Biomedical subjects

Bertrand Perret

Publications and source records attributed to Bertrand Perret.

At least 19 recordsLinked to original sources

PubMed2006

Exosome lipidomics unravels lipid sorting at the level of multivesicular bodies.

Exosomes are part of the family of "bioactive vesicles" and appear to be involved in distal communications between cells. They vehiculate bioactive lipids and lipolytic enzymes and their biogenesis require specific lipids and a membrane reorganisation. Their biogenesis pathway could be a way to secrete enzymes involved in lipid signalling and to generate "particulate agonists". However, this pathway seems also to be used by pathogens such as HIV. This review will consider several aspects of lipidomics studies which might help to understand the fate and role of these fascinating vesicles.

Animals↗

PubMed2006

Influence of dyslipidemia on moxidectin distribution in plasma lipoproteins and on its pharmacokinetics.

PURPOSE: We studied the influence of dyslipemia on the distribution of moxidectin, a potent antiparasitic drug of the macrocyclic lactone (ML) family, in plasma lipoproteins and on its pharmacokinetic behaviour. MATERIALS AND METHODS: Plasma samples from normolipidemic or dyslipidemic subjects were spiked with moxidectin (20 ng/ml). Rabbits fed with standard (n = 5) or cholesterol-enriched diet (n = 5) were injected subcutaneously with moxidectin (300 microg/kg) and blood samples were collected over 32 days. Lipoproteins were separated from plasma samples by ultracentrifugation on density gradients. Moxidectin and lipids were measured in plasma and in lipoproteins and the pharmacokinetic parameters calculated. RESULTS: In normolipidemic subjects or rabbits, the drug bound preferentially to HDL. In hyperlipidemic samples, moxidectin shifted to the VLDL-LDL fraction. In addition, hyperlipidemic rabbits had a 2.8-fold higher area under the plasma concentration versus time curve (AUC) and a lower clearance and volume of distribution when compared with controls. CONCLUSION: Dyslipidemia led to major changes in moxidectin plasma distribution and in drug disposition. Therefore, a high variability in moxidectin disposition might be expected in humans or animals liable to develop dyslipidemia, with a possible impact on the efficacy and safety of this class of drugs.

Animals↗

PubMed2006

The adaptor protein Gab1 couples the stimulation of vascular endothelial growth factor receptor-2 to the activation of phosphoinositide 3-kinase.

Phosphoinositide 3-kinase (PI3K) mediates essential functions of vascular endothelial growth factor (VEGF), including the stimulation of endothelial cell proliferation and migration. Nevertheless, the mechanisms coupling the receptor VEGFR-2 to PI3K remain obscure. We observed that the Grb2-bound adapter Gab1 is tyrosine-phosphorylated and relocated to membrane fractions upon VEGF stimulation of endothelial cells. We could detect the PI3K regulatory subunit p85 in immunoprecipitates of endogenous Gab1, and vice versa, and measure a Gab1-associated lipid kinase activity upon VEGF stimulation. Furthermore, transfection of the Gab1-YF3 mutant lacking all p85-binding sites strongly repressed PI3K activation measured in vitro. Moreover, Gab1-YF3 severely decreased the cellular amount of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) generated in response to VEGF. Furthermore, adenoviral expression of Gab1-YF3 suppressed both Akt phosphorylation and recovery of wounded human umbilical vein endothelial cell monolayers, a VEGF-dependent process involving cell migration and proliferation under PI3K control. Transfection of other Gab1 mutants, lacking Grb2-binding sites or the pleckstrin homology (PH) domain, also prevented Akt activation, further demonstrating Gab1 involvement in PI3K activation. These mutants were also used to show that interactions with both Grb2 and PtdIns(3,4,5)P3 mediate Gab1 recruitment by VEGFR-2. Importantly, Gab1 mobilization was impaired by (i) PI3K inhibitors, (ii) deletion of Gab1 PH domain, (iii) PTEN (phosphatase and tensin homolog deleted on chromosome 10) overexpression to repress PtdIns(3,4,5)P3 production, and (iv) overexpression of a competitor PH domain for PtdIns(3,4,5)P3 binding, which altogether demonstrated that PI3K is also an upstream regulator of Gab1. Gab1 thus appears as a primary actor in coupling VEGFR-2 to PI3K/Akt, recruited through an amplification loop involving PtdIns(3,4,5)P3 and its PH domain.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Selective activation of nuclear phospholipase D-1 by g protein-coupled receptor agonists in vascular smooth muscle cells.

Recent studies highlight the existence of an autonomous nuclear lipid metabolism related to cellular proliferation. However, the importance of nuclear phosphatidylcholine (PC) metabolism is poorly understood. Therefore, we were interested in nuclear PCs as a source of second messengers and, particularly, nuclear phospholipase D (PLD) identification in membrane-free nuclei isolated from pig aorta vascular smooth muscle cells (VSMCs). Using immunoblot experiment, in vitro PLD assay with fluorescent substrate and confocal microscopy analysis, we demonstrated that only PLD1 is expressed in VSMC nucleus, whereas PLD1 and PLD2 are present in VSMC. Inhibition of RhoA and protein kinase Czeta (PKCzeta) by C3-exoenzyme and PKCzeta pseudosubstrate inhibitor, respectively, conducted a decrease of phosphatidylethanol production. On the other hand, treatment of intact VSMCs, but not nuclei, with phosphoinositide 3-kinase (PI3K) inhibitors prevented partially nuclear PLD1 activity, indicating for the first time that PI3K may have a role in nuclear PLD regulation. In addition, lysophosphatidic acid (LPA) or angiotensin II treatment of VSMCs resulted in an increase of intranuclear PLD activity, whereas platelet-derived growth factor and epidermal growth factor have no significant effect. Moreover, pertussis toxin induced a decrease of LPA-stimulated nuclear PLD1 activity, suggesting that heterotrimeric G(i)/G(0) protein involvement in intranuclear PLD1 regulation. We also show that LPA-induced nuclear PLD1 activation implied PI3K/PKCzeta pathway activation and PKCzeta nuclear translocation as well as nuclear RhoA activation. Thus, the characterization of an endogenous PLD1 that could regulate PC metabolism inside VSMC nucleus provides a new role for this enzyme in control of vascular fibroproliferative disorders.

Angiotensin II↗

PubMed2006

Accelerated lipid absorption in mice overexpressing intestinal SR-BI.

Dietary cholesterol absorption contributes to a large part of the circulating cholesterol. However, the mechanism of sterol intestinal uptake is not clearly elucidated. Scavenger receptor class B type I (SR-BI), major component in the control of cholesterol homeostasis, is expressed in the intestine, but its role in this organ remains unclear. We have generated transgenic mice overexpressing SR-BI primarily in the intestine by using the mouse SR-BI gene under the control of intestinal specific "apoC-III enhancer coupled with apoA-IV promoter." We found SR-BI overexpression with respect to the natural protein along the intestine and at the top of the villosities. After a meal containing [(14)C]cholesterol and [(3)H]triolein, SR-BI transgenic mice presented a rise in intestinal absorption of both lipids that was not due to a defect in chylomicron clearance nor to a change in the bile flow or the bile acid content. Nevertheless, SR-BI transgenic mice showed a decrease of total cholesterol but an increase of triglyceride content in plasma without any change in the high density lipoprotein apoA-I level. Thus, we described for the first time a functional role in vivo for SR-BI in cholesterol but also in triglyceride intestinal absorption.

Absorption↗

PubMed2006

Interleukin 6 is associated with subclinical atherosclerosis: a link with soluble intercellular adhesion molecule 1.

BACKGROUND: Among the markers of inflammation, a cytokine, interleukin (IL)-6, promotes the expression of intercellular adhesion molecule 1 (ICAM-1), C-reactive protein (CRP) synthesis, and leads to a series of procoagulant actions with potential major implications on the progression of atherosclerosis. AIM OF THE STUDY: To analyse in a population-based study, the relationship between IL-6 and atherosclerotic lesions and the role of serum ICAM-1 and CRP on this relationship. POPULATION: Among 1015 individuals randomly recruited between 1995 and 1997 in Haute-Garonne, a French region with a low cardiovascular risk, 953 subjects with complete data for all measurements were analysed. Common carotid intima-media thickness (IMT) and the presence of plaques in the carotid and femoral arteries were assessed by ultrasonography. RESULTS: Quartiles of IL-6, serum ICAM-1 and CRP were positively associated with plaques and IMT. After adjustment for traditional risk factors, IL-6 (P < 0.001) and serum ICAM-1 (P < 0.002) remained positively associated with plaques but not CRP (P = 0.20). Neither IL-6, nor serum ICAM-1, nor CRP were independently associated with IMT. When serum ICAM-1 was entered into the model in addition to traditional risk factors and IL-6, the percentage of variance in the number of plaques explained by the model did not increase significantly. CONCLUSION: IL-6 levels are associated with subclinical atherosclerotic lesions independently of traditional risk factors; the influence of IL-6 on ICAM-1 secretion may play a role in this association. These results argue the interest of IL-6 in the stratification of cardiovascular risk.

Adult↗

PubMed2005

Fasting insulin concentrations and coronary heart disease incidence in France and Northern Ireland: the PRIME Study.

BACKGROUND: Reports about the relationships between insulin concentrations and CHD risk are controversial. The objective of this survey was to study the association between insulin levels and CHD risk in middle-aged male participants of the PRIME Study after 5 years of follow-up. METHODS: Our study adopted a nested case-control design including 294 cases of CHD and 536 controls randomly selected among healthy participants from the PRIME cohort. Data were obtained by questionnaires (medical history, lifestyle), standardised clinical measurements (blood pressure, anthropometric measurements), and a blood sample was obtained for biological measurements. Odds-Ratios for associations of four ordered classes of insulin concentration with CHD risk after adjustment for confounding factors were estimated using conditional logistic regression. RESULTS: In Belfast, a significant trend (p<0.03) was observed between insulin classes and CHD risk in bivariate analyses, but this association lost its significance after multiple adjustments. In the French centres, a high risk of CHD (OR=3.24 [1.80-5.85], p<0.0001) was observed only for the second class of insulin concentration (6.5 to 9.9 mIU/l), compared with the reference class (<6.5 mIU/l). After multiple adjustments, this association remained highly significant (OR=2.92 [1.44-5.92], p<0.005). CONCLUSIONS: In Belfast (high-risk population), a significant trend was observed between insulin concentration classes and CHD risk but hyperinsulinaemia lost its association with CHD risk in multivariate analyses. In the French centres (lower risk population), slightly increased insulin concentrations were associated with a high risk of CHD, independently of cardiovascular risk factors and other features of the metabolic syndrome, but very high insulin concentrations were not.

Case-Control Studies↗

PubMed2005

High frequency of endothelial vasomotor dysfunction after acute coronary syndromes in non-culprit and angiographically normal coronary arteries: a reversible phenomenon.

This study aimed to assess endothelium-dependent vasomotor function in non-culprit coronary vessels in patients with recent acute coronary syndrome (ACS). Endothelial dysfunction can also concern peripheral vessels during ACS, but the frequency of this phenomenon at coronary circulation level is unknown. Endothelial function was assessed by infusion of graded doses of acetylcholine (ACh) in a non-culprit coronary artery of normal appearance in 43 patients having recently suffered from non-ST ACS, and reassessed 6 months later. Endothelium-dependent vasoreactivity was impaired at baseline in 81% (35/43) of the patients, and only 19% (8/43) of patients showed a normal response to ACh. Among the 35 subjects with initial dysfunction, 77% showed a significant improvement 6 months later. All patients without initial endothelial dysfunction remained normal. C-reactive protein (CRP) level was elevated at month 0, and had declined at follow-up, tending towards normal levels. At that time, apolipoprotein A-I (apoA-I) levels were correlated with vasomotor improvement in univariate (p < 0.02) and multivariate analysis (p < 0.04). In conclusion, endothelium dysfunction is very frequently observed after recent ACS in non-culprit and angiographically normal coronary arteries, and an improvement occurs within 6 months in most cases. After resolution of the initial inflammation, apoA-I seems to play an important role in endothelial function.

Acetylcholine↗

PubMed2005

Tumor recognition following Vgamma9Vdelta2 T cell receptor interactions with a surface F1-ATPase-related structure and apolipoprotein A-I.

Vgamma9Vdelta2 T lymphocytes, a major gammadelta T lymphocyte subset in humans, display cytolytic activity against various tumor cells upon recognition of yet uncharacterized structures. Here, we show that an entity related to the mitochondrial F1-ATPase is expressed on tumor cell surface and promotes tumor recognition by Vgamma9Vdelta2 T cells. When immobilized, purified F1-ATPase induces selective activation of this lymphocyte subset. The Vgamma9Vdelta2 T cell receptors (TCR) and the F1-ATPase also bind a delipidated form of apolipoprotein A-I (apo A-I), as demonstrated by surface plasmon resonance. Moreover, the presence of apo A-I in the culture medium is required for optimal activation of Vgamma9Vdelta2 T cells by tumors expressing F1-ATPase. This study thus describes an unanticipated tumor recognition mechanism by Vgamma9Vdelta2 lymphocytes and a possible link between gammadelta T cell immunity and lipid metabolism.

Apolipoprotein A-I↗

PubMed2005

Production of lysophosphatidic acid in blister fluid: involvement of a lysophospholipase D activity.

Lysophosphatidic acid (LPA) is present in abundance in serum resulting from platelet activation and is also found in other biological fluids. LPA controls numerous cellular responses and plays a role in specific functions such as wound healing, especially in the skin. Nevertheless, its presence in the skin has never been investigated. Since re-epithelialization occurs after blister rupture, we tested the presence of endogenous LPA in blister fluid and investigated a possible mechanism for its biosynthesis and biological functions. Using a radioenzymatic assay, LPA was detected in 33 blister fluids originating from 24 bullous dermatoses, and at higher concentrations than in plasma. In parallel, blister fluids contained a lysophospholipase D (LPLD) activity but no detectable phospholipase A2 activity. The expressions of the LPLD autotaxin (ATX) and of LPA1-receptor (LPA1-R) were greatly increased in blister skin when compared with normal skin. Finally, LPA was found to have a positive effect on the migration of cultured keratinocytes. These results show that LPA is present in blister fluid synthesized by the LPLD ATX. Due to its ability to enhance keratinocyte migration, LPA in blister fluid could, via the LPA1-R, play an important role in re-epithelialization occurring after blister rupture.

Adult↗

PubMed2004

Sex hormone-binding globulin is a major determinant of the lipid profile: the PRIME study.

The prevalence of coronary heart disease is much higher in men than in women and sex hormones might play a role in these differences through their influence on the lipid profile. The aim of this cross-sectional study was to study the relationship between hormonal markers (total testosterone (TT), estradiol (E2), sex-hormone-binding globulin (SHBG)) and plasma lipids in a population-based sample. Subjects were 352 men, 50-59 years old, selected in France (Lille, Strasbourg and Toulouse) and Northern-Ireland (Belfast) who had questionnaires and a medical examination at baseline of the PRIME prospective study (1991-1993). Pearson correlation coefficients and Student's t tests were used to identify factors associated with plasma lipids. Multiple linear regression models were used for multivariate analyses, using triglycerides (TG) (log-transformed) and high density-lipoprotein cholesterol (HDL-C) as dependent variables. SHBG and TT were negatively correlated with TG (p<0.0001 and p<0.05, respectively) and positively correlated with HDL-C (p<0.0001 and p<0.01). E2 was positively correlated with TG (p<0.05). No significant association was found between sex-hormones and LDL-C. In multiple linear regression analyses, SHBG remained independently associated negatively with TG (p<0.01) and positively with HDL-C (p<0.0001) after adjustment for centre of recruitment, age, body mass index, systolic blood pressure, smoking, alcohol intake and physical activity. After further adjustment for insulin, the association between SHBG and HDL-C remained highly significant (p<0.0001). The association between SHBG and TG was weakened but remained also significant. Our results suggest that SHBG might to be a central protein in the hormonal regulation of the lipid profile.

Biomarkers↗

PubMed2004

A novel role for Gab1 and SHP2 in epidermal growth factor-induced Ras activation.

SHP2 was recently found to down-regulate PI3K activation by dephosphorylating Gab1 but the mechanisms explaining the positive role of the Gab1/SHP2 pathway in EGF-induced Ras activation remain ill defined. Substrate trapping experiments now suggest that SHP2 dephosphorylates other Gab1 phosphotyrosines located within a central region displaying four YXXP motifs. Because these sites are potential docking motifs for Ras-GAP, we tested whether SHP2 dephosphorylates them to facilitate Ras activation. We observed that a Gab1 construct preventing SHP2 recruitment promoted membrane relocation of RasGAP. Moreover, a RasGAP-inactive mutant restored the activation of Ras in cells transfected with SHP2-inactivating Gab1 mutant or in SHP2-deficient fibroblasts, supporting the hypothesis that RasGAP is a downstream target of SHP2. To determine whether Gab1 is a RasGAP-binding partner, a Gab1 mutant deleted of four YXXP motifs was produced. The deletion suppressed RasGAP redistribution and restored the defective Ras activation caused by SHP2-inactivating mutations. Moreover, Gab1 was found to interact with RasGAP SH2 domains, only under conditions where SHP2 is not activated. To identify Ras-GAP-binding sites, Tyr to Phe mutants of Gab1 YXXP motifs were produced. Gab1 constructs mutated on Tyr(317) were severely affected in RasGAP binding and were the most active in compensating for Ras-defective activation and blocking RasGAP redistribution induced by SHP2 inactivation. We have thus localized on Gab1 a Ras-negative regulatory tyrosine phosphorylation site involved in RasGAP binding and showed that an important SHP2 function is to down-regulate its phosphorylation to disengage RasGAP and sustain Ras activation.

Amino Acid Sequence↗

PubMed2004

PLD2 is enriched on exosomes and its activity is correlated to the release of exosomes.

Exosomes are small vesicles secreted by different immune cells and which display anti-tumoral properties. Stimulation of RBL-2H3 cells with ionomycin triggered phospholipase D2 (PLD2) translocation from plasma membrane to intracellular compartments and the release of exosomes. Although exosomes carry the two isoforms of PLD, PLD2 was enriched and specifically sorted on exosomes when overexpressed in cells. PLD activity present on exosomes was clearly increased following PLD2 overexpression. PLD2 activity in cells was correlated to the amount of exosome released, as measured by FACS. Therefore, the present work indicates that exosomes can vehicle signaling enzymes.

Animals↗

PubMed2004

Mast cell- and dendritic cell-derived exosomes display a specific lipid composition and an unusual membrane organization.

Exosomes are small vesicles secreted from multivesicular bodies, which are able to stimulate the immune system leading to tumour cell eradication. We have analysed lipids of exosomes secreted either upon stimulation from rat mast cells (RBL-2H3 cells), or constitutively from human dendritic cells. As compared with parent cells, exosomes displayed an enrichment in sphingomyelin, but not in cholesterol. Phosphatidylcholine content was decreased, but an enrichment was noted in disaturated molecular species as in phosphatidylethanolamines. Lyso(bis)phosphatidic acid was not enriched in exosomes as compared with cells. Fluorescence anisotropy demonstrated an increase in exosome-membrane rigidity from pH 5 to 7, suggesting their membrane reorganization between the acidic multivesicular body compartment and the neutral outer cell medium. NMR analysis established a bilayer organization of exosome membrane, and ESR studies using 16-doxyl stearic acid demonstrated a higher flip-flop of lipids between the two leaflets as compared with plasma membrane. In addition, the exosome membrane exhibited no asymmetrical distribution of phosphatidylethanolamines. Therefore exosome membrane displays a similar content of the major phospholipids and cholesterol, and is organized as a lipid bilayer with a random distribution of phosphatidylethanolamines. In addition, we observed tight lipid packing at neutral pH and a rapid flip-flop between the two leaflets of exosome membranes. These parameters could be used as a hallmark of exosomes.

Animals↗

PubMed2003

Enterophilin-1 interacts with focal adhesion kinase and decreases beta1 integrins in intestinal Caco-2 cells.

Intestinal cell growth and differentiation are tightly regulated by growth factors and extracellular matrix components along the crypt-villus axis. We previously described enterophilin-1 (Ent-1) as a new intestinal protein associated with growth arrest and enterocyte differentiation. Ent-1 interacted with sorting nexin 1 and decreased cell surface epidermal growth factor receptor. Because beta(1) integrins are mostly found in vivo in the proliferative crypt cells, we investigated the role of Ent-1 in the fate of beta(1) integrin subunits. In undifferentiated intestinal Caco-2 cells, overexpression of Ent-1 induces a marked decrease of alpha(5)beta(1) integrin pools, whereas alpha(2)beta(1) integrin is weakly affected. Conversely, overexpression of sorting nexin 1 has no effect on integrin levels despite its ability to interact with Ent-1. Interestingly, we identified focal adhesion kinase as a new Ent-1 partner using yeast two-hybrid screening and co-precipitation experiments. Furthermore by confocal microscopy, we observed that Ent-1 and beta(1) integrins partly co-localize on vesicular structures, suggesting a role for Ent-1 in integrin trafficking. Because focal adhesion kinase is able to bind both Ent-1 and beta(1) integrins, the kinase might act as a molecular bridge between the two proteins. Altogether, these results support a role of Ent-1 in regulating beta(1) integrin expression that could favor intestinal differentiation.

Caco-2 Cells↗

PubMed2003

Effect of apolipoprotein E alleles and angiotensin-converting enzyme insertion/deletion polymorphisms on lipid and lipoprotein markers in middle-aged men and in patients with stable angina pectoris or healed myocardial infarction.

The effects of the apolipoprotein E epsilon and angiotensin-converting enzyme insertion/deletion alleles on lipid levels and hypolipidemic drug treatment was assessed in 400 men with stable angina pectoris or healed myocardial infarction and 338 healthy controls. The data indicate that epsilon4 carriers have increased total and low-density lipoprotein cholesterol levels, that the epsilon4 allele unfavorably decreases the efficiency of statin treatment, and that the angiotensin-converting enzyme insertion/deletion polymorphism exerts no significant effect, with the exception of an increase in apolipoprotein E levels.

Adult↗

PubMed2003

SHIP-2 and PTEN are expressed and active in vascular smooth muscle cell nuclei, but only SHIP-2 is associated with nuclear speckles.

Recently, the control of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3)-dependant signaling by phosphatases has emerged, but there is a shortage of information on intranuclear PtdIns(3,4,5)P3 phosphatases. Therefore, we investigated the dephosphorylation of [32P]PtdIns(3,4,5)P3 specifically labeled on the D-3 position of the inositol ring in membrane-free nuclei isolated from pig aorta vascular smooth muscle cells (VSMCs). In vitro PtdIns(3,4,5)P3 phosphatase assays revealed the production of both [32P]PtdIns(3,4)P2 and inorganic phosphate, demonstrating the presence of PtdIns(3,4,5)P3 5- and 3-phosphatase activities inside the VSMC nucleus, respectively. Both activities presented the same potency in cellular lysates, whereas the nuclear PtdIns(3,4,5)P3 5-phosphatase activity appeared to be the most efficient. Immunoblot experiments showed for the first time the expression of the 5-phosphatase SHIP-2 (src homology 2 domain-containing inositol phosphatase) as well as the 3-phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10) in VSMC nuclei. In addition, immunoprecipitations from nuclear fractions indicated a [32P]PtdIns(3,4,5)P3 dephosphorylation by both SHIP-2 and PTEN. Moreover, confocal microscopy analyses demonstrated that SHIP-2 but not PTEN colocalized with a speckle-specific component, the SC35 splicing factor. These results suggest that SHIP-2 may be the primary enzyme for metabolizing PtdIns(3,4,5)P3 into PtdIns(3,4)P2 within the nucleus, thus producing another second messenger, whereas PTEN could down-regulate nuclear phosphoinositide 3-kinase signaling. Finally, intranuclear PtdIns(3,4,5)P3 phosphatases might be involved in the control of VSMC proliferation and the pathogenesis of vascular proliferative disorders.

Alternative Splicing↗

PubMed2003

Enterophilin-1, a new partner of sorting nexin 1, decreases cell surface epidermal growth factor receptor.

We previously described enterophilin-1 (Ent-1), a new intestinal protein bearing an extended leucine zipper and a B30.2 domain. Ent-1 expression is associated with growth arrest and enterocyte differentiation. To investigate the importance of Ent-1 in the differentiation, we performed a yeast two-hybrid screening. We identified sorting nexin 1 (SNX1) as a novel partner of Ent-1 and confirmed the specificity of interaction by co-immunoprecipitation experiments in mammalian cells. SNX1 is associated with endosomal membranes and triggers the endosome-to-lysosome pathway of epidermal growth factor receptor (EGFR). We observe by immunofluorescence microscopy that Ent-1 and SNX1 are co-localized on vesicular and tubulovesicular structures, which are different from early endosome antigen 1-containing endosomes. By gel filtration chromatography, we show that Ent-1 and SNX1 co-eluted in macromolecular complexes containing part of EGFR. Furthermore, overexpressed Ent-1 decreases cell surface EGFR. Ent-1 and SNX1 co-overexpression strongly extends EGFR diminution, indicating a synergetic effect of both proteins on cell surface EGFR removal. Interestingly, the increase of endogenous Ent-1 expression correlates with the decrease of EGFR during spontaneous differentiation of Caco-2 cells. We thus propose a role of Ent-1 in the trafficking of EGFR to down-regulate intestinal mitogenic signals, highlighting the mechanisms of cell growth arrest associated with enterocytic differentiation.

Animals↗