL and EA.hy 926 cells. While EMSA and antibody supershift assays established that the Ets family member Fli1, but not Ets-1, is capable of binding to the PU.1 element within PrmIP in vitro, ChIP analysis established that neither Fli1 nor Ets-1 bind to that element in vivo. Collectively, these data provide critical insights into the transcriptional regulation of the IP gene in human megakaryocytic and endothelial cells, identifying Sp1, PU.1 and Oct-1 as the critical factors involved in its basal regulation in humans.
    Scopus© Citations 15  668
  • Publication
    Prostaglandin D2 receptor-mediated desensitization of the alpha isoform of the human thromboxane A2 receptor
    Thromboxane (TX) A2 and prostaglandin (PG) D2 mediate opposing actions in platelets and in vascular and non-vascular smooth muscle. Here, we investigated the effects of stimulation of the PGD2 receptor (DP) on signaling by the TXA2 receptor (TP) expressed in human platelets and in human embryonic kidney (HEK) 293 cells over-expressing the individual TPalpha and TPbeta isoforms. In platelets, the selective DP agonist BW245C abolished TP-mediated mobilization of intracellular calcium ([Ca2+]i) and inhibited platelet aggregation in response to the TXA2 mimetic U46619. DP-mediated desensitization of TP signaling in platelets was prevented by pre-treatment with the cAMP-dependent PKA inhibitor, H-89, but was unaffected by the PKC inhibitor GF 109203X. In HEK 293 cells signaling by TPalpha, but not TPbeta, was subject to DP mediated desensitization in a PKA dependent, PKC independent manner. U46619-induced signaling by TP-328, a truncated variant of TP containing only those residues common to TPalpha and TPbeta, was insensitive to prior DP stimulation indicating that the carboxyl terminal tail of TPalpha contains the target site(s) for DP-mediated desensitization. Mutation of Ser329 to Ala329 within a consensus PKA site in TPalpha rendered the mutant TPalphaS329A insensitive to BW245C-mediated desensitization. Whole cell phosphorylation assays established that TPalpha, but not TPbeta or TPalphaS329A, was subject to DP-mediated phosphorylation and that TPalpha phosphorylation was blocked by the PKA inhibitor H-89. These data establish that TPalpha, but not TPbeta, is subject to DP mediated cross desensitization, which occurs through direct PKA mediated phosphorylation of TPalpha at Ser329.
    Scopus© Citations 30  340
  • 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.
    Scopus© Citations 3  540