L'hepatocyte growth factor/scatter factor (HGF/SF) est le ligand du recepteur tyrosine kinase MET, qui induit des reponses de survie, proliferation, motilite et morphogenese sur des cellules epitheliales. La signalisation de l'HGF/SF-MET est essentielle au cours du developpement et sa deregulation peut conduire au developpement tumoral et a la progression metastatique. L'activation du recepteur MET par l'HGF/SF est classiquement associee a la survie cellulaire, en reponse a des stress varies. Cependant, nous avons montre qu'en absence d'HGF/SF, ces memes stress peuvent convertir le recepteur MET en facteur pro-apoptotique capable d'amplifier l'apoptose. En effet, MET est clive de maniere sequentielle par les caspases durant l'apoptose, sur son extremite C-terminale puis dans sa region juxtamembranaire, generant un fragment p40 MET. La fonction apoptotique de p40 MET necessite son activite kinase mais egalement sa dimerisation via son domaine C-terminal. D'autre part, nous avons montre que le recepteur MET est constitutivement clive par des metalloproteases dans son domaine extracellulaire, creant un fragment N-terminal soluble (MET-NTF) et un C-terminal membranaire (MET-CTF). Ce dernier est ensuite proteolyse par le complexe γ-secretase, conduisant a la liberation d'un fragment intracellulaire instable (MET-ICD), qui peut transloquer dans le noyau. De plus, l'inhibition des activites metalloprotease et γ-secretase empeche l'expression de c-Jun induite par l'HGF/SF. Nos resultats demontrent que les clivages proteolytiques du recepteur MET conduisent a la generation de differents fragments actifs qui redeploient le recepteur vers d'autres localisations cellulaires et qui diversifient ses reponses biologiques.
Alzheimer's disease (AD) occurs as either an autosomal dominant inherited disease or sporadically. While familial mutant genes can be expressed in cells or in animal models to assess dysregulated functions, sporadic AD cannot be replicated in models given our lack of understanding of causality. Fur thermore, the study of sporadic forms of AD is difficult given the inaccessibility of brain tissues in living individuals and the manifestation of symptoms years after the onset of disease. Here, the objective was to assess if induced pluripotent stem cell-derived neurons from well-ascertained sporadic AD individuals could represent potential cellular models to determine the underlying molecular mechanisms of disease. We used cryopreserved peripheral blood mononuclear cells from three well-ascertained sporadic AD and three non-cognitively impaired (NCI) individuals of the CIMA-Q cohort to obtain iPSC-derived neurons. Microtubule associated protein 2 was decreased in AD neurons, whereas expression of AD-associated amyloid precursor protein, tau, and amyloid-β peptide was similar in AD and NCI individuals. RNA sequencing identified several upregulated and downregulated mRNAs in AD relative to NCI neurons. Of these, complement Factor H (CFH), signal regulatory protein beta1 (SIRPB1), and insulin like growth factor binding protein 5 (IGFBP5) were previously associated with AD. In addition, several transcription factors not previously associated with AD, but involved in neuronal proliferation and differentiation were differentially expressed. The results identify novel avenues for the study of the underlying causes of sporadic AD and support the establishment of additional lines to identify mechanisms of disease in sporadic AD individuals.
Abstract: Hepatocyte growth factor/scatter factor (HGF/SF) induces scattering, morphogenesis, and survival of epithelial cells through activation of the MET tyrosine kinase receptor. HGF/SF and MET are involved in normal development and tumor progression of many tissues and organs, including the mammary gland. In order to find target genes of HGF/SF involved in its survival function, we used an oligonucleotide microarray representing 1,920 genes known to be involved in apoptosis, transcriptional regulation, and signal transduction. MCF‐10A human mammary epithelial cells were grown in the absence of serum and treated or not with HGF/SF for 2 h. Total RNA was reverse‐transcribed to cDNA in the presence of fluorescent Cy3‐dUTP or Cy5‐dUTP to generate fluorescently labeled cDNA probes. Microarrays were performed and the ratios of Cy5/Cy3 fluorescence were determined. The expression of three apoptotic genes was modified by HGF/SF, with A20 being upregulated, and DAXX and SMAC being downregulated. These changes of expression were confirmed by real‐time quantitative PCR. According to current‐knowledge, A20 is antiapoptotic and SMAC is proapoptotic, while a pro‐ or antiapoptotic function of DAXX is controversial. The fact that HGF/SF upregulates an antiapoptotic gene (A20) and downregulates a proapoptotic gene (SMAC) is in agreement with its survival effect in MCF‐10A cells. This study identified novel apoptotic genes regulated by HGF/SF, which can contribute to its survival effect.
Abstract The receptor tyrosine kinase Met and its ligand, the hepatocyte growth factor/scatter factor, are essential for embryonic development, whereas deregulation of Met signaling pathways is associated with tumorigenesis and metastasis. The presenilin‐regulated intramembrane proteolysis ( PS‐RIP ) is involved in ligand‐independent downregulation of Met. This proteolytic process involves shedding of the Met extracellular domain followed by γ‐secretase cleavage, generating labile intracellular fragments degraded by the proteasome. We demonstrate here that upon shedding both generated Met N‐ and C‐terminal fragments are degraded directly in the lysosome, with C‐terminal fragments escaping γ‐secretase cleavage. PS‐RIP and lysosomal degradation are complementary, because their simultaneous inhibition induces synergistic accumulation of fragments. Met N‐terminal fragments associate with the high‐affinity domain of HGF / SF , confirming its decoy activity which could be reduced through their routing to the lysosome at the expense of extracellular release. Finally, the DN30 monoclonal antibody inducing Met shedding promotes receptor degradation through induction of both PS‐RIP and the lysosomal pathway. Thus, we demonstrate that Met shedding initiates a novel lysosomal degradation which participates to ligand‐independent downregulation of the receptor.
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.
Caspase-6 is highly activated in neuropil threads, neurofibrillary tangles, and neuritic plaques of Alzheimer Disease (AD) brains. Caspase-6 activity restricted to the entorhinal cortex and hippocampal CA1 of aged non-AD individuals, correlates with lower cognitive performance. Expression of active Caspase-6 in mouse brain CA1 is sufficient to cause age-dependent spatial and episodic memory deficits. Caspase-1 activates Caspase-6 in primary cultures of human neurons. Here, we investigated which inflammasome may be responsible for Caspase-1-mediated Caspase-6 activation in human neurons and in AD brains. qRT-PCR and western blots identified the levels of specific inflammasome receptor components in primary cultures of fetal human neurons. Neurons were challenged by serum deprivation or purinergic P 2 X 7 receptor agonist, benzylated ATP to activate the inflammasomes. Caspase-1 and Caspase-6 activities were determined by fluorogenic assays, interleukin-1-beta and tubulin cleaved by Caspase-6 production. A cell free system treated with blocking antibodies identified specific functional inflammasomes. Null mice, injected with the lipopolysaccharide inflammatory agent, LPS, confirmed inflammasome-mediated activation of Caspase-1 and Caspase-6 in vivo. AD inflammasome mRNA and protein were assessed by RT-PCR and immunohistochemistry. Caspase-1 activation appeared before Caspase-6 activation in challenged primary cultures of human neurons. These neurons expressed the Nod-like receptor protein 1 (NLRP1), absent in melanoma 2 (AIM2), and ICE protease activating factor (IPAF-1) receptor components of the inflammasomes, but not NLRP3. Antibodies to NLRP1, but not to AIM2 and IPAF-1, blocked Caspase-1 and Caspase-6 activation in neuronal S100 sub-cellular fractions. LPS-mediated activation of Caspase-1 and Caspase-6 in wild-type mice brains was repressed in NLRP1 -/- and Casp1 -/- mice. NLRP1 mRNA levels and NLRP1-immunopositive neurons were increased in AD brains. We conclude that the NLRP1 inflammasome can regulate Caspase-1-mediated activation of Caspase-6 in human neurons, in mice brains, and in AD. The presence of functional inflammasomes in neurons indicates a mechanism by which neuronal production of interleukin-1-beta could initiate the inflammatory microglial response in AD brains. Our data suggest that the NLRP1 could represent a novel upstream target to prevent both the inflammatory response and Caspase-6-mediated axonal degeneration in AD.
Alzheimer's disease (AD) is an intractable progressive neurodegenerative disease characterized by cognitive decline and dementia. An inflammatory neurodegenerative pathway, involving Caspase-1 activation, is associated with human age-dependent cognitive impairment and several classical AD brain pathologies. Here, we show that the nontoxic and blood-brain barrier permeable small molecule Caspase-1 inhibitor VX-765 dose-dependently reverses episodic and spatial memory impairment, and hyperactivity in the J20 mouse model of AD. Cessation of VX-765 results in the reappearance of memory deficits in the mice after 1 month and recommencement of treatment re-establishes normal cognition. VX-765 prevents progressive amyloid beta peptide deposition, reverses brain inflammation, and normalizes synaptophysin protein levels in mouse hippocampus. Consistent with these findings, Caspase-1 null J20 mice are protected from episodic and spatial memory deficits, neuroinflammation and Aβ accumulation. These results provide in vivo proof of concept for Caspase-1 inhibition against AD cognitive deficits and pathologies.
