Régulation des fonctions du récepteur tyrosine kinase MET par clivages protéolytiques

L'hepatocyte growth factor/scatter factor (HGF/SF) est le ligand du récepteur tyrosine kinase MET, qui induit des réponses de survie, prolifération, motilité et morphogenèse sur des cellules épithéliales. La signalisation de l'HGF/SF-MET est essentielle au cours du développement et sa dérégulation peut conduire au développement tumoral et à la progression métastatique. L'activation du récepteur MET par l'HGF/SF est classiquement associée à la survie cellulaire, en réponse à des stress variés. Cependant, nous avons montré qu'en absence d'HGF/SF, ces mêmes stress peuvent convertir le récepteur MET en facteur pro-apoptotique capable d'amplifier l'apoptose. En effet, MET est clivé de manière séquentielle par les caspases durant l'apoptose, sur son extrémité C-terminale puis dans sa région juxtamembranaire, générant un fragment p40 MET. La fonction apoptotique de p40 MET nécessite son activité kinase mais également sa dimérisation via son domaine C-terminal. D'autre part, nous avons montré que le récepteur MET est constitutivement clivé par des métalloprotéases dans son domaine extracellulaire, créant un fragment N-terminal soluble (MET-NTF) et un C-terminal membranaire (MET-CTF). Ce dernier est ensuite protéolysé par le complexe g-secrétase, conduisant à la libération d'un fragment intracellulaire instable (MET-ICD), qui peut transloquer dans le noyau. De plus, l'inhibition des activités métalloprotéase et g-secrétase empêche l'expression de c-Jun induite par l'HGF/SF. Nos résultats démontrent que les clivages protéolytiques du récepteur MET conduisent à la génération de différents fragments actifs qui redéploient le récepteur vers d'autres localisations cellulaires et qui diversifient ses réponses biologiques.LILLE1-BU (590092102) / SudocSudocFranceF

Expression and activation of caspase-6 in human fetal and adult tissues.

Caspase-6 is an effector caspase that has not been investigated thoroughly despite the fact that Caspase-6 is strongly activated in Alzheimer disease brains. To understand the full physiological impact of Caspase-6 in humans, we investigated Caspase-6 expression. We performed western blot analyses to detect the pro-Caspase-6 and its active p20 subunit in fetal and adult lung, kidney, brain, spleen, muscle, stomach, colon, heart, liver, skin, and adrenals tissues. The levels were semi-quantitated by densitometry. The results show a ubiquitous expression of Caspase-6 in most fetal tissues with the lowest levels in the brain and the highest levels in the gastrointestinal system. Caspase-6 active p20 subunits were only detected in fetal stomach. Immunohistochemical analysis of a human fetal embryo showed active Caspase-6 positive apoptotic cells in the dorsal root ganglion, liver, lung, kidney, ovary, skeletal muscle and the intestine. In the adult tissues, the levels of Caspase-6 were lower than in fetal tissues but remained high in the colon, stomach, lung, kidney and liver. Immunohistological analyses revealed that active Caspase-6 was abundant in goblet cells and epithelial cells sloughing off the intestinal lining of the adult colon. These results suggest that Caspase-6 is likely important in most tissues during early development but is less involved in adult tissues. The low levels of Caspase-6 in fetal and adult brain indicate that increased expression as observed in Alzheimer Disease is a pathological condition. Lastly, the high levels of Caspase-6 in the gastrointestinal system indicate a potential specific function of Caspase-6 in these tissues

Inflammation-induced tumorigenesis in mouse colon is caspase-6 independent.

Caspases play an important role in maintaining tissue homeostasis. Active Caspase-6 (Casp6) is considered a novel therapeutic target against Alzheimer disease (AD) since it is present in AD pathological brain lesions, associated with age-dependent cognitive decline, and causes age-dependent cognitive impairment in the mouse brain. However, active Casp6 is highly expressed and activated in normal human colon epithelial cells raising concerns that inhibiting Casp6 in AD may promote colon carcinogenesis. Furthermore, others have reported rare mutations of Casp6 in human colorectal cancers and an effect of Casp6 on apoptosis and metastasis of colon cancer cell lines. Here, we investigated the role of Casp6 in inflammation-associated azoxymethane/dextran sulfate sodium (AOM/DSS) colon cancer in Casp6-overexpressing and -deficient mice. In wild-type mice, AOM/DSS-induced tumors had significantly higher Casp6 mRNA, protein and activity levels compared to normal adjacent colon tissues. Increased human Casp6 or absence of Casp6 expression in mice colon epithelial cells did not change colonic tumor multiplicity, burden or distribution. Nevertheless, the incidence of hyperplasia was slightly reduced in human Casp6-overexpressing colons and increased in Casp6 null colons. Overexpression of Casp6 did not affect the grade of the tumors while all tumors in heterozygous or homozygous Casp6 null colons were high grade compared to only 50% high grade in wild-type mice. Casp6 levels did not alter cellular proliferation and apoptosis. These results suggest that Casp6 is unlikely to be involved in colitis-associated tumors

Phosphorylation of the MET receptor on juxtamembrane tyrosine residue 1001 inhibits its caspase-dependent cleavage

International audienceThe MET tyrosine kinase is the hepatocyte growth factor/scatter factor (HGF/SF) receptor, which elicits multiple biological responses in epithelial cells, including cell survival. We previously demonstrated that in stress conditions, the MET receptor is cleaved by caspases within its juxtamembrane region, generating a pro-apoptotic intracellular fragment of 40 kDa. The caspase cleavage site at aspartic acid D1000 is adjacent to tyrosine Y1001, which when phosphorylated upon MET activation, is involved in CBL recruitment, allowing receptor ubiquitination and down regulation. Scanning mutagenesis of the MET juxtamembrane region led us to demonstrate that V999 and D1000 are essential for the caspase cleavage, while D1000 and Y1001 are essential for CBL recruitment. By examining whether overlapping of these sites leads to a functional interference, an inverse relationship was found between generation of p40 MET and phosphorylation of MET, with a direct involvement of phosphorylated Y1001 in protecting MET against its caspase cleavage. A molecular modeling analysis of caspase 3 interaction with the juxtamembrane region of MET confirmed that phosphorylation of this tyrosine is not compatible with its recognition by active caspase 3. These data demonstrate a direct protection mechanism of an activated phosphorylated MET receptor, against its caspase-dependent cleavage

Steady state levels of Casp6, Casp1, and Casp3 in human adult tissues.

<p><b>A</b>. Representative western blot (series #2) of 100 µg of total proteins from adult tissues with Upstate anti-Casp6 antisera (ProCasp6) and β-Actin antibody. <b>B</b>. Age of adult tissues used for the study. <b>C</b>. Bar graph of the average of proCasp6 levels relative to that in colon arbitrarily placed at 100. Data represent the individual values as well as mean and SEM of 3 independent experiments. <b>D</b>. Coomassie stained gel of proteins from tissues in series 2.</p

Micrographs of immunohistochemical analyses of adult normal and cancerous colon with 1277 neoepitope antisera against the active p20 subunit of Casp6.

<p>Normal colonic mucosa (A) and crypt openings (B), colonic adenocarcinomas (C) and crypt openings (D) immunostained with anti-p20Casp6 neoepitope antisera. E. Larger magnification of normal colon showing active Casp6-positive epithelial cell sloughing off in the lumen. F. Western blot of 100 µg of total proteins from normal and cancerous colon tissues with Upstate anti-Casp6 antisera (ProCasp6) and β-Actin antibody.</p

Micrographs of immunohistochemical analyses of fetal tissues with 1277 neoepitope antisera detecting the active p20 subunit of Casp6.

<p>A. At low power (original magnification, 100x), immunopositive cells (arrows) in the dorsal root ganglion (DRG), in the anterior horn (AH), and in the dorsal root entry zone (DREZ) of the spinal cord white matter (WM). B. High power view of dorsal root ganglion (DRG: original magnification, 400×) indicating mitotic activity (solid arrowhead), features of early neuronal differentiation (open arrowheads) and several apoptotic immunopositive cells (arrows). C. High power view of anterior horn (AH: original magnification, 400×) indicating dividing cells (solid arrow heads), early maturation of cells (open arrowheads), and an immunopositive apoptotic precursor cell (arrow). WM; white matter). D. High power view of dorsal spinal cord (original magnification, 400×). Immunopositive finely granular (synaptic) pattern in the dorsal root entry zone (DREZ) of the spinal cord white matter (WM). DRG, dorsal root ganglion; DR, dorsal root; PH, posterior horn). E. Extensive extramedullary hematopoiesis (EMH) in the sinusoids that separate the cords of hepatocytes (HC) in the liver (original magnification 400×). In the hematopoietic islands, there are many immunopositive apoptotic cells (arrows).</p