Sylvie G. Bernier

Modulation of Pro-inflammatory Gene Expression by Nuclear Lysophosphatidic Acid Receptor Type-1

Lysophosphatidic acid (LPA) is a bioactive molecule involved in inflammation, immunity, wound healing, and neoplasia. Its pleiotropic actions arise presumably by interaction with their cell surface G protein-coupled receptors. Herein, the presence of the specific nuclear lysophosphatidic acid receptor-1 (LPA1R) was revealed in unstimulated porcine cerebral microvascular endothelial cells (pCMVECs), LPA1R stably transfected HTC4 rat hepatoma cells, and rat liver tissue using complementary approaches, including radioligand binding experiments, electron- and cryomicroscopy, cell fractionation, and immunoblotting with three distinct antibodies. Coimmunoprecipitation studies in enriched plasmalemmal fractions of unstimulated pCMVEC showed that LPA1Rs are dually sequestrated in caveolin-1 and clathrin subcompartments, whereas in nuclear fractions LPA1R appeared primarily in caveolae. Immunofluorescent assays using a cell-free isolated nuclear system confirmed LPA1R and caveolin-1 co-localization. In pCMVEC, LPA-stimulated increases in cyclooxygenase-2 and inducible nitric-oxide synthase RNA and protein expression were insensitive to caveolea-disrupting agents but sensitive to LPA-generating phospholipase A2 enzyme and tyrosine kinase inhibitors. Moreover, LPA-induced increases in Ca2+ transients and/or iNOS expression in highly purified rat liver nuclei were prevented by pertussis toxin, phosphoinositide 3-kinase/Akt inhibitor wortmannin and Ca2+ chelator and channel blockers EGTA and SK&F96365, respectively. This study describes for the first time the nucleus as a potential organelle for LPA intracrine signaling in the regulation of pro-inflammatory gene expression.

Regulation of eNOS Expression in Brain Endothelial Cells by Perinuclear EP3 Receptors

We reported upregulation of endothelial nitric oxide synthase (eNOS) by PGE2 in tissues and presence of perinuclear PGE2 receptors (EP). We presently studied mechanisms by which PGE2 induces eNOS expression in cerebral microvessel endothelial cells (ECs). 16,16-Dimethyl PGE2 and selective EP3 receptor agonist M&B28767 increased eNOS expression in ECs and the NO-dependent vasorelaxant responses induced by substance P on cerebral microvessels. These effects could be prevented by prostaglandin transporter blocker bromcresol green and actinomycin D. EP3 immunoreactivity was confirmed on plasma and perinuclear membrane of ECs. M&B28767 increased eNOS RNA expression in EC nuclei, and this effect was augmented by overexpression of EP3 receptors. M&B28767 also induced increased phosphorylation of Erk-1/2 and Akt, as well as changes in membrane potential revealed by the potentiometric fluorescent dye RH421, which were prevented by iberiotoxin; perinuclear KCa channels were detected, and their functionality corroborated by NS1619-induced Ca2+ signals and nuclear membrane potential changes. Moreover, pertussis toxin, Ca2+ chelator, and channel blockers EGTA, BAPTA, and SK&F96365, as well as KCa channel blocker iberiotoxin, protein-kinase inhibitors wortmannin and PD 98059, and NF-&kgr;B inhibitor pyrrolidine dithiocarbamate prevented M&B28767-induced increase in Ca2+ transients and/or eNOS expression in EC nuclei. We describe for the first time that PGE2 through its access into cell by prostaglandin transporters induces eNOS expression by activating perinuclear EP3 receptors coupled to pertussis toxin-sensitive G proteins, a process that depends on nuclear envelope KCa channels, protein kinases, and NF-&kgr;B; the roles for nuclear EP3 receptors seem different from those on plasma membrane.

Proinflammatory Gene Induction by Platelet-Activating Factor Mediated Via Its Cognate Nuclear Receptor

It has been postulated that intracellular binding sites for platelet-activating factor (PAF) contribute to proinflammatory responses to PAF. Isolated nuclei from porcine cerebral microvascular endothelial cells (PCECs) produced PAF-molecular species in response to H2O2. Using FACS analysis, we demonstrated the expression of PAF receptors on cell and nuclear surfaces of PCECs. Confocal microscopy studies performed on PCECs, Chinese hamster ovary cells stably overexpressing PAF receptors, and isolated nuclei from PCECs also showed a robust nuclear distribution of PAF receptors. Presence of PAF receptors at the cell nucleus was further revealed in brain endothelial cells by radioligand binding experiments, immunoblotting, and in situ in brain by immunoelectron microscopy. Stimulation of nuclei with methylcarbamate-PAF evoked a decrease in cAMP production and a pertussis toxin-sensitive rise in nuclear calcium, unlike observations in plasma membrane, which exhibited a pertussis toxin-insensitive elevation in inositol phosphates. Moreover, on isolated nuclei methylcarbamate-PAF evoked the expression of proinflammatory genes inducible nitric oxide synthase and cyclooxygenase-2 (COX-2) and was associated with augmented extracellular signal-regulated kinase 1/2 phosphorylation and NF-κB binding to the DNA consensus sequence. COX-2 expression was prevented by mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 and NF-κB inhibitors. This study describes for the first time the nucleus as a putative organelle capable of generating PAF and expresses its receptor, which upon stimulation induces the expression of the proinflammatory gene COX-2.

Altered Vascular Function in Fetal Programming of Hypertension

Background and Purpose— Reduced endothelium-dependent vasorelaxation partly due to loss of nitric oxide (NO) bioavailability occurs in most cases of chronic hypertension. Intrauterine nutritional deprivation has been associated with increased risk for hypertension and stroke, associated with relaxant dysfunction and decreased vascular compliance, but the underlying mechanisms are not known. The present studies were undertaken to investigate whether endothelial dysfunction associated with altered NO-dependent vasodilatation pathways is also observed in a model of in utero programming of hypertension. Methods— Pregnant Wistar rats were fed a normal (18%), low (9%), or very low (6%) protein isocaloric diet during gestation. Vasomotor response of resistance cerebral microvessels (<50 &mgr;m) was studied in adult offspring of dams fed the 18% and 9% protein diets by a video imaging technique. Endothelial NOS (eNOS), soluble guanylate cyclase (sGC), and KCa channel expression were measured by Western blot. NO synthase (NOS) activity was measured enzymatically as well as in situ by NADPH diaphorase staining. Results— Litter size and survival to adulthood were not affected by the diets. Birth weights of offspring of dams fed the 6% diet were markedly lower than those of dams fed the 9% diet, which were marginally lower than those of controls. Systolic blood pressures of adult offspring of mothers in the 6% and 9% groups were comparably greater (156±2 and 155±1 mm Hg, respectively) than that of control offspring (137±1 mm Hg); we therefore focused on the 9% and 18% groups. Cerebral microvessel constriction to thromboxane A2 mimetic and dilation to carba-prostaglandin I2 did not differ between diet groups. In contrast, vasorelaxation to the NO-dependent agents substance P and acetylcholine was diminished by 50% in low protein-exposed offspring, but eNOS expression and activity were similar between the 2 diet groups. Vasorelaxant response to the NO donor sodium nitroprusside was also decreased and was associated with reduced (by 50% to 65%) cGMP levels and sGC expression. cGMP analogues caused comparable vasorelaxation in the 2 groups. Expression of KCa (another important mediator of NO action) and relaxation to the KCa opener NS1619 were unchanged by antenatal diet. Conclusions— Maternal protein deprivation, which leads to hypertension in the offspring, is associated with diminished NO-dependent relaxation of major organ (cerebral) microvasculature, which seems to be largely attributed to decreased sGC expression and cGMP levels. The study provides an additional explanation for abnormal vasorelaxation in nutrient-deprived subjects in utero.

N- and C-terminal structure-activity study of angiotensin II on the angiotensin AT2 receptor

The predominant angiotensin II receptor expressed in the human myometrium is the angiotensin AT2 receptor. This preparation was used for a structure-activity relationship study on angiotensin II analogues modified in positions 1 and 8. The angiotensin AT2 receptor present on human myometrium membranes displayed a high affinity (pKd = 9.18) and was relatively abundant (53-253 fmol/mg of protein). The pharmacological profile was typical of an angiotensin AT2 receptor with the following order of affinities: (angiotensin III > or = angiotensin II > angiotensin I > PD123319 > angiotensin-(1-7) > angiotensin-(1-6) approximately angiotensin IV >> Losartan). Modifications of the N-terminal side chain and of the primary amine of angiotensin II were evaluated. Neutralisation of the methylcarboxylate (Asp) to a methylcarboxamide (Asn) or to a hydroxymethyl (Ser) or substitution for a methylsulfonate group (cysteic acid) improved the affinity. Extension from methylcarboxylate (Asp) to ethylcarboxylate (Glu) did not affect the affinity. Introduction of larger side chains such as the bulky p-benzoylphenylalanine (p-Bpa) or the positively charged Lys did not substantially affect the affinity. Complete removal of the side chain (angiotensin III), however, resulted in a significant affinity increase. Removal or acetylation of the primary amine of angiotensin II did not noticeably influence the affinity. Progressive alkylation of the primary amine significantly increased the affinity, betain structures being the most potent. It appears that quite important differences exist between the angiotensin AT1 and AT2 receptors concerning their pharmacological profile towards analogues of angiotensin II modified in position 1. On position 8 of angiotensin II, a structure-activity relationship on the angiotensin AT2 receptor was quite similar to that observed with angiotensin AT1 receptor. Bulky, hydrophobic aromatic residues displayed affinities similar to or even better than [Sarcosine1]angiotensin II. Aliphatic residues, especially those of reduced size, caused a significant decrease in affinity especially [Sarcosine1, Gly8]angiotensin II who showed a 30-fold decrease. Introduction of a positive charge (Lys) at position 8 reduced the affinity even further. Stereoisomers in position 8 (L-->D configuration) also induced lower affinities. The angiotensin AT2 receptor display a structure-activity relationship similar to that observed on the AT1 receptor for the C-terminal position of the peptide hormone. Position 1 structure-activity relationships are however fundamentally different between the angiotensin AT1 and AT2 receptor.

Adenosine induces cyclic‐AMP formation and inhibits endothelin‐1 production/secretion in guinea‐pig tracheal epithelial cells through A2B adenosine receptors

The adenosine receptor subtype mediating adenosine 3′ : 5′‐cyclic monophosphate (cyclic AMP) formation and the effect of its activation on endothelin‐1 (ET‐1) secretion were studied in primary cultures of tracheal epithelial cells. Adenosine analogues showed the following rank order of potency (pD2 value) and intrinsic activity on the generation of cyclic AMP by tracheal epithelial cells: 5′‐N‐ethylcarboxyamidoadenosine (NECA, A1/A2A/A2B, pD2: 5.44±0.16)>adenosine (ADO, non selective, pD2: 4.99±0.09; 71±9% of NECA response) 2‐Cl‐adenosine (2CADO, non selective, pD2: 4.72±0.14; 65±9% of NECA response)>>>CGS21680 (A2A; inactive at up to 100 μM). Cyclic AMP formation stimulated by NECA in guinea‐pig tracheal epithelial cells was inhibited by adenosine receptor antagonist with the following order of apparent affinity (pA2 value): Xanthine amine congeners (XAC, A2A/A2B, 7.89±0.22)>CGS15943 (A2A/A2B, 7.24±0.26)>ZM241385 (A2A, 6.69±0.14)>DPCPX (A1, 6.51±0.14)>3n‐propylxanthine (weak A2B, 4.30±0.10). This rank order of potency is typical for A2B‐adenosine receptor. Adenosine decreased basal and LPS‐stimulated irET production in a concentration‐dependent manner. Moreover, NECA but not CGS21680 inhibited LPS‐induced irET production. The inhibitory effect of NECA on LPS‐induced irET production was reversed by XAC (pA2=8.84±0.12) and DPCPX (pA2=8.10±0.22). These results suggested that adenosine increased cyclic AMP formation and inhibited irET production/secretion by guinea‐pig tracheal epithelial cells through the activation of a functional adenosine receptor that is most likely the A2B subtype. This adenosine receptor may be involved in the regulation of the level of ET‐1 production/secretion by guinea‐pig tracheal epithelial cells in physiological as well as in pathophysiological conditions.

Platelet Activating Factor Receptors

Platelet activating factor (PAF) is a potent pro-inflammatory lipid mediator. Its effects are mediated through cell surface G protein-coupled receptors (GPCRs) that are distributed on numerous cells notably on endothelium [1]. An intracrine mode of action for PAF is proposed based on evidence for intracellular PAF binding sites [2] and retention of newly generated PAF within its producing cells [3]. Separate functions for the intracellular and cell surface receptors are suggested using agents which putatively distinguish them [4, 5]; immediate effects are mediated by cell surface receptors whereas regulation of gene expression are dependent upon intracellular receptors consistent with presence of signaling effectors in nuclei including G proteins, Ca++, kinases and NF-κB [6,6,8]; but intracellular PAF receptors; especially on nuclei, which may explain their presumed involvement in gene regulation [5] has never been explicitly demonstrated. We speculated that PAF receptors exist at the cell nucleus where they induce major pro-inflammatory gene cyclooxygenase-2 (COX-2) expression.