Karen Douville

Acceleration of Cardiovascular Disease by a Dysfunctional Prostacyclin Receptor Mutation Potential Implications for Cyclooxygenase-2 Inhibition

Recent increased adverse cardiovascular events observed with selective cyclooxygenase-2 inhibition led to the withdrawal of rofecoxib (Vioxx) and valdecoxib (Bextra), but the mechanisms underlying these atherothrombotic events remain unclear. Prostacyclin is the major end product of cyclooxygenase-2 in vascular endothelium. Using a naturally occurring mutation in the prostacyclin receptor, we report for the first time that a deficiency in prostacyclin signaling through its G protein–coupled receptor contributes to atherothrombosis in human patients. We report that a prostacyclin receptor variant (R212C) is defective in adenylyl cyclase activation in both patient blood and in an in vitro COS-1 overexpression system. This promotes increased platelet aggregation, a hallmark of atherothrombosis. Our analysis of patients in 3 separate white cohorts reveals that this dysfunctional receptor is not likely an initiating factor in cardiovascular disease but that it accelerates the course of disease in those patients with the greatest risk factors. R212C was associated with cardiovascular disease only in the high cardiovascular risk cohort (n=980), with no association in the low-risk cohort (n=2293). In those at highest cardiovascular risk, both disease severity and adverse cardiovascular events were significantly increased with R212C when compared with age- and risk factor–matched normal allele patients. We conclude that for haploinsufficient mutants, such as the R212C, the enhanced atherothrombotic phenotype is likely dependent on the presence of existing atherosclerosis or injury (high risk factors), analogous to what has been observed in the cyclooxygenase-2 inhibition studies or prostacyclin receptor knockout mice studies. Combining both biochemical and clinical approaches, we conclude that diminished prostacyclin receptor signaling may contribute, in part, to the underlying adverse cardiovascular outcomes observed with cyclooxygenase-2 inhibition.

Consequences of altered TGF-β expression and responsiveness in breast cancer: evidence for autocrine and paracrine effects

To characterize the impact of increased production of TGF-β in a xenograft model of human breast cancer, TGF-β-responsive MDA-231 cells were genetically modified by stable transfection so as to increase their production of active TGF-β1. Compared with control cells, cells that produced increased amounts of TGF-β proliferated in vitro more slowly. In vivo, however, tumors derived from these cells exhibited increased proliferation and grew at an accelerated pace. To evaluate the role of autocrine TGF-β signaling, cells were also transfected with a dominant-negative truncated type II TGF-β receptor (TβRII). Disruption of autocrine TGF-β signaling in the TGF-β-overexpressing cells reduced their in vivo growth rate. Co-inoculation of Matrigel with the TGF-β-overexpressing cells expressing the truncated TβRII compensated for their diminished in vivo growth capacity, compared with the TGF-β-overexpressing cells with an intact autocrine loop. Tissue invasion by the tumor was a distinctive feature of the TGF-β-overexpressing cells, whether or not the autocrine loop was intact. Furthermore, tumors derived from TGF-β-overexpressing cells, irrespective of the status of the autocrine TGF-β-signaling pathway, had a higher incidence of lung metastasis. Consistent with the suggestion that TGF-βs enhancement of invasion and metastasis is paracrine-based, we observed no significant differences among the cell clones in an in vitro invasion assay. Thus, in this experimental model system in vitro assays of cell proliferation and invasion do not accurately reflect in vivo observations, perhaps due to autocrine and paracrine effects of TGF-β that influence the important in vivo-based phenomena of tumor growth, invasion, and metastasis.

Versatility and Differential Roles of Cysteine Residues in Human Prostacyclin Receptor Structure and Function

Prostacyclin plays important roles in vascular homeostasis, promoting vasodilatation and inhibiting platelet thrombus formation. Previous studies have shown that three of six cytoplasmic cysteines, particularly those within the C-terminal tail, serve as important lipidation sites and are differentially conjugated to palmitoyl and isoprenyl groups (Miggin, S. M., Lawler, O. A., and Kinsella, B. T. (2003) J. Biol. Chem. 278, 6947-6958). Here we report distinctive roles for extracellular- and transmembrane-located cysteine residues in human prostacyclin receptor structure-function. Within the extracellular domain, all cysteines (4 of 4) appear to be involved in disulfide bonding interactions (i.e. a highly conserved Cys-92-Cys-170 bond and a putative non-conserved Cys-5-Cys-165 bond), and within the transmembrane (TM) region there are several cysteines (3 of 8) that maintain critical hydrogen bonding interactions (Cys-118 (TMIII), Cys-251 (TMVI), and Cys-202 (TMV)). This study highlights the necessity of sulfhydryl (SH) groups in maintaining the structural integrity of the human prostacyclin receptor, as 7 of 12 extracellular and transmembrane cysteines studied were found to be differentially indispensable for receptor binding, activation, and/or trafficking. Moreover, these results also demonstrate the versatility and reactivity of these cysteine residues within different receptor environments, that is, extracellular (disulfide bonds), transmembrane (H-bonds), and cytoplasmic (lipid conjugation).

New insights into human prostacyclin receptor structure and function through natural and synthetic mutations of transmembrane charged residues

The human prostacyclin receptor (hIP), a G‐protein coupled receptor (GPCR) expressed mainly on platelets and vascular smooth muscle cells, plays important protective roles in the cardiovascular system. We hypothesized that significant insights could be gained into the structure and function of the hIP through mutagenesis of its energetically unfavourably located transmembrane charged residues.

Comprehensive biochemical analysis of rare prostacyclin receptor variants: Study of association of signaling with coronary artery obstruction

Currently, pharmacogenetic studies are at an impasse as the low prevalence (<2%) of most variants hinder their pharmacogenetic analysis with population sizes often inadequate for sufficiently powered studies. Grouping rare mutations by functional phenotype rather than mutation site can potentially increase sample size. Using human population-based studies (n = 1,761) to search for dysfunctional human prostacyclin receptor (hIP) variants, we recently discovered 18 non-synonymous mutations, all with frequencies less than 2% in our study cohort. Eight of the 18 had defects in binding, activation, and/or protein stability/folding. Mutations (M113T, L104R, and R279C) in three highly conserved positions demonstrated severe misfolding manifested by impaired binding and activation of cell surface receptors. To assess for association with coronary artery disease, we performed a case-control study comparing coronary angiographic results from patients with reduced cAMP production arising from the non-synonymous mutations (n = 23) with patients with non-synonymous mutations that had no reduction in cAMP (n = 17). Major coronary artery obstruction was significantly increased in the dysfunctional mutation group in comparison with the silent mutations. We then compared the 23 dysfunctional receptor patients with 69 age- and risk factor-matched controls (1:3). This verified the significantly increased coronary disease in the non-synonymous dysfunctional variant cohort. This study demonstrates the potential utility of in vitro functional characterization in predicting clinical phenotypes and represents the most comprehensive characterization of human prostacyclin receptor genetic variants to date.

Novel signaling pathways promote a paracrine wave of prostacyclin-induced vascular smooth muscle differentiation.

The important athero-protective role of prostacyclin is becoming increasingly evident as recent studies have revealed adverse cardiovascular effects in mice lacking the prostacyclin receptor, in patients taking selective COX-2 inhibitors, and in patients in the presence of a dysfunctional prostacyclin receptor genetic variant. We have recently reported that this protective mechanism includes the promotion of a quiescent differentiated phenotype in human vascular smooth muscle cells (VSMC). Herein, we address the intriguing question of how localized endothelial release of the very unstable eicosanoid, prostacyclin, exerts a profound effect on the vascular media, often 30 cell layers thick. We report a novel PKA-, Akt-1- and ERK1/2-dependent prostacyclin-induced prostacyclin release that appears to play an important role in propagation of the quiescent, differentiated phenotype through adjacent arterial smooth muscle cells in the vascular media. Treating VSMC with the prostacyclin analog iloprost induced differentiation (contractile protein expression and contractile morphology), and also up-regulated COX-2 expression, leading to prostacyclin release by VSMC. This paracrine prostacyclin release, in turn, promoted differentiation and COX-2 induction in neighboring VSMC that were not exposed to iloprost. Using siRNA and pharmacologic inhibitors, we report that this positive feedback mechanism, prostacyclin-induced prostacyclin release, is mediated by cAMP/PKA signaling, ERK1/2 activation, and a novel prostacyclin receptor signaling pathway, inhibition of Akt-1. Furthermore, these pathways appear to be regulated by the prostacyclin receptor independently of one another. We conclude that prevention of de-differentiation and proliferation through a paracrine positive feedback mechanism is a major cardioprotective function of prostacyclin.

BRCA1 splice variants exhibit overlapping and distinct transcriptional transactivation activities.

The global changes in gene expression induced by transient increased expression of full length BRCA1 as well as the spliced variant BRCA1S were evaluated by cDNA expression array in a human non‐tumorigenic mammary epithelial cell line, MCF10A. Over 30 genes were identified that displayed an altered expression pattern in response to the expression of BRCA1 splice variants. The expression of NFκB inducing kinase was markedly down‐regulated in BRCA1L transfected cells. However, a NFκB‐responsive promoter construct yielded increased basal activity in BRCA1L transfected cells, as well as following treatment with tumor necrosis factor‐α or lymphotoxin. In addition, nuclear extracts from BRCA1L transfected cells displayed increased DNA binding to the κB consensus site. The transcriptional activity of a panel of promoter constructs was evaluated following expression of wild type or mutant BRCA1. Full length BRCA1 transactivated the estrogen receptor‐α (ERα) and BCL2 promoters as well as AP‐1, SRE, and CRE containing promoters. Transactivation activity of the exon 11‐deleted BRCA1S was more limited and usually of lower magnitude. The ability of a pathogenic mutation, 5382insC, to abrogate the transcriptional transactivation by BRCA1L and BRCA1S was also investigated. Mutant BRCA1 retained wild type levels of transcriptional activity for the ERα promoter as well as for the NFκB, AP‐1, and CRE‐responsive promoters but had reduced or no activity with the BCL2 and SRE promoters. These results show that BRCA1 isoforms have both overlapping and distinct transcriptional transactivation activity, and that a mutant form of BRCA1 implicated in carcinogenesis is not devoid of all activity. J. Cell. Biochem. 89: 120–132, 2003.

Human Thromboxane A2 Receptor Genetic Variants: In Silico, In Vitro and “In Platelet” Analysis

Thromboxane and its receptor have emerged as key players in modulating vascular thrombotic events. Thus, a dysfunctional hTP genetic variant may protect against (hypoactivity) or promote (hyperactivity) vascular events, based upon its activity on platelets. After extensive in silico analysis, six hTP-α variants were selected (C68S, V80E, E94V, A160T, V176E, and V217I) for detailed biochemical studies based on structural proximity to key regions involved in receptor function and in silico predictions. Variant biochemical profiles ranged from severe instability (C68S) to normal (V217I), with most variants demonstrating functional alteration in binding, expression or activation (V80E, E94V, A160T, and V176E). In the absence of patient platelet samples, we developed and validated a novel megakaryocyte based system to evaluate human platelet function in the presence of detected dysfunctional genetic variants. Interestingly, variant V80E exhibited reduced platelet activation whereas A160T demonstrated platelet hyperactivity. This report provides the most comprehensive in silico, in vitro and “in platelet” evaluation of hTP variants to date and highlightscurrent inherent problems in evaluating genetic variants, with possible solutions. The study additionally provides clinical relevance to characterized dysfunctional hTP variants.