Glycogen Synthase Kinase-3β Inhibition Links Mitochondrial Dysfunction, Extracellular Matrix Remodelling and Terminal Differentiation in Chondrocytes

Abstract

Following inflammatory stimuli, GSK3 inhibition functions as a hub with pleiotropic effects leading to cartilage degradation. However, little is known about the effects triggered by its direct inhibition as well as the effects on mitochondrial pathology, that contributes to osteoarthritis pathogenesis. To this aim we assessed the molecular mechanisms triggered by GSK3β inactivating stimuli on 3-D (micromass) cultures of human articular chondrocytes. Stimuli were delivered either at micromass seeding (long term) or after maturation (short term) to explore “late” effects on terminal differentiation or “early” mitochondrial effects, respectively. GSK3β inhibition significantly enhanced mitochondrial oxidative stress and damage and endochondral ossification based on increased nuclear translocation of Runx-2 and β-catenin, calcium deposition, cell death and enhanced remodelling of the extracellular matrix as demonstrated by the increased collagenolytic activity of supernatants, despite unmodified (MMP-1) or even reduced (MMP-13) collagenase gene/protein expression. Molecular dissection of the underlying mechanisms showed that GSK3β inhibition achieved with pharmacological/silencing strategies impacted on the control of collagenolytic activity, via both decreased inhibition (reduced TIMP-3) and increased activation (increased MMP-10 and MMP-14). To conclude, the inhibition of GSK3β enhances terminal differentiation via concerted effects on ECM and therefore its activity represents a tool to keep articular cartilage homeostasis.

Chondrocyte-Specific RUNX2 Overexpression Accelerates Post-traumatic Osteoarthritis Progression in Adult Mice

“…Glycogen synthase kinase 3 beta (GSK3β) can also directly phosphorylate RUNX2 in osteoblasts, but on residues that inhibit transcriptional transactivation . Further, treatment of chondrocyte cultures with GSK3β inhibitors was shown to promote nuclear localization of RUNX2 while inhibition of GSK3β in vivo enhanced cartilage degeneration in a murine model of PTOA . With regard to protein‐protein interactions, RUNX2 binds to numerous other transcription factors, co‐activators, and co‐repressors that modulate its ability to affect gene expression .…”

“…(80) Further, treatment of chondrocyte cultures with GSK3β inhibitors was shown to promote nuclear localization of RUNX2 while inhibition of GSK3β in vivo enhanced cartilage degeneration in a murine model of PTOA. (81,82) With regard to protein-protein interactions, RUNX2 binds to numerous other transcription factors, co-activators, and co-repressors that modulate its ability to affect gene expression. (71) CCAAT enhancer binding protein beta (C/ EBPβ), for example, is a potent transcriptional partner of RUNX2 in regulation of Mmp13 expression and is only expressed in articular chondrocytes following joint injury in a hypoxia-inducible factor 2 alpha (HIF-2α)-dependent manner.…”

Spermidine rescues the deregulated autophagic response to oxidative stress of osteoarthritic chondrocytes

“…Phosphorylation of H2AX is one of the earlier cellular response to DSBs that accumulates in DNA foci and activates complex molecular pathways [43] collectively known as the DNA damage response (DDR) [44] to obtain rapid and efficient error correction and to preserve genomic stability. 8-oxo-dG is a known marker of oxidative damage to DNA [45,46] that in chondrocytes mostly stains mitochondrial DNA [47], more susceptible to oxidative damage and less protected compared to genomic DNA [48]. Furthermore, 8-oxo-dG staining can also indicate oxidative damage to RNA that severely hampers RNA synthesis and protein translation [49].…”

Chondrocyte-specific RUNX2 Overexpression Causes Chondrodysplasia During Development, but is Not Sufficient to Induce OA-like Articular Cartilage Degeneration in Adult Mice Without Injury

“…GSK3 can also directly phosphorylate RUNX2 in osteoblasts, but on residues that inhibit transcriptional transactivation (78) . Further, treatment of chondrocyte cultures with GSK3 inhibitors was shown to promote nuclear localization of RUNX2 while inhibition of GSK3 in vivo enhanced cartilage degeneration in a murine model post-traumatic OA (79,80) . With regard to protein-protein interactions, RUNX2 binds to numerous other transcription factors, co-activators, and co-repressors that modulate its ability to affect gene expression (69) .…”