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. 2022 Oct 13;17(10):e0268663.
doi: 10.1371/journal.pone.0268663. eCollection 2022.

RORβ modulates a gene program that is protective against articular cartilage damage

Mi Ra Chang  1 , Patrick R Griffin  1
Affiliations

RORβ modulates a gene program that is protective against articular cartilage damage

Mi Ra Chang et al. PLoS One. .

Abstract

Osteoarthritis (OA) is the most prevalent chronic joint disease which increases in frequency with age eventually impacting most people over the age of 65. OA is the leading cause of disability and impaired mobility, yet the pathogenesis of OA remains unclear. Treatments have focused mainly on pain relief and reducing joint swelling. Currently there are no effective treatments to slow the progression of the disease and to prevent irreversible loss of cartilage. Here we demonstrate that stable expression of RORβ in cultured cells results in alteration of a gene program that is supportive of chondrogenesis and is protective against development of OA. Specifically, we determined that RORβ alters the ratio of expression of the FGF receptors FGFR1 (associated with cartilage destruction) and FGFR3 (associated with cartilage protection). Additionally, ERK1/2-MAPK signaling was suppressed and AKT signaling was enhanced. These results suggest a critical role for RORβ in chondrogenesis and suggest that identification of mechanisms that control the expression of RORβ in chondrocytes could lead to the development of disease modifying therapies for the treatment of OA.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Selection of MG63 clone which is consistently over expressing hRORβ in MG63 cells.
hRORβ was subcloned into pLPCX vector which was transfected into MG63 cells and selected by 1ug/mL of puromycin. hRORβ was consistently overexpressing in MG63 that was confirmed by a) protein expression level and b) relative mRNA expression level. WT: mock vector (pLPCX) transfected MG63 cells, OE: Consistently over expressing of hRORβ in MG63 cells.
Fig 2
Fig 2. Differential gene expression sequence.
A) principle component analysis (PCA) of wild type (213–1, 213–2, 213–3) and over expressing RORβ clone (213–4, 213–5, 213–6) in NextSeq 500. Each condition is labeled with a separate color allowing for a visual method for identifying sample outliers. RORβOE cells and WT cells are colored in orange and green, respectively. B) Heatmap of the sample to sample distance. The scale is the Euclidean distance between two samples in multi-dimensional space which is also represented in different shades of blue. Each sample is most closely related to itself (with a Euclidian distance of 0) which explains the perfect relatedness (dark blue) along the diagonal. c) Suppression of osteoarthritis signaling pathway. mRNAseq data was analyzed using IPA program. The green color of genes represents significantly repressed fold changing genes on OE compared with WT and the red color of genes represent significantly increased fold changing genes on OE compared with WT in OA pathway signaling.
Fig 3
Fig 3. mRNA expression level for cartilage structure genes and cartilage destruction related genes.
a) Relative mRNA expression for ADAMTS4, MMP3, and IL6 which are direct/indirect cartilage destructive genes and b) the protein expression of MMP3 in OE/MG63 clone. c) Relative mRNA expression for aggrecan (ACAN) and COL2A1 which is main structure of cartilage maintain and d) the protein expression of ACAN on OE/MG63 clone. TC28α2 cells were used for positive control and MG63 cells were used for negative control. Graph with gray color is isotype control.
Fig 4
Fig 4. Reduction of FGFR1 expression and Erk signaling and enhanced FGFR3 expression and Akt signaling in RORβ OE cells.
a) Relative mRNA expression of FGFR1 and FGFR3 along with RORβ. b) The expression of phosphorylated Erk1/2 and phosphorylated Akt in RORβ over expressing MG63 cells.
Fig 5
Fig 5. Suppression of IL1β stimulated genes in hRORβ/MG63 clone.
Inflammatory cytokine IL1β was treated into WT or OE RORβ/MG63 cells for 24 hr with 2nM. The mRNA expression level of IL6, MMP3 and ADAMTS4 was significantly increased by IL1 stimulation but it was protected in RORβ OE cells. The RORβ expression did not affect by IL1β stimulation.
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