engleski

Role of Axin2/β-catenin in chondrocyte de/re-differentiation

Cell-based cartilage therapies as autologous chondrocyte implantation rely on high number of chondrocytes and demand their two-dimensional (2D) expansion. Culturing chondrocytes in a 2D environment causes their dedifferentiation where they loose their characteristic phenotype seen in native cartilage and therefore it is crucial to understand what the underlying mechanisms that control this effect are. A common indication of chondrocytic phenotype is the expression of cartilage-specific extracellular matrix components such as collagen II. 3D culturing, hypoxia and inhibition of RhoA are known to induce re-differentiation of chondrocytes followed by upregulation of collagen II gene. We have shown that expression of Axin2 - a downstream target of the Wnt/β-catenin pathway, correlates with collagen II expression in all these different re-differentiation-inducing environments. The aim of this thesis was to explore possible causalities accompanying this correlation and to understand how Wnt/β-catenin and RhoA pathways are regulated in the context of chondrocyte de-differentiation on 2D and redifferentiation in 3D alginate hydrogels and hypoxia. We have investigated the expression of Axin2, β-catenin, TAZ, RhoA, collagen II and collagen I on protein and gene levels in the course of 2D passaging of chondrocytes as well as 3D and hypoxic culturing for re-differentiation. We have found that during de-differentiation of chondrocytes β-catenin and its downstream targets are upregulated accompanied by a nuclear localization of RhoA whereas Axin2 and Collagen 2 were downregulated. We observed that Axin2 and TAZ are upregulated during 3D re-differentiation and hypoxia contributes to a further increase in the levels of these proteins. On the other hand β-catenin decreases during re-differentiation and is clearly downregulated in hypoxia together with RhoA. We conducted small molecule inhibition studies to confirm the role and effect of all the markers we investigated in 3D and hypoxia and their possible interplay. In conclusion re-differentiation of chondrocytes in 3D hydrogels is highly regulated by Wnt/β-catenin and RhoA pathways with distinct roles and interplay of downstream effectors Axin2 and TAZ with even more pronounced effects under hypoxic conditions suggesting new insights on the effect of oxygen tension on chondrocytic phenotype.

chondrocyte ; axin2 ; β-catenin

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