Henrik Laurell

Chronic Estradiol Administration In Vivo Promotes the Proinflammatory Response of Macrophages to TLR4 Activation: Involvement of the Phosphatidylinositol 3-Kinase Pathway

Short-term exposure to 17β-estradiol (E2) in vitro has been reported to decrease the production of proinflammatory cytokines by LPS-activated macrophages through estrogen receptor α (ERα)-dependent activation of the PI3K pathway. In the present study, we confirm that in vitro exposure of mouse peritoneal macrophages to E2 enhanced Akt phosphorylation and slightly decreased LPS-induced cytokine production. In striking contrast, we show that chronic administration of E2 to ovariectomized mice markedly increases the expression of IL-1β, IL-6, IL-12p40, and inducible NO synthase by resident peritoneal macrophages in response to LPS ex vivo. These results clearly indicate that short-term E2 treatment in vitro does not predict the long-term effect of estrogens in vivo on peritoneal macrophage functions. We show that this in vivo proinflammatory effect of E2 was mediated through ERα. Although the expression of components of the LPS-recognition complex remained unchanged, we provided evidences for alterations of the TLR4 signaling pathway in macrophages from E2-treated mice. Indeed, E2 treatment resulted in the inhibition of PI3K activity and Akt phosphorylation in LPS-activated macrophages, whereas NF-κB p65 transcriptional activity was concomitantly increased. Incubation of macrophages with the PI3K inhibitor wortmanin enhanced proinflammatory cytokine gene expression in response to TLR4 activation, and abolishes the difference between cells from placebo- or E2-treated mice, demonstrating the pivotal role of the PI3K/Akt pathway. We conclude that the macrophage activation status is enhanced in vivo by E2 through ERα and, at least in part, by the down-modulation of the PI3K/Akt pathway, thereby alleviating this negative regulator of TLR4-signaling.

The transactivating function 1 of estrogen receptor α is dispensable for the vasculoprotective actions of 17β-estradiol

Full-length 66-kDa estrogen receptor α (ERα) stimulates target gene transcription through two activation functions (AFs), AF-1 in the N-terminal domain and AF-2 in the ligand binding domain. Another physiologically expressed 46-kDa ERα isoform lacks the N-terminal A/B domains and is consequently devoid of AF-1. Previous studies in cultured endothelial cells showed that the N-terminal A/B domain might not be required for estradiol (E2)-elicited NO production. To evaluate the involvement of ERα AF-1 in the vasculoprotective actions of E2, we generated a targeted deletion of the ERα A/B domain in the mouse. In these ERαAF-10 mice, both basal endothelial NO production and reendothelialization process were increased by E2 administration to a similar extent than in control mice. Furthermore, exogenous E2 similarly decreased fatty streak deposits at the aortic root from both ovariectomized 18-week-old ERαAF-1+/+ LDLr−/− (low-density lipoprotein receptor) and ERαAF-10 LDLr −/− mice fed with a hypercholesterolemic diet. In addition, quantification of lesion size on en face preparations of the aortic tree of 8-month-old ovariectomized or intact female mice revealed that ERα AF-1 is dispensable for the atheroprotective action of endogenous estrogens. We conclude that ERα AF-1 is not required for three major vasculoprotective actions of E2, whereas it is necessary for the effects of E2 on its reproductive targets. Thus, selective ER modulators stimulating ERα with minimal activation of ERα AF-1 could retain beneficial vascular actions, while minimizing the sexual effects.

Hormone-sensitive Lipase Is Structurally Related to Acetylcholinesterase, Bile Salt-stimulated Lipase, and Several Fungal Lipases BUILDING OF A THREE-DIMENSIONAL MODEL FOR THE CATALYTIC DOMAIN OF HORMONE-SENSITIVE LIPASE

Hormone-sensitive lipase is the key enzyme in the mobilization of fatty acids from adipose tissue, thereby playing a crucial role in the overall energy homeostasis in mammals. Its activity is stimulated by catecholamines through cAMP-dependent phosphorylation of a single serine, a process that is prevented by insulin. This regulatory property is unique to this enzyme among all known lipases and has been acquired during evolution through insertion of a regulatory module into an ancestral lipase. Sequence alignments have failed to detect significant homology between hormone-sensitive lipase and the rest of the mammalian lipases and esterases, to which this enzyme is only very distantly related. In the present work, we report the finding of a remarkable secondary structure homology between hormone-sensitive lipase and the enzymes from a superfamily of esterases and lipases that includes acetylcholinesterase, bile salt-stimulated lipase, and several fungal lipases. This finding, based on the identification of the secondary structure elements in the hormone-sensitive lipase sequence, has allowed us to construct a three-dimensional model for the catalytic domain of hormone-sensitive lipase. The model reveals the topological organization, predicts the components of the catalytic triad, suggests a three-dimensional localization of the regulatory module, and provides a valuable tool for the future study of structural and functional aspects of this metabolically important enzyme.

Endothelial Estrogen Receptor-α Plays a Crucial Role in the Atheroprotective Action of 17β-Estradiol in Low-Density Lipoprotein Receptor–Deficient Mice

Background— The prevention of early atheroma by estrogens has been clearly demonstrated in all animal models and appears to be mediated through a direct action on the arterial wall rather than through an effect on the lipoprotein profile. The goal of the present study was to evaluate which cellular target is crucial in this beneficial action of estradiol. Methods and Results— We first confirmed the key role of estrogen receptor-&agr; (ER&agr;) in the atheroprotective effect of estradiol, because this action was completely abolished in mice deficient in both the low-density lipoprotein receptor (LDLr) and ER&agr;. Second, using chimeric mice with an ER&agr; deficiency in the hematopoietic lineage, we showed the persistence of the protective action of estradiol, which suggests the involvement of extrahematopoietic ER&agr;. Third, we showed that loxP-flanked ER&agr; mice (ER&agr;flox/flox) bred with Tie2-Cre+ mice on an LDLr−/− background had complete inactivation of ER&agr; in most hematopoietic and all endothelial cells. Remarkably, in this mouse model, the atheroprotective effect of estradiol was completely abolished. Fourth, the atheroprotective effect of estradiol remained abolished in Tie2-Cre+ ER&agr;flox/flox LDLr−/− mice transplanted with either Tie2-Cre+ ER&agr;flox/flox or ER&agr;−/− bone marrow, whereas it was present in analogous chimeric Tie2-Cre− ER&agr;flox/flox LDLr−/− receivers expressing endothelial ER&agr;. Conclusions— We demonstrate directly and for the first time that endothelial ER&agr; represents a key target of the atheroprotective effect of estradiol, whereas hematopoietic ER&agr; is dispensable. Selective estrogen receptor modulators that mimic the endothelial action of estradiol should now be considered in atheroprotection.

Estrogens in vascular biology and disease: where do we stand today?

Purpose of review Whereas hormone therapy may increase the risk of coronary heart disease and stroke in menopausal women, epidemiological studies (protection in premenopausal women) suggest and experimental studies (prevention of fatty streak development in animals) demonstrate a major atheroprotective action of estradiol. There is also evidence for a thrombogenic effect of oral estrogens. An understanding of the deleterious and beneficial effects of estrogens is thus required. Recent findings The immuno-inflammatory system plays a key role in the development of fatty streak deposit as well as in the rupture of the atherosclerotic plaque. Whereas estradiol favors an anti-inflammatory effect in vitro (cultured cells), it rather elicits a pro-inflammatory response in vivo involving several subpopulations of the immuno-inflammatory system, which could contribute to plaque destabilization. Endothelium is another important target for estrogens, since estradiol potentiates endothelial nitric oxide and prostacyclin production. The respective actions of estrogens on these cell populations may be influenced by the timing of hormonal therapy initiation, hormone regimens, status of the vessel wall and expression of isoforms of estrogen receptors α and β. Summary A better understanding of the balance between the deleterious and beneficial effects of estrogens is required and should help to improve hormonal therapy safety and to optimize the prevention of cardiovascular disease after menopause.

Translokin is an intracellular mediator of FGF-2 trafficking

Basic fibroblast growth factor (bFGF or FGF-2) exerts its pleiotropic activities both as an exogenous and an intracellular factor. FGF-1 and FGF-2 are prototypes for this dual signalling, but the mechanisms of their intracellular actions remain unknown. Here we show that Translokin, a cytoplasmic protein of relative molecular mass 55,000 (Mr 55K), interacts specifically with the 18K form of FGF-2. Translokin is ubiquitously expressed and colocalizes with the microtubular network. As Translokin does not interact with FGF-1, we used a strategy based on FGF-1–FGF-2 chimaeras to map the interacting regions in FGF-2 and to generate Nb1a2, a non-interacting variant of FGF-2. Although most of the FGF-2 properties are preserved in Nb1a2, this variant is defective in intracellular translocation and in stimulating proliferation. The fusion of a nuclear localization signal to Nb1a2 restores its mitogenic activity and its nuclear association. Inhibiting Translokin expression by RNA interference reduces the translocation of FGF-2 without affecting the intracellular trafficking of FGF-1. Our data show that the nuclear association of internalized FGF-2 is essential for its mitogenic activity and that Translokin is important in this translocation pathway.

Human Hormone-Sensitive Lipase: Genetic Mapping, Identification of a New Dinucleotide Repeat, and Association With Obesity and NIDDM

NIDDM and obesity are complex metabolic disorders with a significant genetic component (1). The two conditions are frequently associated and share many metabolic abnormalities suggesting that they may also share some susceptibility genes. However, their multifactorial nature makes it difficult to dissect the genetic determinants. Despite numerous studies, only a few candidate genes accounting for a minor part of the genetic background have been implicated, usually in particular populations. Hormone-sensitive lipase (HSL), which plays a critical role in the control of energy homeostasis by catalyzing the hydrolysis of adipose tissue triglycerides into free fatty acids, appears as a good candidate gene. Indeed, this enzyme may be involved in the loss of insulin sensitivity since there is increasing evidence that free fatty acids are involved in the development of insulin resistance (2). The adipose tissue isoform of HSL is encoded by a single gene composed of nine exons (3). The gene locus symbolized LIPE has been assigned to chromosome 19ql3.1-13.2 (4). However, no genetic mapping studies have been carried out for LIPE to date. This report describes the characterization of a novel polymorphism in the HSL gene coupled with its genetic mapping. Moreover, we report association studies of this novel microsatellite with NIDDM and obesity within a Caucasian population. First, we characterized a (CA)21 repeat, named HSIi6[CA]n, found within HSL intron six after sequencing of genomic clone hHSLrllll (3). Polymorphism of this repeat was

Antiangiogenic Properties of Fibstatin, an Extracellular FGF-2–Binding Polypeptide

By using the two-hybrid system with basic fibroblast growth factor (FGF-2) as bait, we isolated and characterized fibstatin, an endogenous Mr 29,000 human basement membrane-derived inhibitor of angiogenesis and tumor growth. Fibstatin, a fragment containing the type III domains 12–14 of fibronectin, was produced as a recombinant protein and was shown to inhibit the proliferation, migration, and differentiation of endothelial cells in vitro. Antiangiogenic activity of fibstatin was confirmed in a Matrigel angiogenesis assay in vivo, and electrotransfer of the fibstatin gene into muscle tissue resulted in reduced B16F10 tumor growth. Taken together, these results suggest that fibstatin could act as a powerful molecule for antiangiogenic therapy.

Understanding the oestrogen action in experimental and clinical atherosclerosis

Whereas hormone replacement/menopause therapy (HRT) in postmenopausal women increases the coronary artery risk, epidemiological studies (protection in premenopaused women) suggest and experimental studies (prevention of the development of fatty streaks in animals) demonstrate a major atheroprotective action of oestradiol (E2). The understanding of the deleterious and beneficial effects of oestrogens is thus required. The immuno‐inflammatory system plays a key role in the development of fatty streak deposit as well as in the rupture of the atherosclerotic plaque. Whereas E2 favours an anti‐inflammatory effect in vitro (cultured cells), it rather elicits in vivo a proinflammation at the level of several subpopulations of the immuno‐inflammatory system, which could contribute to plaque destabilization. Endothelium is another important target for E2, as it potentiates endothelial NO and prostacyclin production, thus promoting the beneficial effects as vasorelaxation and inhibition of platelet aggregation. Prostacyclin, but not NO, appears to be involved in the atheroprotective effect of E2. E2 also accelerates endothelial regrowth, thus favouring vascular healing. Finally, most of these effects of E2 are mediated by oestrogen receptor α, and are independent of oestrogen receptor β. In summary, a better understanding of the mechanisms of oestrogen action not only on the normal and atheromatous arteries, but also on innate and adaptive immune responses is required and should help to optimize the prevention of cardiovascular disease after menopause. These mouse models should help to screen existing and future selective oestrogen receptor modulators.

Identification of an upstream promoter of the human somatostatin receptor, hSSTR2, which is controlled by epigenetic modifications.

Somatostatin is a neuropeptide that inhibits exocrine and endocrine secretions of several hormones and negatively regulates cell proliferation. These events are mediated through somatostatin engagement on one of five G protein-coupled receptors named SSTR1 to STTR5. Somatostatin binding to SSTR2 mediates predominantly antisecretory and antiproliferative effects; two important biological activities in the gastroenteropancreatic endocrine and exocrine system. Herein we demonstrate novel regulatory sequences for human (h) SSTR2 transcription. By genomic DNA sequence analysis, we reveal two CpG islands located 3.8 kb upstream from the transcription start site. We identify a novel transcription start site and a promoter region within one of these CpG islands. We demonstrate that two epigenetic modifications, DNA methylation and histone acetylation, regulate the activation of hSSTR2 upstream promoter. Furthermore, we show that the transcription from this upstream promoter region directly correlates to hSSTR2 mRNA expression in various human cell lines. A combined treatment of a demethylating agent, 5-aza-2-deoxycytidine and a histone deacetylase inhibitor, trichostatin A, leads to increased expression of hSSTR2 mRNA in cell lines in which the CpG island is methylated. The epigenetic regulation of this promoter region results in differential expression of hSSTR2 mRNA in human cell lines. This study reveals the existence of a novel upstream promoter for the hSSTR2 gene that is regulated by epigenetic modifications, suggesting for complex control of the hSSTR2 transcription.