Matthias Canault

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.

Human CalDAG-GEFI gene (RASGRP2) mutation affects platelet function and causes severe bleeding.

First case of a human RASGRP2 mutation affecting Rap1 activation in platelets and causing severe bleeding.

Vitamin D protects against diet-induced obesity by enhancing fatty acid oxidation

Prospective studies reported an inverse correlation between 25-hydroxyvitamin D [25(OH)D] plasma levels and prevalence of obesity and type 2 diabetes. In addition, 25(OH)D status may be a determinant of obesity onset. However, the causality between these observations is not yet established. We studied the preventive effect of vitamin D3 (VD3) supplementation (15,000 IU/kg of food for 10 weeks) on onset of obesity in a diet-induced obesity mouse model. We showed that the VD3 supplementation limited weight gain induced by high-fat diet, which paralleled with an improvement of glucose homeostasis. The limitation of weight gain could further be explained by an increased lipid oxidation, possibly due to an up-regulation of genes involved in fatty acid oxidation and mitochondrial metabolism, leading to increased energy expenditure. Altogether, these data show that VD3 regulates energy expenditure and suggest that VD3 supplementation may represent a strategy of preventive nutrition to fight the onset of obesity and associated metabolic disorders.

Upregulation of TNF-α-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‐α)‐ and interleukin‐1 alpha (IL1‐α)‐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 β‐adrenergic receptors did not alter the TNF‐α‐induced ICAM‐1 surface expression. Pretreatment of HUVEC with pertussis toxin, which is known to activate AC through Giα inhibition, upregulated mRNA levels and surface expression of ICAM‐1 induced by TNF‐α. 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.

Procoagulant Platelets Form an α-Granule Protein-covered “Cap” on Their Surface That Promotes Their Attachment to Aggregates

Background: Phosphatidylserine-expressing platelets do not have active integrin αIIbβ3 but somehow retain fibrinogen. Results: The adhesive α-granule proteins fibrinogen and thrombospondin are concentrated in a fibrin polymerization-dependent “cap” on phosphatidylserine-expressing platelets that promotes their incorporation into thrombi. Conclusion: This suggests a revised model for the adhesive properties of phosphatidylserine-expressing platelets. Significance: The role of phosphatidylserine-expressing platelets in thrombus formation and its mechanism are re-evaluated. Strongly activated “coated” platelets are characterized by increased phosphatidylserine (PS) surface expression, α-granule protein retention, and lack of active integrin αIIbβ3. To study how they are incorporated into thrombi despite a lack of free activated integrin, we investigated the structure, function, and formation of the α-granule protein “coat.” Confocal microscopy revealed that fibrin(ogen) and thrombospondin colocalized as “cap,” a single patch on the PS-positive platelet surface. In aggregates, the cap was located at the point of attachment of the PS-positive platelets. Without fibrin(ogen) retention, their ability to be incorporated in aggregates was drastically reduced. The surface fibrin(ogen) was strongly decreased in the presence of a fibrin polymerization inhibitor GPRP and also in platelets from a patient with dysfibrinogenemia and a fibrinogen polymerization defect. In contrast, a fibrinogen-clotting protease ancistron increased the amount of fibrin(ogen) and thrombospondin on the surface of the PS-positive platelets stimulated with collagen-related peptide. Transglutaminases are also involved in fibrin(ogen) retention. However, platelets from patients with factor XIII deficiency had normal retention, and a pan-transglutaminase inhibitor T101 had only a modest inhibitory effect. Fibrin(ogen) retention was normal in Bernard-Soulier syndrome and kindlin-3 deficiency, but not in Glanzmann thrombasthenia lacking the platelet pool of fibrinogen and αIIbβ3. These data show that the fibrin(ogen)-covered cap, predominantly formed as a result of fibrin polymerization, is a critical mechanism that allows coated (or rather “capped”) platelets to become incorporated into thrombi despite their lack of active integrins.

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. J. Cell. Physiol. 214: 687–693, 2008.

A Novel Leukocyte Adhesion Deficiency III Variant: Kindlin-3 Deficiency Results in Integrin- and Nonintegrin-Related Defects in Different Steps of Leukocyte Adhesion

Leukocyte adhesion deficiency type III is a recently described condition involving a Glanzmann-type bleeding syndrome and leukocyte adhesion deficiency. This was ascribed to a defect of the FERMT3 gene resulting in abnormal expression of kindlin-3, a protein expressed in hematopoietic cells with a major role in the regulation of integrin activation. In this article, we describe a patient with a new mutation of FERMT3 and lack of kindlin-3 expression in platelets and leukocytes. We assayed quantitatively the first steps of kindlin-3–defective leukocyte adhesion, namely, initial bond formation, bond strengthening, and early spreading. Initial bond formation was readily stimulated with neutrophils stimulated by fMLF, and neutrophils and lymphocytes stimulated by a phorbol ester or Mn2+. In contrast, attachment strengthening was defective in the patient’s lymphocytes treated with PMA or Mn2+, or fMLF-stimulated neutrophils. However, attachment strengthening was normal in patient’s neutrophils treated with phorbol ester or Mn2+. In addition, the patient’s T lymphocytes displayed defective integrin-mediated spreading and a moderate but significant decrease of spreading on anti-CD3–coated surfaces. Patient’s neutrophils displayed a drastic alteration of integrin-mediated spreading after fMLF or PMA stimulation, whereas signaling-independent Mn2+ allowed significant spreading. In conclusion, the consequences of kindlin-3 deficiency on β2 integrin function depend on both cell type and the stimulus used for integrin activation. Our results suggest looking for a possible kindlin-3 involvement in membrane dynamical event independent of integrin-mediated adhesion.

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. J. Cell. Physiol. 208: 363–372, 2006.

Progression of atherosclerosis in ApoE-deficient mice that express distinct molecular forms of TNF-alpha†

TNFα (TNF) critically regulates inflammation‐driven atherosclerosis. Because the transmembrane (tmTNF) and soluble (sTNF) forms of TNF possess distinct immuno‐modulatory properties, we hypothesized that they might differentially regulate atherosclerosis progression. Three groups of male ApoE−/− mice were studied: one expressing wild‐type TNF (WT‐TNF); one expressing exclusively a mutated non‐cleavable form of TNF (KI‐TNF); and one deficient in TNF (KO‐TNF). Mice aged 5 weeks were fed the high‐fat diet for 5 (T5) and 15 weeks (T15) or a standard chow diet for 15 weeks. At T5, in mice fed the high‐fat diet, no significant differences in lesion area were observed among the three groups, either in valves or in aortas. At T15, lesion areas in valves were significantly lower in KO‐TNF mice compared with those in WT‐TNF mice, whereas in KI‐TNF mice, they were intermediate between KO‐ and WT‐TNF mice but not significantly different from these two groups. In aortas, lesions in KI‐TNF were comparable to those of KO‐TNF, both being significantly lower than those in WT‐TNF. Theses differences were not linked to circulating lipids, or to macrophage, actin, and collagen contents of lesions. At T15, in mice fed the chow diet, lesion areas in valves and the aortic arch were not significantly different between the three groups. Levels of IL‐6, IFNγ, IL‐10, and Foxp3 mRNAs in spleens and production of IL‐6, IL‐10, MCP‐1, RANTES, and TNFR‐2 by peritoneal macrophages at T15 of the high‐fat diet showed a decrease in pro‐inflammatory status, more marked in KO‐TNF than in KI‐TNF mice. Apoptosis was reduced only in KO‐TNF mice. In conclusion, these data show that TNF effects on atherosclerosis development are detectable at stages succeeding fatty streaks and that wild‐type TNF is superior to tmTNF alone in promoting atherosclerosis. TNF‐dependent progression of atherosclerosis is probably linked to the differential production of pro‐inflammatory mediators whether tmTNF is preponderant or essentially cleaved. Copyright

Two Types of Procoagulant Platelets Are Formed Upon Physiological Activation and Are Controlled by Integrin α IIb β 3

Objective—Phosphatidylserine (PS) externalization by platelets upon activation is a key event in hemostasis and thrombosis. It is currently believed that strong stimulation of platelets forms 2 subpopulations, only 1 of which expresses PS. Methods and Results—Here, we demonstrate that physiological stimulation leads to the formation of not 1 but 2 types of PS-expressing activated platelets, with dramatically different properties. One subpopulation sustained increased calcium level after activation, whereas another returned to the basal low-calcium state. High-calcium PS-positive platelets had smaller size, high surface density of fibrin(ogen), no active integrin &agr;IIb&bgr;3, depolarized mitochondrial membranes, gradually lost cytoplasmic membrane integrity, and were poorly aggregated. In contrast, the low-calcium PS-positive platelets had normal size, retained mitochondrial membrane potential and cytoplasmic membrane integrity, and combined retention of fibrin(ogen) with active &agr;IIb&bgr;3 and high proaggregatory function. Formation of low-calcium PS-positive platelets was promoted by platelet concentration increase or shaking and was decreased by integrin &agr;IIb&bgr;3 antagonists, platelet dilution, or in platelets from kindlin-3–deficient and Glanzmann thrombasthenia patients. Conclusion—Identification of a novel PS-expressing platelet subpopulation with low calcium regulated by integrin &agr;IIb&bgr;3 can be important for understanding the mechanisms of PS exposure and thrombus formation.