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HU308 Mitigates Osteoarthritis by Stimulating Sox9-Related Networks of Carbohydrate Metabolism

ABSTRACT: Osteoarthritis (OA) is characterized by progressive, irreversible erosion of articular cartilage accompanied with severe pain and immobility. This study aimed to assess the effect and mechanism of action of HU308- a selective cannabinoid receptor type 2 (CB2) agonist, in potentially preventing OA-related joint damage. To test this assumption, Cn2r null mice and WT mice were aged to reach 20 months, and analyzed for joint structural features. OA was induced in Wild type (WT) mice via ageing or post-traumatic procedure, followed by HU308 systemic (intraperitoneal) or local (intra-articular) administration, respectively. Additional analysis of time and dose courses for HU308 were carried out in human primary chondrocytes, analyzed by RNA-sequencing, RT-PCR, ChIP and immunoblotting. Our results show that Cnr2r null mice exhibited enhanced age-related OA severity and synovitis compared to age-matched WT mice. Systemic administration of HU308 to 16-month mice improved pain sensitivity and maintained joint integrity, which was consistent with the intra-articular administration of HU308 in post-traumatic OA mice. When assessing human chondrocytes treated with HU308, we uncovered a dose- and time related increase in ACAN and COL2A1 expression, which was preceded by increased SOX9 expression due to pCREB transcriptional activation of the SOX9 gene. Finally, transcriptomic analysis of patient-derived human chondrocytes, identified HU308 populations exhibiting enhanced SOX9 expression, accompanied by enriched gene networks related to carbohydrate metabolism. Collectively, the results show that HU308 reduced trauma and age-induced OA, via CB2-pCREB dependent activation of SOX9, contributing to augmented genes networks related to metabolism.

ORGANISM(S): Homo sapiens

PROVIDER: GSE210826 | GEO | 2022/11/27

REPOSITORIES: GEO

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Project description:Autologous chondrocyte transplantation (ACT) is a routine technique to regenerate focal cartilage lesions. However, patients with osteoarthritis (OA) are lacking an appropriate long-lasting treatment alternative, partly since it is not known if chondrocytes from OA patients have the same chondrogenic differentiation potential as chondrocytes from donors not affected by OA. Articular chondrocytes from patients with OA undergoing total knee replacement (Mankin Score >3, AhlbÃ¤ck Score >2) and from patients undergoing ACT, here referred to as normal donors (ND), were isolated applying protocols used for ACT. Their chondrogenic differentiation potential was evaluated both in high-density pellet and scaffold (Hyaff-11) cultures by histological proteoglycan assessment (Bern Score) and immunohistochemistry for collagen types I and II. Chondrocytes cultured in monolayer and scaffolds were subjected to gene expression profiling using genome-wide oligonucleotide microarrays. Expression data were verified by using quantitative RT-PCR. Chondrocytes from ND and OA donors demonstrated accumulation of comparable amounts of cartilage matrix components, including sulphated proteoglycans and collagen types I and II. The mRNA expression of cartilage markers (COL2A1, COMP, aggrecan, CRTL1, SOX9) and genes involved in matrix synthesis (biglycan, COL9A2, COL11A1, TIMP4, CILP2) was highly induced in 3D cultures of chondrocytes from both donor groups. Genes associated with hypertrophic or OA cartilage (COL10A1, RUNX2, periostin, ALP, PTHR1, MMP13, COL1A1, COL3A1) were not significantly regulated between the two groups of donors. The expression of 661 genes, including COMP, FN1, and SOX9, were differentially regulated between OA and ND chondrocytes cultured in monolayer. During scaffold culture, the differences diminished between the OA and ND chondrocytes, and only 184 genes were differentially regulated. Only few genes were differentially expressed between OA and ND chondrocytes in Hyaff-11 culture. The risk of differentiation into hypertrophic cartilage does not seem to be increased for OA chondrocytes. Our findings suggest that the chondrogenic capacity is not significantly affected by OA and OA chondrocytes fulfill the requirements for matrix-associated ACT. Experiment Overall Design: Gene expression profiles of monolayer cultures (ML; passage 2) and Hyaff-11 scaffold cultures (3D; 14 days in vitro) of chondrocytes from 3 normal donors (ND; underwent ACT treatment) and 3 donors suffering from Osteoarthritis (OA; underwent knee replacement surgery) were determined. Comparative analyses between 3D and ML cultures (3D vs. ML) were performed to assess differentiation capacity of ND and OA chondrocytes. Furthermore, OA-related differences were determined comparing OA and ND monolayers as well as scaffold cultures (each OA vs. ND).

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HU308 Mitigates Osteoarthritis by Stimulating Sox9-Related Networks of Carbohydrate Metabolism

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