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Hepatocyte growth factor (HGF), a factor which stimulates cell growth, morphogenesis and migration, was found in hepatocytes and other epithelial cells. HGF was found to induce cell scattering, so that it was previously named as ¡§scatter factor¡¨ (SF), which turned out to be the same protein. HGF has two chains, a-chain, which contains N-terminal domain and four kringle domains and b-chain, a serine protease-like domain linked with a-chain by disulfide bond. The size of b-chain is 34 kDa after glycosylation in human cells. The function of HGFb was not unknown until recently that HGFb was shown 1000-fold lower affinity to c-Met than HGFa. Thus this project is to create biotechnological approaches for quick and large scale production of HGFb. The DNA fragment amplified by PCR was transferred to pET-22b(+) for expression, and showed that large amount of recombinant HGFb was produced both in periplasmic space and culture supernatant. Assays for cancer proliferation and migration with the HGFb showed that the recombinant protein inhibited cancer cells in a dose-independent manner. Our experiment showed clearly that 10 nM of the E. coli-produced HGFb inhibited proliferation and migration of cancer cells in comparison with the medium and pET controls, 1000-fold higher affinity to c-Met than reported.

Le cancer spinocellulaire (CSC) est le cancer de la peau le plus fréquent chez l’homme. Son étiologie est liée à l'exposition aux rayonnements ultraviolets (UV). Le xeroderma pigmentosum C (XP-C) est une maladie génétique caractérisée par l’absence de la protéine XPC entrainant une déficience dans la réparation des lésions dans l’ADN induites par les UV. La persistance de ces lésions chez ces patients entraine l’apparition précoce de CSC particulièrement agressifs. Les fibroblastes cutanés XP-C présentent un phénotype ressemblant à celui des fibroblastes associés aux cellules cancéreuses, suggérant un rôle promoteur dans le développement précoce des CSC XP-C. Nous avons étudié les effets des fibroblastes XP-C sur l’invasion de cellules de carcinomes. Dans des cultures organotypiques de peau, les fibroblastes XP-C favorisent l'invasion des cellules de CSC. De plus, ex vivo, la cicatrisation des cellules CSC est plus rapide en présence de surnageants de culture de fibroblastes XP-C et est due à un effet mitogénique des fibroblastes XP-C qui augmente la proportion des cellules de CSC dans la phase G2-M du cycle. Les fibroblastes XP-C surexpriment le facteur de croissance HGF qui active le récepteur c-Met et les voies de signalisation p38 et JNK dans les cellules de CSC. Le blocage de HGF entraîne l’inactivation de c-Met, p38 et JNK et bloque l'invasion des cellules CSC. De plus, nous montrons que les fibroblastes XP-C jouent un rôle de cellules « leader » dans l’invasion des CSC. Les fibroblastes XP-C créent un microenvironnement permissif à l'invasion des CSC. Des thérapies ciblant les fibroblastes XP-C pourraient permettre le contrôle de l’invasion des CSC chez les XP. / Squamous cell carcinoma (SCC) is the most frequent metastatic skin cancer. His etiology is linked to exposure to ultraviolet radiation (UVR). Xeroderma pigmentosum C (XP-C) is a genetic disorder characterized by a severe susceptibility to aggressive SCCs following minimal exposure to UVR. XP-C cells are deficient in nucleotide excision repair (NER) of UV-induced DNA lesions. XP-C dermal fibroblasts expresse a phenotype resembling that of stromal fibroblasts associated to cancer cells with accumulation of reactive oxygen species and over expression of matrix metalloproteinase 1 (MMP1). We explored the effects of XP-C fibroblasts on migration and invasion of SCC cells. In organotypic skin cultures, XP-C fibroblasts promote the invasion of SCC cells. Also, scratch healing of SCC cells is enhanced with culture supernatants of XP-C fibroblasts through a mitogenic effect connected to increased ratio of SCC cells in the G2-M phase of the cell cycle. We show that XP-C fibroblasts overexpress the hepatocyte growth factor/scatter factor (HGF/SF) and activate the c-Met receptor and the p38 and JNK pathways in SCC cells. Blockage of HGF inhibits c-Met, p38 and JNK activation and prevented invasiveness of SCC cells within dermal equivalents. Spheroid assays show that XP-C fibroblasts lead SCC invasions. Our data indicate for the first time that XP-C fibroblasts are responsible for the formation of a permissive microenvironment towards SCC cells proliferation and invasion. Therapies targeting XP-C fibroblasts may be considered as a way to control aggressive cancer in XP-C patients.

No description available.

Using patch clamp and Ca2+ imaging techniques, we have studied Ca2+ entry pathways in human hepatoblastoma (HepG2) cells. These cells express the mRNA of TRPV1, TRPV2, TRPV3 and TRPV4 channels, but not those of TRPV5 and TRPV6. Functional assessment showed that capsaicin (10 μM), 4α-phorbol-12,13-didecanoate (4αPDD, 1 μM), arachidonic acid (10 μM), hypotonic stress, and heat all stimulated increases in [Ca2+]i within minutes. The increase in [Ca2+]i depended on extracellular Ca2+ and on the transmembrane potential, which indicated that both driving forces affected Ca2+ entry. Capsaicin also stimulated an increase in [Ca2+]i in nominally Ca2+-free solutions, which was compatible with the receptor functioning as a Ca2+ release channel. Hepatocyte growth factor/scatter factor (HGF/SF) modulated Ca2+ entry. Ca2+ influx was greater in HepG2 cells incubated with HGF/SF (20 ng/ml for 20 h) compared with non-stimulated cells, but this occurred only in those cells with a migrating phenotype as determined by presence of a lamellipodium and trailing footplate. The effect of capsaicin on [Ca2+]i was greater in migrating HGF/SF-treated cells, and this was inhibited by capsazepine. The difference between control and HGF/SF-treated cells was not found in Ca2+-free solutions. 4αPDD also had no greater effect on HGF/SF-treated cells. We conclude that TRPV1 and TRPV4 channels provide Ca2+ entry pathways in HepG2 cells. HGF/SF increases Ca2+ entry via TRPV1, but not via TRPV4. This rise in [Ca2+]i may constitute an early response of a signalling cascade that gives rise to cell locomotion and the migratory phenotype.

Glycosaminoglycans (GAGs) such as heparin, heparan sulfate (HS), chondroitin sulfate (CS) and dermatan sulfate (DS) are sulfated polysaccharides that exist on animal cell surfaces and in the extracellular matrix. GAGs are important in providing structural and hydrating support and interaction points for proteins of varied functions, for example growth factors and homeostasis regulatory proteins. Hepatocyte Growth Factor (HGF) is a protein growth factor that regulates cell growth, survival, proliferation, chemotaxis, cell morphology, tissue regeneration and angiogenesis. It is involved in embryogenesis, wound healing and many cancers. In this project, the interactions between the GAG binding N and NK -domains of HGF (HGF-N and HGF-NK) and different types of GAGs are characterised with biophysical techniques. GAG oligosaccharides were produced by enzymatic digestion and purified by preparative gel filtration and ion exchange chromatography. Different constructs of HGF were cloned from human cDNA, expressed with the Pichia pastoris expression system, purified to homogeneity and characterised by mass spectrometry and nuclear magnetic resonance (NMR). The dissociation constants between the different HGF protein constructs, different heparin oligosaccharide lengths and the drug Fondaparinux were shown by isothermal calorimetry (ITC) to vary between 0.35 and 9.26 μM. It was found that the entropy contribution was favourable for short oligosaccharides and disfavourable for long oligosaccharides and that the enthalpy contribution was less important for shorter oligosaccharides than for longer oligosaccharides. NMR titrations of CS, DS, heparin, Fondaparinux and sulfated maltose into 15N labelled protein samples showed that all ligands bind to the same HGF-N binding site, but different binding modes exists. The binding site consists of three regions, with the α2-helix and L2 loops playing key roles (residues 70-84). All GAGs also utilise the N-terminal residues 32-42, whereas long heparin oligosaccharides can also utilise a binding region formed mainly by the β2-strand (residues 59-64, 66, 95, 96). The GAG binding mode changes if HGF-N has an N-terminal truncation and the β2- strand residues become more important, emphasising the role of the N-terminal residues in the HGF-GAG interaction. Spin-labelled fully sulfated heparin-derived hexasaccharide was used to determine its binding direction on the HGF-N surface. Affinity chromatography confirmed the importance of the N-terminal residues and that HGF binds to all investigated GAGs. The oligomeric states of HGF-N and HGF-NK were investigated by AUC, gel filtration and ITC. The results suggest that the proteins oligomerise like beads on a string for long oligosaccharides. An HGF-N self-associating dimer with a slow on/off rate was characterised by affinity chromatography, gel filtration and NMR.

Hepatocyte growth factor (HGF) is a multifunctional protein, which secrets via Golgi complex after synthesized, and is hydrolyzed into an active heterodimer containing an £\ and a £] chain by extracellular protease. It is known that HGF functions through surface domain of Met, and thus induces mitosis and metastasis. The interaction domain of HGF is believed to be located in the £\-chain. In order to study these findings structurally and functionally, we designed and constructed four different recombinant coding regions of the gene (NK1, NK2, NK3, and NK4) which was then successfully expressed in E. coli. Purification of these four different recombinant proteins with glutathione-agarose column showed that all of the four constructs had been successfully expressed with some degradations. Cell proliferation assay showed that the recombinant proteins inhibited the growth of breast cancer cells to some extent. The assay also showed that GST-NK1 and GST-NK2 were better inhibitors than GST-NK3 and GST-NK4 to the cancer cells. It is concluded that E. coli expression is an appropriate system for achieving functional HGF.

Le Syndrome de Détresse Respiratoire Aiguë (SDRA) est la forme la plus sévère d’agression alvéolaire aiguë. Il estcaractérisé par un dommage alvéolaire diffus, suivi d’une phase de réparation nécessaire à la guérison. Bien queles monocytes/macrophages soient des acteurs incontournables de la pathogénie et de la résolution du SDRA, leurpolarisation et leur rôle dans la réparation alvéolaire restent mal connus chez l’Homme. L’hypothèse défendue parcette thèse est que l’environnement alvéolaire module la différenciation et la polarisation desmonocytes/macrophages au cours du SDRA, et que les macrophages alvéolaires ainsi polarisés participentactivement à la réparation et à sa régulation. Les principaux résultats de nos travaux ont permis d’établir que : 1)l’environnement alvéolaire de SDRA inhibe la différenciation des monocytes en fibrocytes (précurseursmésenchymateux associés à la fibroprolifération et à pronostic péjoratif). L’inhibition est majoritairement due à laSerum Amyloid protein P (SAP), provenant en partie du relargage de ses stocks matriciels pulmonaires à la faveurde la lésion alvéolaire ; 2) l’environnement alvéolaire de SDRA induit une polarisation anti-inflammatoire desmacrophages se rapprochant de la polarisation induite in vitro par l’IL-10 ; 3) les macrophages anti-inflammatoirespolarisés par le lavage broncho-alvéolaire (LBA) de patients SDRA favorisent la réparation alvéolaire épithéliale viala production polarisation-dépendante d’Hepatocyte Growth Factor (HGF). Cette production macrophagique d’HGFest amplifiée via une boucle autocrine PTGS2/PGE2 chez l’Homme ; 4) ces résultats semblent étayés par lesdonnées obtenues sur une cohorte clinique qui montrent que les concentrations de sCD163 (marqueur depolarisation anti-inflammatoire) et d’HGF rapportées au nombre de macrophages alvéolaires sont plus élevéeschez les patients survivants que chez les patients décédés de SDRA. L’ensemble de nos travaux démontrent pour lapremière fois chez l’Homme le rôle bénéfique de l’environnement alvéolaire sur les monocytes/macrophages,d’une part en inhibant leur différenciation en fibrocytes contribuant ainsi à limiter la fibroprolifération pulmonaire,et d’autre part en induisant un phénotype macrophagique anti-inflammatoire, contribuant à limiter l’inflammationengendrée par la lésion alvéolaire initiale et favorisant la réparation épithéliale via la production d’HGF. Lesdonnées physiopathologiques obtenues pourraient permettre d’envisager la reprogrammation anti-inflammatoiredes macrophages comme une cible thérapeutique du SDRA en cas d’excès d’inflammation ou de fibro-proliférationavec réparation aberrante. / Acute Respiratory Distress Syndrome (ARDS) is the most severe form of acute lung injury. ARDS is characterized bydiffuse alveolar damage followed by a phase of alveolar repair necessary to recovery. Althoughmonocytes/macrophages are key actors of pathogenicity and resolution of ARDS, little is known about theirpolarization and role on alveolar repair during human ARDS. The hypothesis of our studies was that ARDS alveolarenvironment modulates differentiation and polarization of monocytes and macrophages, and that polarizedmacrophages are involved in alveolar repair and its regulation. The main results of our work have shown that: 1)ARDS alveolar environment inhibited monocytes differentiation into fibrocytes (mesenchymal progenitorsassociated with fibroprolifération and a poor prognosis), mainly through its Serum Amyloid P (SAP) content,originating, at least in part, from the release of SAP associated with lung connective tissue during ARDS; 2) ARDSalveolar environment drove an anti-inflammatory macrophage polarization, close to that induced by IL-10 in vitro;3) anti-inflammatory macrophages polarized by broncho-alveolar lavage (BAL) from ARDS patients favored alveolarepithelial repair through a polarization-dependent production of Hepatocyte Growth Factor (HGF). This HGFproduction is amplified by an autocrine PTGS2/PGE2 dependent loop in human macrophages; 4) these results mayhave clinical relevance, since sCD163 (a marker of anti-inflammatory polarization) and HGF concentrations,expressed relatively to BAL macrophage count, were higher in ARDS survivors than non-survivors. Taken together,our works demonstrate for the first time the beneficial role of the ARDS alveolar environment onmonocytes/macrophages, inhibiting their differentiation into fibrocytes, thus limiting excessive lungfibroproliferation, and inducing an anti-inflammatory macrophage polarization, thus limiting the inflammationgenerated by the initial alveolar damage and favoring epithelial repair through HGF production. The datapresented in this thesis may allow considering anti-inflammatory macrophage repolarization as a potential newtherapeutic target of ARDS with excessive inflammation or fibro-proliferation with aberrant repair.

No description available.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.