Perturbation of adhesion molecule-mediated chondrocyte-matrix interactions by 4-hydroxynonenal binding: implication in osteoarthritis pathogenesis

ABSTRACT: INTRODUCTION: Objectives were to investigate whether interactions between human osteoarthritic chondrocytes and 4-hydroxynonenal (HNE)-modified type II collagen (Col II) affect cell phenotype and functions and to determine the protective role of carnosine (CAR) treatment in preventing these effects. METHODS: Human Col II was treated with HNE at different molar ratios (MR) (1:20 to 1:200; Col II:HNE). Articular chondrocytes were seeded in HNE/Col II adduct-coated plates and incubated for 48 hours. Cell morphology was studied by phase-contrast and confocal microscopy. Adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and alpha1beta1 integrin at protein and mRNA levels were quantified by Western blotting, flow cytometry and real-time reverse transcription-polymerase chain reaction. Cell death, caspases activity, prostaglandin E2 (PGE2), metalloproteinase-13 (MMP-13), mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-kappaB) were assessed by commercial kits. Col II, cyclooxygenase-2 (COX-2), MAPK, NF-kappaB-p65 levels were analyzed by Western blotting. The formation of alpha1beta1 integrin-focal adhesion kinase (FAK) complex was revealed by immunoprecipitation. RESULTS: Col II modification by HNE at MR approximately 1:20, strongly induced ICAM-1, alpha1beta1 integrin and MMP-13 expression as well as extracellular signal-regulated kinases 1 and 2 (ERK1/2) and NF-kappaB-p65 phosphorylation without impacting cell adhesion and viability or Col II expression. However, Col II modification with HNE at MR approximately 1:200, altered chondrocyte adhesion by evoking cell death and caspase-3 activity. It inhibited alpha1beta1 integrin and Col II expression as well as ERK1/2 and NF-kappaB-p65 phosphorylation, but, in contrast, markedly elicited PGE2 release, COX-2 expression and p38 MAPK phosphorylation. Immunoprecipitation assay revealed the involvement of FAK in cell-matrix interactions through the formation of alpha1beta1 integrin-FAK complex. Moreover, the modification of Col II by HNE at a 1:20 or approximately 1:200 MR affects parameters of the cell shape. All these effects were prevented by CAR, an HNE-trapping drug. CONCLUSIONS: Our novel findings indicate that HNE-binding to Col II results in multiple abnormalities of chondrocyte phenotype and function, suggesting its contribution in osteoarthritis development. CAR was shown to be an efficient HNE-snaring agent capable of counteracting these outcomes

Regulation of cell signalling in the control of tumor cell invasion by tenascin-X, an extracellular matrix glycoprotein

La ténascine-X (TNX) est une glycoprotéine de la matrice extracellulaire. Son expression est fortement réprimée dans de nombreux cancers et l’invasion tumorale est accrue chez des souris TNX-/-. La TNX apparaît donc comme un répresseur potentiel du développement des tumeurs. L’objectif de notre travail est d’étudier cet effet présumé et d’en comprendre les mécanismes, en analysant in vitro le rôle de la TNX sur la croissance et la migration de cellules de fibrosarcome HT-1080 dans des modèles de culture bi- et surtout tridimensionnels, plus représentatifs de l’environnement cellulaire in vivo. Nos résultats montrent que la TNX inhibe la croissance des cellules tumorales, sans induire de mort apoptotique ou nécrotique. Des observations par microscopie confocale ont montré que la présence de TNX réduit l’étalement des cellules ainsi que leur efficacité de migration. Nous avons pu mettre en évidence que la TNX provoque un ralentissement de la migration des cellules tumorales ainsi qu’une diminution de la directionnalité de leurs trajectoires. L’observation de la protéolyse du collagène de type I par les cellules en migration montre qu’elle est inhibée en présence de TNX. Par ailleurs, la TNX réduit l’expression et l’activation des MMP 2, MMP-9, et MT1 MMP. Certaines voies de signalisation associées ont été étudiées : la TNX inhibe la phosphorylation de FAK sur sa tyrosine 397, ainsi que l’activation des GTPases RhoA et Rac, sans affecter celle de Cdc42. Par une régulation fine de ces molécules, qui sont impliquées dans le contrôle de la croissance et de la migration cellulaire, la TNX se caractérise comme un inhibiteur extracellulaire de l’invasion tumoraleTenascin-X (TNX) is involved not only in the organisation of the extracellular matrix architecture but also in the regulation of cell behaviour. This matrix glycoprotein is down-regulated in many tumor types, while tumor invasion is promoted in TNX-deficient mice. In order to decipher the mechanisms by which TNX modulates tumor cell growth and migration, we compared the behaviour of HT1080 fibrosarcoma cells in conventionnal 2D culture model or embedded in 3D collagen gels, both containing or not recombinant TNX. Some experimentations have permit us to demonstrate that TNX inhibits tumor cells growth, without inducing apoptotic or necrotic cell death. Laser confocal microscopy observations demonstrated that the presence of TNX reduces cell spreading and migration efficency. Moreover, video time-lapse analysis showed that TNX reduces both velocity and directionnality of cell migration. This result is partly due to a decrease of pericellular proteolysis, as observed in situ using FITC-collagen-containing gels. Besides, we showed that TNX led to a decrease of MMP 2, MMP-9, MT1 MMP expression and activity. Then, we determined that both FAK phosphorylation on tyrosine 397 and activation of Rac1/2/3 and RhoA small GTPases were inhibited in TNX conditions. An inactivation of these small GTPases of the Rho family is known to deregulate cell cycle and highly decrease tumor cell spreading and migration efficiency in 3D environment. Taken together, these results indicate that TNX is an extracellular inhibitor of cell invasion, which acts by downregulating the main signalling pathways responsible for cell growth and motility in 3D-collagen gel

Tenascin-X increases the stiffness of collagen gels without affecting fibrillogenesis

Tenascin-X is an extracellular matrix protein whose absence leads to an Ehlers-Danlos Syndrome in humans, mainly characterised by connective tissue defects including the disorganisation of fibrillar networks, a reduced collagen deposition, and modifications in the mechanical properties of dense tissues. Here we tested the effect of tenascin-X on in vitro collagen fibril formation. We observed that the main parameters of fibrillogenesis were unchanged, and that the diameter of fibrils was not significantly different when they were formed in the presence of tenascin-X. Interestingly, mechanical analysis of collagen gels showed an increased compressive resistance of the gels containing tenascin-X, indicating that this protein might be directly involved in determining the mechanical properties of collagen-rich tissues in vivo.</p

DISC761102_Supplementary_Material – Supplemental material for MyoScreen, a High-Throughput Phenotypic Screening Platform Enabling Muscle Drug Discovery

<p>Supplemental material, DISC761102_Supplementary_Material for MyoScreen, a High-Throughput Phenotypic Screening Platform Enabling Muscle Drug Discovery by Joanne Young, Yoran Margaron, Mathieu Fernandes, Eve Duchemin-Pelletier, Joris Michaud, Mélanie Flaender, Oana Lorintiu, Sébastien Degot and Pauline Poydenot in SLAS Discovery</p