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Pregnane X receptor knockout mice display aging-dependent wearing of articular cartilage.

Azuma K, Casey SC, Urano T, Horie-Inoue K, Ouchi Y, Blumberg B, Inoue S - PLoS ONE (2015)

Bottom Line: Here we report that systemic deletion of PXR results in aging-dependent wearing of articular cartilage of knee joints.Histomorphometrical analysis showed remarkable reduction of width and an enlarged gap between femoral and tibial articular cartilage in PXR knockout mice.We hypothesized that genes induced by SXR in chondrocytes have a protective effect on articular cartilage and identified Fam20a (family with sequence similarity 20a) as an SXR-dependent gene induced by the known SXR ligands, rifampicin and vitamin K2.

View Article: PubMed Central - PubMed

Affiliation: Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan.

ABSTRACT
Steroid and xenobiotic receptor (SXR) and its murine ortholog, pregnane X receptor (PXR), are nuclear receptors that are expressed at high levels in the liver and the intestine where they function as xenobiotic sensors that induce expression of genes involved in detoxification and drug excretion. Recent evidence showed that SXR and PXR are also expressed in bone tissue where they mediate bone metabolism. Here we report that systemic deletion of PXR results in aging-dependent wearing of articular cartilage of knee joints. Histomorphometrical analysis showed remarkable reduction of width and an enlarged gap between femoral and tibial articular cartilage in PXR knockout mice. We hypothesized that genes induced by SXR in chondrocytes have a protective effect on articular cartilage and identified Fam20a (family with sequence similarity 20a) as an SXR-dependent gene induced by the known SXR ligands, rifampicin and vitamin K2. Lastly, we demonstrated the biological significance of Fam20a expression in chondrocytes by evaluating osteoarthritis-related gene expression of primary articular chondrocytes. Consistent with epidemiological findings, our results indicate that SXR/PXR protects against aging-dependent wearing of articular cartilage and that ligands for SXR/PXR have potential role in preventing osteoarthritis caused by aging.

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Consensus SXR/PXR responsive element motifs in the first introns of SXR and PXR genes.(A) Consensus SXR/PXR responsive element motifs, variant direct repeat 5, were identified in the first intron of both murine PXR gene (chromosome 11) and human SXR gene (chromosome 17). The bold letters indicate consensus SXR/PXR binding motif. (B) Generation of reporter plasmid containing three copies of PXR responsive element (PXRE) and PXRE with mutation (PXREmut). Underlined letters indicate mutated nucleotides. (C) Cos7 cells were transfected with PXR or SXR expression vector and reporter plasmid containing murine PXR responsive element or mutated PXR responsive element, and β-galactosidase expression vector (β-gal). The cells were then treated with indicated concentrations of PXR agonist pregnane-16α-carbonitrile (PCN) or SXR agonist rifampicin (RIF) or vehicles: DMSO for PCN and ethanol (Et) for RIF. Data are shown as relative light units (R.L.U.) normalized by β-galactosidase activity. ** P < 0.01, ***P < 0.001 in Dunnett’s test with vehicle treated group as a control.
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pone.0119177.g004: Consensus SXR/PXR responsive element motifs in the first introns of SXR and PXR genes.(A) Consensus SXR/PXR responsive element motifs, variant direct repeat 5, were identified in the first intron of both murine PXR gene (chromosome 11) and human SXR gene (chromosome 17). The bold letters indicate consensus SXR/PXR binding motif. (B) Generation of reporter plasmid containing three copies of PXR responsive element (PXRE) and PXRE with mutation (PXREmut). Underlined letters indicate mutated nucleotides. (C) Cos7 cells were transfected with PXR or SXR expression vector and reporter plasmid containing murine PXR responsive element or mutated PXR responsive element, and β-galactosidase expression vector (β-gal). The cells were then treated with indicated concentrations of PXR agonist pregnane-16α-carbonitrile (PCN) or SXR agonist rifampicin (RIF) or vehicles: DMSO for PCN and ethanol (Et) for RIF. Data are shown as relative light units (R.L.U.) normalized by β-galactosidase activity. ** P < 0.01, ***P < 0.001 in Dunnett’s test with vehicle treated group as a control.

Mentions: We previously demonstrated that SXR is expressed in human primary chondrocytes at almost the same levels as in human primary osteoblasts [19]. Therefore, we hypothesized that SXR/PXR expressed in chondrocytes has a physiological role in maintaining articular cartilage and used the murine chondrocytic cell line ATDC5 for our initial investigations. Because ATDC5 cells express low amounts of endogenous PXR, we overexpressed human SXR in ATDC5 cells using an adenovirus vector. These cells were stimulated with the SXR ligands rifampicin, vitamin K2, or vehicle (ethanol) for 24 h. Gene expression was analyzed using microarrays, and hierarchical clustering analysis was performed. Clusters including SXR-dependent ligand-induced genes are shown in Fig. 3A and S1 Table. Among several candidate genes, induction of Fam20a (family with sequence similarity 20a) was validated by quantitative RT-PCR (Fig. 3B). A lower amount of Fam20a expression was detected in primary articular chondrocytes derived from PXR knockout mice compared with chondrocytes from wild-type mice (Fig. 3C), further supporting the SXR/PXR-dependent induction of Fam20a. We found a consensus SXR/PXR responsive element motif, variant direct repeat 5 [1], on the anti-sense strand in the first intron of both human and murine Fam20a genes (Fig. 4A). Transient transfection assays were performed using luciferase reporters containing three copies of the putative responsive element or three copies of a mutated element (Fig. 4B). Both the PXR agonist, pregnane-16α-carbonitrile (PCN), and the SXR agonist, rifampicin, increased the reporter gene activity in experiments using the wild-type element whereas they elicited no activity of the mutated element (Fig. 4C). This result indicated Fam20a is a primary responsive gene of SXR/PXR.


Pregnane X receptor knockout mice display aging-dependent wearing of articular cartilage.

Azuma K, Casey SC, Urano T, Horie-Inoue K, Ouchi Y, Blumberg B, Inoue S - PLoS ONE (2015)

Consensus SXR/PXR responsive element motifs in the first introns of SXR and PXR genes.(A) Consensus SXR/PXR responsive element motifs, variant direct repeat 5, were identified in the first intron of both murine PXR gene (chromosome 11) and human SXR gene (chromosome 17). The bold letters indicate consensus SXR/PXR binding motif. (B) Generation of reporter plasmid containing three copies of PXR responsive element (PXRE) and PXRE with mutation (PXREmut). Underlined letters indicate mutated nucleotides. (C) Cos7 cells were transfected with PXR or SXR expression vector and reporter plasmid containing murine PXR responsive element or mutated PXR responsive element, and β-galactosidase expression vector (β-gal). The cells were then treated with indicated concentrations of PXR agonist pregnane-16α-carbonitrile (PCN) or SXR agonist rifampicin (RIF) or vehicles: DMSO for PCN and ethanol (Et) for RIF. Data are shown as relative light units (R.L.U.) normalized by β-galactosidase activity. ** P < 0.01, ***P < 0.001 in Dunnett’s test with vehicle treated group as a control.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4352085&req=5

pone.0119177.g004: Consensus SXR/PXR responsive element motifs in the first introns of SXR and PXR genes.(A) Consensus SXR/PXR responsive element motifs, variant direct repeat 5, were identified in the first intron of both murine PXR gene (chromosome 11) and human SXR gene (chromosome 17). The bold letters indicate consensus SXR/PXR binding motif. (B) Generation of reporter plasmid containing three copies of PXR responsive element (PXRE) and PXRE with mutation (PXREmut). Underlined letters indicate mutated nucleotides. (C) Cos7 cells were transfected with PXR or SXR expression vector and reporter plasmid containing murine PXR responsive element or mutated PXR responsive element, and β-galactosidase expression vector (β-gal). The cells were then treated with indicated concentrations of PXR agonist pregnane-16α-carbonitrile (PCN) or SXR agonist rifampicin (RIF) or vehicles: DMSO for PCN and ethanol (Et) for RIF. Data are shown as relative light units (R.L.U.) normalized by β-galactosidase activity. ** P < 0.01, ***P < 0.001 in Dunnett’s test with vehicle treated group as a control.
Mentions: We previously demonstrated that SXR is expressed in human primary chondrocytes at almost the same levels as in human primary osteoblasts [19]. Therefore, we hypothesized that SXR/PXR expressed in chondrocytes has a physiological role in maintaining articular cartilage and used the murine chondrocytic cell line ATDC5 for our initial investigations. Because ATDC5 cells express low amounts of endogenous PXR, we overexpressed human SXR in ATDC5 cells using an adenovirus vector. These cells were stimulated with the SXR ligands rifampicin, vitamin K2, or vehicle (ethanol) for 24 h. Gene expression was analyzed using microarrays, and hierarchical clustering analysis was performed. Clusters including SXR-dependent ligand-induced genes are shown in Fig. 3A and S1 Table. Among several candidate genes, induction of Fam20a (family with sequence similarity 20a) was validated by quantitative RT-PCR (Fig. 3B). A lower amount of Fam20a expression was detected in primary articular chondrocytes derived from PXR knockout mice compared with chondrocytes from wild-type mice (Fig. 3C), further supporting the SXR/PXR-dependent induction of Fam20a. We found a consensus SXR/PXR responsive element motif, variant direct repeat 5 [1], on the anti-sense strand in the first intron of both human and murine Fam20a genes (Fig. 4A). Transient transfection assays were performed using luciferase reporters containing three copies of the putative responsive element or three copies of a mutated element (Fig. 4B). Both the PXR agonist, pregnane-16α-carbonitrile (PCN), and the SXR agonist, rifampicin, increased the reporter gene activity in experiments using the wild-type element whereas they elicited no activity of the mutated element (Fig. 4C). This result indicated Fam20a is a primary responsive gene of SXR/PXR.

Bottom Line: Here we report that systemic deletion of PXR results in aging-dependent wearing of articular cartilage of knee joints.Histomorphometrical analysis showed remarkable reduction of width and an enlarged gap between femoral and tibial articular cartilage in PXR knockout mice.We hypothesized that genes induced by SXR in chondrocytes have a protective effect on articular cartilage and identified Fam20a (family with sequence similarity 20a) as an SXR-dependent gene induced by the known SXR ligands, rifampicin and vitamin K2.

View Article: PubMed Central - PubMed

Affiliation: Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan.

ABSTRACT
Steroid and xenobiotic receptor (SXR) and its murine ortholog, pregnane X receptor (PXR), are nuclear receptors that are expressed at high levels in the liver and the intestine where they function as xenobiotic sensors that induce expression of genes involved in detoxification and drug excretion. Recent evidence showed that SXR and PXR are also expressed in bone tissue where they mediate bone metabolism. Here we report that systemic deletion of PXR results in aging-dependent wearing of articular cartilage of knee joints. Histomorphometrical analysis showed remarkable reduction of width and an enlarged gap between femoral and tibial articular cartilage in PXR knockout mice. We hypothesized that genes induced by SXR in chondrocytes have a protective effect on articular cartilage and identified Fam20a (family with sequence similarity 20a) as an SXR-dependent gene induced by the known SXR ligands, rifampicin and vitamin K2. Lastly, we demonstrated the biological significance of Fam20a expression in chondrocytes by evaluating osteoarthritis-related gene expression of primary articular chondrocytes. Consistent with epidemiological findings, our results indicate that SXR/PXR protects against aging-dependent wearing of articular cartilage and that ligands for SXR/PXR have potential role in preventing osteoarthritis caused by aging.

Show MeSH
Related in: MedlinePlus