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Fibroblast Growth Factor 23 drives MMP13 expression in human osteoarthritic chondrocytes in a Klotho-independent manner A. Bianchi, M. Guibert, F. Cailotto, A. Gasser, N. Presle, D. Mainard, P. Netter, H. Kempf, J.-Y. Jouzeau, P. Reboul Osteoarthritis and Cartilage Volume 24, Issue 11, Pages (November 2016) DOI: /j.joca Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions
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Fig. 1 FGF23, FGFRs and Klotho expression in healthy vs OA primary chondrocytes. Total RNA was extracted from primary cultured human chondrocytes collected from healthy donors (H, n = 7) or OA patients (OA, n = 18) then reverse transcribed into cDNA and analysed by real-time PCR. The relative abundance of FGF23, Klotho and FGFR mRNAs was normalized to RPS29 mRNA. We indicated the mean of Ct values of genes on each panel. Comparison was made by using the ΔΔCt method with the fold value of reference = 1). The lines show the average values ± 95% CI. Statistics were calculated with respect to healthy cells. Exact P values were given unless P < 0.001. Osteoarthritis and Cartilage , DOI: ( /j.joca ) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions
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Fig. 2 FGF23, FGFR1 and Klotho expression in human cartilage. A) FGF23 and FGFR1 immunohistochemistry in healthy vs OA human cartilage. Safranin O-Fast Green staining and immunohistochemical detection of FGF23 and FGFR1 in representative sections from cartilage biopsies obtained from healthy donors (H, n = 10) and OA patients (OA, n = 22). Magnification ×10. Graphs represent the average of the number of positive cells counted by field under light microscope. The lines show the average values ± 95% CI. Statistics were calculated with respect to Mild zones. Exact P values were given unless P < B) Differential expression of FGF23, FGFR1, Klotho, MMP13 and Col10A1 in OA human cartilage according to the severity of cartilage degradation. The OA cartilage from tibial plateau was obtained from patients after knee joint replacement and assigned, according to macroscopic observation, into two groups of either preserved (Mild) or damaged (Severe) cartilage. Expression was analysed by RT-real-time PCR. Comparison was made by using the ΔΔCt method with the fold value of reference (fold = 1) assigned to mild samples. The lines show the average values ± 95% CI. Statistics were calculated with respect to Mild zones. Exact P values were given unless P < 0.001, n = 6 patients. Osteoarthritis and Cartilage , DOI: ( /j.joca ) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions
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Fig. 3 Effect of rhFGF23 on the expression of FGF23 and markers of hypertrophy in OA chondrocytes: Implication of FGFR1 and Klotho. Human OA chondrocytes were stimulated with increasing concentrations of rhFGF23. A) RT – real-time PCR analysis was performed 24 h after exposure of chondrocytes to rhFGF23. B) Collagenase 3 activity was determined in 48 h cultured supernatants using a fluorometric assay. C–F) Human OA chondrocytes were transfected with 10 nM of either FGFR1 or Klotho or scramble (Scr) siRNAs. FGF23, MMP13, Col10 and VEGF mRNAs were analysed by RT – real-time PCR. The lines show the average values ± 95% CI. Statistics were calculated with respect to Scr alone or Scr+100 ng/ml rhFGF23. Exact P values were given unless P < 0.001, n = 4 for all panels. Osteoarthritis and Cartilage , DOI: ( /j.joca ) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions
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Fig. 4 Identification of rhFGF23 signalling pathways in human OA chondrocytes. A) Kinetics of signalling events induced by 100 ng/ml rhFGF23. Total proteins were extracted from human chondrocytes and subjected to MAPK antibody arrays. Up and low blue circles: p38α and p38δ isoforms, respectively. Up and low red circles: ERK1 and ERK2 isoforms, respectively. Up, middle and low magenta circles: AKT1, AKT2 isoforms and panAKT activation, respectively. B) Immunoblotting studies of rhFGF23-induced signalling events. Experiments were performed using anti-phospho-ERK1/2, anti-phospho-p38-MAPK, anti-phospho-PI3K and anti-phospho-AKT antibodies. Loadings were controlled with β-actin detection. Osteoarthritis and Cartilage , DOI: ( /j.joca ) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions
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Fig. 5 Contribution of signalling pathways to rhFGF23-induced MMP13 expression. After 1 h of pre-treatment with selective inhibitors of each signalling pathway, human chondrocytes were stimulated for 24 h with 100 ng/ml of rhFGF23. A) MMP13 expression was analysed by RT – real–time PCR. B) Collagenase 3 activity was measured using a fluorometric assay. LY: LY294002, PI3K inhibitor (10 μM); PD: PD98059, MEK-1 inhibitor (30 μM); SB: SB203580, p38-MAPK inhibitor (10 μM). Data are expressed as mean ± SEM (n = 4 patients). The lines show the average values ± 95% CI. Statistics were calculated with respect to vehicle alone or vehicle + rhFGF23. Exact P values were given unless P < 0.001, n = 4. Osteoarthritis and Cartilage , DOI: ( /j.joca ) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions
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Supplemental Fig. 1 Efficacy of RNA silencing against FGFR1 and Klotho. Human OA chondrocytes were transfected with 10 nM of either FGFR1 or Klotho or scramble (Scr) siRNAs. A & C) FGFR1 and B & D) Klotho mRNAs were analysed by RT – real-time PCR or western blot, respectively. The lines show the average values ± 95% CI. Statistics were calculated with respect to Scr alone. Exact P values were given unless P < 0.001, n = 4. Osteoarthritis and Cartilage , DOI: ( /j.joca ) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions
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