The Transcriptional Effects of PCB118 and PCB153 on the Liver, Adipose Tissue, Muscle and Colon of Mice: Highlighting of Glut4 and Lipin1 as Main Target Genes for PCB Induced Metabolic Disorders.

Epidemiological studies have associated environmental exposure to polychlorinated biphenyls (PCBs) with an increased risk of type 2 diabetes; however, little is known about the underlying mechanisms involved in the metabolic side-effects of PCB. Our study evaluated the transcriptional effects of a subchronic exposure (gavage at Day 0 and Day 15 with 10 or 100 µmol/Kg bw) to PCB118 (dioxin-like PCB), PCB153 (non-dioxin-like PCB), or an equimolar mixture of PCB118 and PCB153 on various tissues (liver, visceral adipose tissue, muscle, and colon) in mice. Our results showed that a short-term exposure to PCB118 and/or PCB153 enhanced circulating triglyceride levels but did not affect glycemia. Among the studied tissues, we did not observe any modification of the expression of inflammation-related genes, such as cytokines or chemokines. The main transcriptional effects were observed in visceral adipose and liver tissues. We found a downregulation of lipin1 and glut4 expression in these two target organs. In adipose tissue, we also showed a downregulation of Agpat2, Slc25a1, and Fasn. All of these genes are involved in lipid metabolism and insulin resistance. In muscles, we observed an induction of CnR1 and Foxo3 expression, which may be partly involved in PCB metabolic effects. In summary, our results suggest that lipin1 and glut4, notably in adipose tissue, are the main targeted genes in PCB-induced metabolic disorders, however, further studies are required to fully elucidate the mechanisms involved.

[Endocrine disruptors and metabolic diseases: a major public health challenge]. / Perturbateurs endocriniens et maladies métaboliques: un défi majeur en santépublique.

The purpose of this research note is to summarize the current state of knowledge on the impact of environmental exposures on the development of obesity and diabetes. In France, the incidence of diabetes almost doubled between 2000 and 2008 (+ 93%), affecting 1.8 million people. The prevalence of obesity has almost doubled in 10 years. Since the 1980s, the focus of the fight against obesity and diabetes has been on hygienic and dietary measures. However, it is now clear that these measures have failed to reverse the trend. Chronic diseases such as obesity and diabetes have resulted in a dramatic increase in public health spending. A growing body of data has highlighted the important role of chemical pollution in the epidemic of metabolic diseases, particularly substances classified as endocrine disrupting chemicals (EDCs). By altering the endocrine function of organs such as adipose tissue, liver, pancreas, and skeletal muscle, EDCs disrupt the metabolism of carbohydrates and lipids, leading in turn to insulin resistance and diabetes and obesity, which both increase the risk of cardiovascular complications. The traditional toxicological principle that "the dose makes the poison" does not reflect the complexity of the deleterious effect of EDCs either alone or in combination. EDC-induced patho-physiological alterations can be programmed during the fetal period, before appearing later in life. A new paradigm evaluating the toxicological properties of EDCs is needed to support new recommendations in terms of prevention policy, at both national and European levels.

Microparticles from human atherosclerotic plaques promote endothelial ICAM-1-dependent monocyte adhesion and transendothelial migration.

RATIONALE AND OBJECTIVE: Membrane-shed submicron microparticles (MPs) released following cell activation or apoptosis accumulate in atherosclerotic plaques, where they stimulate endothelial proliferation and neovessel formation. The aim of the study was to assess whether or not MPs isolated from human atherosclerotic plaques contribute to increased endothelial adhesion molecules expression and monocyte recruitment. METHOD AND RESULTS: Human umbilical vein and coronary artery endothelial cells were exposed to MPs isolated from endarterectomy specimens (n=62) and characterized by externalized phosphatidylserine. Endothelial exposure to plaque, but not circulating, MPs increased ICAM-1 levels in a concentration-dependant manner (3.4-fold increase) without affecting ICAM-1 mRNA levels. Plaque MPs harbored ICAM-1 and transferred this adhesion molecule to endothelial cell membrane in a phosphatidylserine-dependent manner. MP-borne ICAM-1 was functionally integrated into cell membrane as demonstrated by the increased ERK1/2 phosphorylation following ICAM-1 ligation. Plaque MPs stimulated endothelial monocyte adhesion both in culture and in isolated perfused mouse carotid. This effect was also observed under flow condition and was prevented by anti-LFA-1 and anti-ICAM-1 neutralizing antibodies. MPs isolated from symptomatic plaques were more potent in stimulating monocyte adhesion than MPs from asymptomatic patients. Plaque MPs did not affect the release of interleukin-6, interleukin-8, or MCP-1, nor the expression of VCAM-1 and E-selectin. CONCLUSION: These results demonstrate that MPs isolated from human atherosclerotic plaques transfer ICAM-1 to endothelial cells to recruit inflammatory cells and suggest that plaque MPs promote atherosclerotic plaque progression.

[The two sides of ADAM17 in inflammation: implications in atherosclerosis and obesity]. / Les deux visages d'ADAM17 dans l'inflammation: implications dans l'athérosclérose et l'obésité

ADAM17 was initially characterized as the TNF Alpha Converting Enzyme (TACE) and, until now, has been the most studied member of the ADAM family. It is a type I transmembrane metalloproteinase involved in the shedding of the extracellular domain of several transmembrane proteins (at least 40) such as cytokines, growth factors, receptors or adhesion molecules. As a consequence, depending on the transmembrane molecule cleaved, one may expect possible opposite effects of ADAM17 activity on inflammation (e.g. TNF and its receptors). The role of ADAM17 in regulating inflammatory cellular processes is clearly demonstrated in cells deficient in active ADAM17 or expressing substrates mutated for the ADAM17 cleavage site. As ADAM17-deficient mice died at birth, mice overexpressing the mutated uncleavable form of some substrates and recently conditional knock-out of ADAM17 are used to approach in vivo the role of this metalloprotease in regulating inflammation. Arguments are provided that ADAM17 plays a role in atherosclerosis, in adipose tissue metabolism, insulin resistance and diabetes. The multitude of substrates cleaved by ADAM17 makes this enzyme an attractive candidate to study its role in inflammation-driven pathologies.

HDLs activate ADAM17-dependent shedding.

The tumor necrosis factor-alpha (TNF) converting enzyme (ADAM17) is a metalloprotease that cleaves several transmembrane proteins, including TNF and its receptors (TNFR1 and TNFR2). We recently showed that the shedding activity of ADAM17 is sequestered in lipid rafts and that cholesterol depletion increased the shedding of ADAM17 substrates. These data suggested that ADAM17 activity could be regulated by cholesterol movements in the cell membrane. We investigated if the membrane cholesterol efflux induced by high-density lipoproteins (HDLs) was able to modify the shedding of ADAM17 substrates. HDLs added to different cell types, increased the ectodomain shedding of TNFR2, TNFR1, and TNF, an effect reduced by inhibitors active on ADAM17. The HDLs-stimulated TNF release occurred also on cell-free isolated plasma membranes. Purified apoA1 increased the shedding of TNF in an ABCA1-dependent manner, suggesting a role for the cholesterol efflux in this phenomenon. HDLs reduced the cholesterol and proteins (including ADAM17) content of lipid rafts and triggered the ADAM17-dependent cleavage of TNF in the non-raft region of the membrane. In conclusion, these data demonstrate that HDLs alter the lipid raft structure, which in turn activates the ADAM17-dependent processing of transmembrane substrates.

Microparticles of human atherosclerotic plaques enhance the shedding of the tumor necrosis factor-alpha converting enzyme/ADAM17 substrates, tumor necrosis factor and tumor necrosis factor receptor-1.

Human atherosclerotic plaques express the metalloprotease tumor necrosis factor (TNF)-alpha converting enzyme (TACE/ADAM-17), which cleaves several transmembrane proteins including TNF and its receptors (TNFR-1 and TNFR-2). Plaques also harbor submicron vesicles (microparticles, MPs) released from plasma membranes after cell activation or apoptosis. We sought to examine whether TACE/ADAM17 is present on human plaque MPs and whether these MPs would affect TNF and TNFR-1 cellular shedding. Flow cytometry analysis detected 12,867 +/- 2007 TACE/ADAM17(+) MPs/mg of plaques isolated from 25 patients undergoing endarterectomy but none in healthy human internal mammary arteries. Plaque MPs harbored mainly mature active TACE/ADAM17 and dose dependently cleaved a pro-TNF mimetic peptide, whereas a preferential TACE/ADAM17 inhibitor (TMI-2) and recombinant TIMP-3 prevented this cleavage. Plaque MPs increased TNF shedding from the human cell line ECV-304 overexpressing TNF (ECV-304(TNF)), as well as TNFR-1 shedding from activated human umbilical vein endothelial cells or ECV-304(TNF) cells, without affecting TNF or TNFR-1 synthesis. MPs also activated the shedding of the endothelial protein C receptor from human umbilical vein endothelial cells. All these effects were inhibited by TMI-2. The present study shows that human plaque MPs carry catalytically active TACE/ADAM17 and significantly enhance the cell surface processing of the TACE/ADAM17 substrates TNF, TNFR-1, and endothelial protein C receptor, suggesting that TACE/ADAM17(+) MPs could regulate the inflammatory balance in the culprit lesion.

MRI follow-up of TNF-dependent differential progression of atherosclerotic wall-thickening in mouse aortic arch from early to advanced stages.

OBJECTIVES: An optimized, longitudinal in vivo magnetic resonance vessel wall-imaging protocol was evaluated regarding its capability of detecting differences in the time-dependent atherosclerotic lesion progression in the aortic arch between ApoE(-/-) and double-deficient ApoE(-/-)/TNF(-/-) mice at comparatively early plaque development stages. MATERIALS AND METHODS: Seven ApoE(-/-) and seven ApoE(-/-)/TNF(-/-) female mice underwent MRI at 11.75 teslas at four stages up to 26 weeks of age. A double-gated spin-echo MRI sequence was used with careful perpendicular slice positioning to visualize the vessel wall of the ascending aortic arch. RESULTS: Wall-thickness progression measured with MRI was significant at 11 weeks of age in ApoE(-/-) mice, but only at 26 weeks in ApoE(-/-)/TNF(-/-) mice. A significant correlation was found between MRI wall-thickness and lesion area determined on histology. CONCLUSION: MRI was shown to be sensitive enough to reveal subtle genetically-induced differences in lesion progression at ages earlier than 25 weeks.

The shedding activity of ADAM17 is sequestered in lipid rafts.

The tumor necrosis factor-alpha (TNF) converting enzyme (ADAM17) is a metalloprotease-disintegrin responsible for the cleavage of several biologically active transmembrane proteins. However, the substrate specificity of ADAM17 and the regulation of its shedding activity are still poorly understood. Here, we report that during its transport through the Golgi apparatus, ADAM17 is included in cholesterol-rich membrane microdomains (lipid rafts) where its prodomain is cleaved by furin. Consequently, ADAM17 shedding activity is sequestered in lipid rafts, which is confirmed by the fact that metalloproteinase inhibition increases the proportion of ADAM17 substrates (TNF and its receptors TNFR1 and TNFR2) in lipid rafts. Membrane cholesterol depletion increases the ADAM17-dependent shedding of these substrates demonstrating the importance of lipid rafts in the control of this process. Furthermore, ADAM17 substrates are present in different proportions in lipid rafts, suggesting that the entry of each of these substrates in these particular membrane microdomains is specifically regulated. Our data support the idea that one of the mechanisms regulating ADAM17 substrate cleavage involves protein partitioning in lipid rafts.

FHL2 interacts with both ADAM-17 and the cytoskeleton and regulates ADAM-17 localization and activity.

ADAM-17 is a metalloprotease-disintegrin responsible for the ectodomain shedding of several transmembrane proteins. Using the yeast two-hybrid system, we showed that ADAM-17 interacts with the Four and Half LIM domain 2 protein (FHL2), a LIM domain protein that is involved in multiple protein-protein interaction. We demonstrated that this interaction involved the amino-acid sequence of ADAM-17 from position 721 to739. In the cardiomyoblast cells H9C2, ADAM-17 and FHL2 colocalize with the actin-based cytoskeleton and we showed that FHL2 binds both ADAM-17 and the actin-based cytoskeleton. We found that mainly the mature form of ADAM-17 associates with the cytoskeleton, although the maturation of ADAM-17 by furin is not necessary for its binding to the cytoskeleton. Interestingly, less ADAM-17 was detected at the surface of wild-type mouse macrophages compared to FHL2 deficient macrophages. However, wild-type cells have a higher ability to release ADAM-17 substrates under PMA stimulation. Altogether, these results demonstrate a physical and functional interaction between ADAM-17 and FHL2 that implies that FHL2 has a role in the regulation of ADAM-17.

The TNF alpha converting enzyme (TACE/ADAM17) is expressed in the atherosclerotic lesions of apolipoprotein E-deficient mice: possible contribution to elevated plasma levels of soluble TNF alpha receptors.

TNF alpha converting enzyme (TACE) critically regulates the inflammatory processes as it releases from the cell surface several transmembrane proteins, including TNFalpha (TNF) and its receptors TNFR1 and TNFR2. We investigated the expression of TACE in atherosclerotic lesions of apolipoproteinE-deficient (apoE (-/-)) mice. Five-week-old apoE(-/-) male mice were fed a high-fat diet and examined at 5, 10, 15 and 25 weeks of age. A group of wild-type C57BL/6 mice (WT) fed the high-fat diet for 25 weeks was included. In apoE(-/-) mice, lesions progressed with time in both aortic sinus and arch, in which TACE immunostaining also increased particularly between 5 and 15 weeks. TACE expression was also observed in human atherosclerotic plaques. The plasma levels of soluble TNFR1 and TNFR2 rose with atherosclerosis. In the 25-week-old WT mice, no lesions were observed and the plasma levels of TNFRs were 17% of those of age-matched apoE(-/-) mice. Incubated aortas of 25-week-old apoE(-/-) mice released much higher amounts of sTNF and sTNFRs than did aortas of 5-week-old apoE(-/-) mice or 25-week-old WT mice. Active TACE was expressed at the surface of macrophages isolated from apoE(-/-) mice. In conclusion, TACE expression is associated with lesions in atherosclerosis-prone sites. Our data suggest that atherosclerotic lesions-expressing TACE may contribute to the elevated levels of circulating sTNFRs.

Proteasome inhibition activates the transport and the ectodomain shedding of TNF-alpha receptors in human endothelial cells.

Binding of tumor necrosis factor-alpha (TNF-alpha) to its transmembrane receptors (TNFRs) mediates proinflammatory, apoptotic and survival responses in several cell types including vascular endothelial cells. Because ectodomain shedding of cell surface molecules can be modified by proteasome activity, we studied in human endothelial cells whether the TNF-alpha-TNFRs axis can be regulated by the cleavage of their transmembrane forms in a proteasome-dependent manner. We show that proteasome inhibition increases the release of TNF-alpha and TNFRs from human endothelial cells and decreases their cellular and cell surface expression. This phenomenon involves the transient activation of mitogen-activated protein kinase p42/p44 that triggers the dispersion of TNF-alpha and TNFRs from their intracellular Golgi-complex-associated pool towards the plasma membrane. This results in their enhanced cleavage by TNF-alpha converting enzyme (TACE) because it is reduced by synthetic metalloprotease inhibitors, recombinant TIMP-3 and by a dominant negative form of TACE. In the presence of TACE inhibitor, proteasome inhibition increases the cell surface expression of TNFRs and enhances the sensitivity of these cells to the proapoptotic effect of recombinant TNF-alpha. In conclusion, our data provide evidence that proteasome inhibitors increase TACE-dependent TNFR-shedding in endothelial cells, supporting the use of these molecules in inflammatory disorders. In association with TACE inhibitor, proteasome inhibitors increase the amount of TNFRs at the cell surface and enhance the sensitivity to the proapoptotic effect of TNF-alpha, which might be of interest in the antitumor therapy.

Upregulation of TNF-alpha-induced ICAM-1 surface expression by adenylate cyclase-dependent pathway in human endothelial cells.

The level of adhesion molecules expressed at the endothelial cell surface is critical in the control of inflammation. Adenylate cyclase (AC) activity allowing cyclic adenosine monophosphate (cAMP) production can modulate the inflammatory process. We investigated the AC-dependent modulation of ICAM-1 surface expression in human umbilical venous endothelial cells (HUVEC). Pretreatment of HUVEC with forskolin significantly upregulated tumor necrosis factor alpha (TNF-alpha)- and interleukin-1 alpha (IL1-alpha)-induced ICAM-1 surface expression exclusively after a prolonged time of incubation with forskolin (at least 7-8 h). A poorly metabolizable analog of cAMP, dibutyryl-cAMP, mimicked forskolin effect on ICAM-1 surface expression. Protein kinase A (PKA) inhibitor H89 prevented forskolin effect on ICAM-1 surface expression. Upregulation of ICAM-1 surface level occurred through the increase in its mRNA levels and also to a subsequent activation of ICAM-1 intracellular trafficking towards cell surface. Stimulation by agonists of beta-adrenergic receptors did not alter the TNF-alpha-induced ICAM-1 surface expression. Pretreatment of HUVEC with pertussis toxin, which is known to activate AC through Gialpha inhibition, upregulated mRNA levels and surface expression of ICAM-1 induced by TNF-alpha. This effect was serum-dependent, since ICAM-1 expression was no more upregulated by pertussis toxin in cells cultured in 1% instead of 20% serum-enriched medium. However, forskolin treatment of HUVEC did not modify their overall adhesive properties. In conclusion, a persistent cAMP level elevation activating PKA is able to enhance ICAM-1 expression at the cell surface of endothelial cells placed under pro-inflammatory conditions. Combination of activation of gene transcription and membrane targeting may account for this augmentation.

Proinflammatory properties of murine aortic endothelial cells exclusively expressing a non cleavable form of TNFalpha. Effect on tumor necrosis factor alpha receptor type 2.

Soluble (sTNF) and transmembrane (tmTNF) forms of TNFalpha (TNF) have distinct proinflammatory effects. We investigated whether tmTNF altered the synthesis of some proinflammatory proteins involved in atherothrombosis, in murine aortas and aortic endothelial cells (MAEC). Samples were obtained from wild-type (WT) mice and TNF-deficient mice that express a mutated non cleavable tmTNF transgene (tmTNFnc). The levels of secreted MCP-1, RANTES, IL-6, PAI-1, soluble ICAM-1, and soluble TNF receptor type 1 (TNFR1; CD120a) antigens, MMP-9 activity and of cell surface ICAM-1 were not significantly different between the two types of MAEC. The magnitude of endotoxin-stimulated production of RANTES, MCP-1 and IL-6 was similar in the two types of cells. Of note, the amount of synthesized TNF receptor type 2 (TNFR2; CD120b), measured by its secreted (in aorta and MAEC), intracellular and mRNA levels (in MAEC), was significantly 4-fold lower in tmTNFnc than in WT mice, both in basal and endotoxin-stimulated conditions. A neutralizing anti-TNF antibody or the recombinant murine TNF did not modify the magnitude of the difference in TNFR2 production between the two types of cells, suggesting a preponderant role of tmTNF in the down-regulation of TNFR2 synthesis. Macrophages of tmTNFnc mice also produced less TNFR2 than WT macrophages (-30%). Plasmas of tmTNFnc mice contained significantly less sTNFR2 than WT mice (-75%). In conclusion, an increase in tmTNF levels, rather than the lack of sTNF, significantly down-modulated TNFR2 synthesis in aortic endothelial cells, but had no major influence on the synthesis of some major pro-inflammatory and pro-atherothrombotic proteins.

Exclusive expression of transmembrane TNF-alpha in mice reduces the inflammatory response in early lipid lesions of aortic sinus.

We investigated the effect of transmembrane form of tumor necrosis factor-alpha (TNF) on atherosclerosis in mice. We compared the development of early atherosclerotic lesions in the aortic sinus of (1) TNF-deficient mice that express only a non-cleavable transmembrane form of TNF (tmTNF), (2) wild-type (WT) C57BL/6 mice, and (3) TNF-deficient mice (TNF(-/-)). All mice were fed an atherogenic diet for 20 weeks. Lipid deposition was the most prominent in WT mice (25030 +/- 5693 microm2), tended to be lower in tmTNF mice (13640+/- 2190 microm2, P > 0.05 versus WT mice) and rare in TNF(-/-) mice (1408 +/- 513 microm2, P < 0.05 versus tmTNF and P < 0.01 versus WT). Macrophage accumulation was five-fold lower (P < 0.05) in tmTNF than in WT mice. In addition, the alpha-actin immuno-reactivity of medial smooth muscle cells remained intact in tmTNF mice but not in WT mice. In WT mice, the plasma lipid profile was significantly more atherogenic than that of TNF(-/-) mice (P < 0.05), but not significantly different from that of tmTNF mice (P > 0.05). These results indicated that in contrast to TNF(-/-) mice, mice expressing exclusively tmTNF were not completely protected from early atherosclerotic lesion formation, although their lesions have a less inflammatory state than those of WT mice, which underlines the stronger proinflammatory potential of soluble TNF.

Modulation of PAI-1 and proMMP-9 syntheses by soluble TNFalpha and its receptors during differentiation of the human monocytic HL-60 cell line.

During phorbol ester-induced differentiation of HL-60 monocytic cells, tumor necrosis factoralpha (TNFalpha) synthesis and secretion are increased, which contributes to the autocrine regulation of TNFalpha-responsive genes. We investigated how, during phorbol ester-induced differentiation of HL-60 cells, the secreted TNFalpha modulated plasminogen activator inhibitor type I (PAI-1) and gelatinase B (MMP-9) syntheses, two proteins involved in pericellular proteolysis. The differentiation-induced release of TNFalpha, was abolished by the hydroxamate-based matrix metalloproteinase (MMP) inhibitor, RU36156. RU36156 or a neutralizing anti-TNFalpha significantly down-regulated PAI-1 synthesis exclusively during the early phases of differentiation (from promyelocyte to monocytic-like cells), which underlined the activating role of autocrine TNFalpha during this time range. As cells progressed to monocyte/macrophage phenotype, they still released TNFalpha, but RU36156 or anti-TNFalpha no longer had an effect on PAI-1 synthesis. This lack of effect was not due to a default of TNFalpha signaling since PAI-1 synthesis was still stimulated in response to exogenous TNFalpha. TNFalpha receptor RI was also actively released and was shown to reduce TNFalpha activity which may account for the inability of soluble TNFalpha to up-regulate PAI-1 synthesis. In later mature stage, cells became susceptible to exogenous TNFalpha-induced apoptosis and rapidly lost their ability to respond to TNFalpha. The MMP-9 synthesis followed similar regulation as PAI-1. Isolated human blood monocytes-derived macrophages behave like HL-60-derived macrophages. In conclusion, these results show that during leukocyte differentiation, time windows exist during which the autocrine TNFalpha is active and then down-regulated by RI, which may temper a continuous up-regulation of the synthesis of proteins involved in pericellular proteolysis.

Intracellular maturation and transport of tumor necrosis factor alpha converting enzyme.

The tumor necrosis factor alpha converting enzyme (TACE) activity is required for the shedding of a variety of biologically active membrane bound precursors. The activation of TACE necessitates the proteolytic cleavage of its prodomain, a process that was suggested to be catalyzed by the proprotein convertase furin. However, the involvement of furin in this activation process has never been experimentally demonstrated. We have shown that the furinlike cleavage site (R-V-K-R(214)) localized between the prodomain and the metalloprotease domain of TACE is the sole site that can be in vitro cleaved by furin. In Cos7 cells, the release of TACE-processed substrates was reduced by the overexpression of the furin-specific proprotein convertase inhibitor Portland alpha1-antitrypsin inhibitor, but the release of TACE-processed substrates was increased by overexpression of furin in LoVo cells (deficient in furin activity) in which a mature form of TACE was identified. The immature form of TACE was detected at the surface of LoVo cells and at the surface of Cos7 and HT29 cells upon proprotein convertase inhibition. These results suggest that furin is the major proprotein convertase involved in the maturation/activation of TACE which is not a prerequisite for its cell-surface expression.

Identification of SAP97 as an intracellular binding partner of TACE.

Tumor necrosis factor alpha converting enzyme (TACE) is the metalloprotease-disintegrin responsible for the ectodomain shedding of several proteins, including tumor necrosis factor alpha. Using the yeast two-hybrid system, we identified the scaffolding protein synapse associated protein 97 (SAP97) as a binding partner of the cytoplasmic domain of TACE. By deletions and site-directed mutagenesis, we demonstrated that this interaction involved the PDZ3 domain of SAP97 and the extreme C-terminal amino-acid sequence of TACE. This interaction as well as the identification of the specific domains involved was confirmed in vitro by affinity purification and in mammalian cells by co-immunoprecipitation and alteration of localization analyzed by immunofluorescence microscopy. In addition, confocal microscopy showed that endogenous TACE and SAP97 colocalized in some intracellular areas of COS-7 cells and CACO-2 cells. Furthermore, overexpression of SAP97, unlike that of a mutant form of SAP97 deleted for its PDZ3 domain, altered the ability of TACE to release its substrates. Altogether, these results demonstrate an interaction between TACE and SAP97, which may have a functional implication for the regulation of TACE shedding activity.

Effect of atorvastatin on adhesive phenotype of human endothelial cells activated by tumor necrosis factor alpha.

We studied the effect of atorvastatin on the adhesive phenotype of human endothelial cells (HUVEC) stimulated by tumor necrosis factor (TNF)-alpha. Surface expression of adhesion molecules on HUVEC was examined by flow cytometry and confocal microscopy, and adhesion of monocytes (human THP-1 cell line) was measured in vitro under flow conditions. In TNF-alpha-activated HUVEC, atorvastatin significantly enhanced surface expression of vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1, E-selectin, and fractalkine, when compared with TNF-alpha stimulation alone. This enhancement was reversed by mevalonate or geranylgeranyl pyrophosphate (GGPP) and was mimicked by an inhibitor of geranylgeranylation. The enhancing effect of atorvastatin was restricted to TNF-alpha-inducible adhesion molecule and was the reflect of an increased protein synthesis (mRNA and protein) and not of a reduced shedding. Confocal microscopy examination showed that atorvastatin also altered the surface distribution of adhesion molecules. Adhesion of human THP-1 cells on TNF-alpha-activated HUVEC was significantly reduced by atorvastatin (-42% at 1 microM). Mevalonate or GGPP restored the TNF-alpha-induced adhesive potential. These results show that atorvastatin, by inhibiting prenylation of G proteins, enhances the TNF-alpha-induced expression of adhesion molecules at the endothelial cell surface and also alters their surface distribution which may account for the reduced binding of monocytes.