Hypoxia regulates RhoA and Wnt/β-catenin signaling in a context-dependent way to control re-differentiation of chondrocytes

Abstract

Cartilage tissue is avascular and hypoxic which regulates chondrocyte phenotype via stabilization of HIFs. Here, we investigated the role of hypoxia and HIFs in regulation of Rho and canonical Wnt signaling in chondrocytes. Our data demonstrates that hypoxia controls the expression of RhoA in chondrocytes in a context-dependent manner on the culturing conditions. Within a 3D microenvironment, hypoxia suppresses RhoA on which hypoxia-driven expression of chondrogenic markers depends. Conversely, hypoxia leads to upregulation of RhoA in chondrocytes on 2D with a failure in re-expression of chondrogenic markers. Similarly to RhoA, hypoxic regulation of Wnt/β-catenin signaling depends on the microenvironment. Hypoxia downregulates β-catenin within 3D hydrogels whereas it causes a potent increase on 2D. Hypoxia-induced suppression of canonical Wnt signaling in 3D contributes to the promotion of chondrogenic phenotype as induction of Wnt signaling abrogates the hypoxic re-differentiation of chondrocytes. Inhibiting Wnt/β-catenin signaling via stabilization of Axin2 leads to a synergistic enhancement of hypoxia-induced expression of chondrogenic markers. The effects of hypoxia on Rho and Wnt/β-catenin signaling are HIF-dependent as stabilizing HIFs under normoxia revealed similar effects on chondrocytes. The study reveals important insights on hypoxic signaling of chondrocytes and how hypoxia regulates cellular mechanisms depending on the cellular microenvironment.

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“…Additionally, the frequency of MSC divisions increases at low oxygen concentrations with the intact genotype maintained [ 105 ]. An increase in the chondrogenic potential of MSCs caused by hypoxia has been reported [ 104 , 106 ], although—as with chondrocytes—the direction of MSCs differentiation depends on the oxygen concentration but also on other factors, such as the substrate used [ 107 , 108 ].…”

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“…In addition to preventing nuclear accumulation of MRTF‐A, CCG‐1423 has also been implicated in inhibition of RhoA‐mediated transcription . RhoA is another member of the Rho GTPase family which plays a context‐dependent role in regulating the chondrocyte phenotype and has been shown to intersect with Wnt effectors beta‐catenin and Yes‐associated protein (YAP) and transcriptional co‐activator with PDZ‐binding motif (TAZ) . YAP/TAZ are two structurally similar actin‐regulated transcription factors involved in the canonical Hippo pathway and non‐canonical pathways.…”

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“…However, it has been indicated that 2D monolayer cell culture systems do not fully recapitulate the biological stimulations since chondrocytes tend to dedifferentiate and lose their chondrogenic phenotype into fibroblastic-like cells ( 24 ). It is also indicated that chondrocytes in 2D have higher activity of Wnt/β-catenin signaling that results with accumulation of β-catenin ( 25 ). This leads to decreased synthesis of Col-II, aggrecan and proteoglycan to form cartilage ECM ( 26 , 27 ).…”