The forkhead box O (FOXO) proteins are transcription factors involved in the differentiation of many cell types. Type II collagen (Col2) Cre-Foxo1-knockout and Col2-Cre-Foxo1,3,4 triple-knockout mice exhibit growth plate malformation. Moreover, recent studies have reported that in some cells, the expressions and activities of FOXOs are promoted by transforming growth factor β1 (TGFβ1), a growth factor playing a key role in chondrogenic differentiation. Here, using a murine chondrogenic cell line (ATDC5), mouse embryos, and human mesenchymal stem cells, we report the mechanisms by which FOXOs affect chondrogenic differentiation. FOXO1 expression increased along with chondrogenic differentiation, and FOXO1 inhibition suppressed chondrogenic differentiation. TGFβ1/SMAD signaling promoted expression and activity of FOXO1. In ATDC5, FOXO1 knockdown suppressed expression of sex-determining region Y box 9 (Sox9), a master regulator of chondrogenic differentiation, resulting in decreased collagen type II α1 (Col2a1) and aggrecan (Acan) expression after TGFβ1 treatment. On the other hand, chemical FOXO1 inhibition suppressed Col2a1 and Acan expression without suppressing Sox9. To investigate the effects of FOXO1 on chondrogenic differentiation independently of SOX9, we examined FOXO1's effects on the cell cycle. FOXO1 inhibition suppressed expression of p21 and cell-cycle arrest in G0/G1 phase. Conversely, FOXO1 overexpression promoted expression of p21 and cell-cycle arrest. FOXO1 inhibition suppressed expression of nascent p21 RNA by TGFβ1, and FOXO1 bound the p21 promoter. p21 inhibition suppressed expression of Col2a1 and Acan during chondrogenic differentiation. These results suggest that FOXO1 is necessary for not only SOX9 expression, but also cell-cycle arrest during chondrogenic differentiation via TGFβ1 signaling.

Objective NF‐κB–dependent signaling is an important modulator in osteoarthritis (OA), and G protein–coupled receptor kinase 5 (GRK5) regulates the NF‐κB pathway. This study was undertaken to investigate the functional involvement of GRK5 in OA pathogenesis. Methods GRK5 expression in normal and OA human knee joints was analyzed immunohistochemically. Gain‐ or loss‐of‐function experiments were performed using human and mouse chondrocytes. OA was induced in GRK5‐knockout mice by destabilization of the medial meniscus, and histologic examination was performed. OA was also induced in wild‐type mice, which were then treated with an intraarticular injection of amlexanox, a selective GRK5 inhibitor, every 5 days for 8 weeks. Results GRK5 protein expression was increased in human OA cartilage. In vitro, expression levels of OA‐related factors and NF‐κB transcriptional activation were down‐regulated by suppression of the GRK5 gene in human OA chondrocytes (3.49‐fold decrease in IL6 [P < 0.01], 2.43‐fold decrease in MMP13 [P < 0.01], and 2.66‐fold decrease in ADAMTS4 [P < 0.01]). Conversely, GRK5 overexpression significantly increased the expression of OA‐related catabolic mediators and NF‐κB transcriptional activation. On Western blot analysis, GRK5 deletion reduced IκBα phosphorylation (up to 4.4‐fold decrease [P < 0.05]) and decreased p65 nuclear translocation (up to 6.4‐fold decrease [P < 0.01]) in mouse chondrocytes. In vivo, both GRK5 deletion and intraarticular amlexanox protected mouse cartilage against OA. Conclusion Our results suggest that GRK5 regulates cartilage degradation through a catabolic response mediated by NF‐κB signaling, and is a potential target for OA treatment. Furthermore, amlexanox may be a major compound in relevant drugs.

No abstract

ObjectivesOsteoarthritis (OA) features ageing-related defects in cellular homeostasis mechanisms in articular cartilage. These defects are associated with suppression of forkhead box O (FoxO) transcription factors. FoxO1 or FoxO3 deficient mice show early onset OA while FoxO1 protects against oxidative stress in chondrocytes and promotes expression of autophagy genes and the essential joint lubricant proteoglycan 4 (PRG4). The objective of this study was to identify small molecules that can increase FoxO1 expression.MethodsWe constructed a reporter cell line with FoxO1 promoter sequences and performed high-throughput screening (HTS) of the Repurposing, Focused Rescue and Accelerated Medchem (ReFRAME) library . Hits from the HTS were validated and function was assessed in human chondrocytes, meniscus cells and synoviocytes and following administration to mice. The most promising hit, the histone deacetylase inhibitor (HDACI) panobinostat was tested in a murine OA model.ResultsAmong the top hits were HDACI and testing in human chondrocytes, meniscus cells and synoviocytes showed that panobinostat was the most promising compound as it increased the expression of autophagy genes and PRG4 while suppressing the basal and IL-1β induced expression of inflammatory mediators and extracellular matrix degrading enzymes. Intraperitoneal administration of panobinostat also suppressed the expression of mediators of OA pathogenesis induced by intra-articular injection of IL-1β. In a murine OA model, panobinostat reduced the severity of histological changes in cartilage, synovium and subchondral bone and improved pain behaviours.ConclusionPanobinostat has a clinically relevant activity profile and is a candidate for OA symptom and structure modification.