EUCUREOA

SirT1 Regulation of Aggrecan Expression in Human Chondrocytes

 Partecipanti

Mappa

 Word cloud

Esplora la "nuvola delle parole (Word Cloud) per avere un'idea di massima del progetto.

 Obiettivo del progetto (Objective)

'Osteoarthritis (OA) is a degenerative joint disease of the articular cartilage (AC) characterized by accelerated extracellular matrix (ECM) degradation and severe pain- dysfunction. The chromatin modifying enzyme, SirT1 has been shown to affect cartilage-type ECM gene expression. More specifically, SirT1 forms complexes with Sp1/Sox9 transcription factors (TFs), and induces collagen IIa1 (Coll-II) transcription. Similar to Coll-II, aggrecan has also been established to positively correlate with SirT1 expression. In AC, Coll-II and aggrecan are co-expressed and both genes present Sp1 and Sox9 binding sequences. I therefore hypothesize that Sp1 and/or Sox9 are involved in SirT1-aggrecan proximal promoter- Exon1 (APP/Ex1) association and aggrecan expression. Accordingly, my specific aims are to (1) characterize Sox9 and Sp1 binding to human APP via chromatin-immunoprecipitation (ChIP) analysis and Luciferase assays; (2) establish SirT1 association with Sp1/Sox9 and SirT1-Sp1/Sox9 complex localization on APP/Ex1 using reverse immunoprecipitation (revIP) and Sequential ChIP (SeqChIP), respectively; (3) test if SirT1- Sp1/Sox9 complex is essential for APP/Ex1-driven gene expression. This will be achieved by co-transfecting a luciferase-tagged-APP/Ex1 with SirT1 and Sp1/Sox9, followed by quantitative luminescence; (4) elucidate the presence of known SirT1-associated CMEs (i.e. GCN5 and SET7/9) and their respective histone hallmarks AcH4K5 and 3MeH3K4. This aim will be achieved by means of ChIP analyses of the actively transcribed APP/Ex1. Despite the high incidence of OA in developed countries, there is no treatment for this debilitating disease. Expectdly, the proposed study in humans will provide a regulatory mechanism for aggrecan production, thus identifying novel targets for the treatment of OA and the early diagnosis of susceptible individuals. This last notion is highly relevant, as susceptible subjects may be advised to prevent overloading of their joints.'

Introduzione (Teaser)

Osteoarthritis (OA) is a degenerative joint disease, affecting articular joints. Researchers are investigating the effect of SIRT1 enzyme in maintaining cartilage gene expression and integrity.

Descrizione progetto (Article)

OA causes severe pain-dysfunction as extracellular matrix (ECM) of articular cartilage gradually degrades with advanced age. The EU-funded 'SIRT1 regulation of aggrecan expression in human chondrocytes' (EUCUREOA) project investigated the molecular mechanism by which the SIRT1 enzyme regulates cartilage-specific gene expression of aggrecan (ACAN) and collagen 2A1 (COL2A1).

Aggrecan is a critical component for maintaining cartilage structure and joint resilience, while COL2A1 fibers bestow the joint with the capacity to withstand tensile forces. Since SIRT1 is augmented in healthy cartilage cells (i.e. chondrocytes) and correlates with high expression levels of ACAN and COL2A1, the researchers investigated whether SIRT1 directly induces the expression of both anabolic genes. The results point out that active SIRT1 is required for the expression of ACAN and COL2A1.

Moreover, given that inflammation contributes to cartilage destruction during OA, the team investigated whether inflammation could impact SIRT1 activity. Results showed that in the presence of inflammatory cytokines, SIRT1 was cleaved by cathepsin B in chondrocytes. SIRT1 cleavage reduced its enzymatic activity and capacity to activate its gene targets, as COL2A1. Paradoxically, the cleaved fragment of SIRT1 enabled the cells to survive an inflammatory environment, at the expense of their anabolic functions.

Further research into the exact role of SIRT1 and its impact on cartilage formation and maintenance will be applicable in drug development to combat OA through control of the SIRT1s activity.