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Expression of proteinase-activated receptors (PAR)-2 in articular chondrocytes is modulated by IL-1β, TNF-α and TGF-β  Y. Xiang, M.D., K. Masuko-Hongo,

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Presentation on theme: "Expression of proteinase-activated receptors (PAR)-2 in articular chondrocytes is modulated by IL-1β, TNF-α and TGF-β  Y. Xiang, M.D., K. Masuko-Hongo,"— Presentation transcript:

1 Expression of proteinase-activated receptors (PAR)-2 in articular chondrocytes is modulated by IL-1β, TNF-α and TGF-β  Y. Xiang, M.D., K. Masuko-Hongo, M.D., Ph.D., T. Sekine, Ph.D., H. Nakamura, M.D., Ph.D., K. Yudoh, M.D., Ph.D., K. Nishioka, M.D., Ph.D., T. Kato, M.D., Ph.D.  Osteoarthritis and Cartilage  Volume 14, Issue 11, Pages (November 2006) DOI: /j.joca Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

2 Fig. 1 Expression of the PAR family in human articular chondrocytes. RT-PCR was performed to detect PARs mRNA in the target cells. This was repeated three times for each sample, and representative results are shown. β-Actin and PAR-1 to -4 were amplified from the cDNA of target cells. Total RNA was extracted from the following samples: human pulmonary epithelial cells (A549, line 1), HUVEC (line 2), primary synovial fibroblast-like cells from three normal synovial tissues (NFLS, line 3; a representative result) and from eight patients with OA (OFLS, line 4; a representative result), uncultured fresh chondrocytes separated from three normal articular cartilage tissues (NCT, line 5; a representative result) and from eight OA patients (OCT, line 6; a representative result), primary chondrocytes from three individual normal cartilage samples (NC-1 to NC-3, lines 7–9) and from eight individual OA cartilage samples (OC-1 to OC-3, lines 10–12; three representative results). Water was used in place of cDNA from target cells as a negative control (water, line 13). Osteoarthritis and Cartilage  , DOI: ( /j.joca ) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

3 Fig. 2 Comparison of PAR-2 mRNA levels between synovial fibroblast-like cells and chondrocytes. (a) To determine the copy number level of each real-time PCR product, standard curves were generated by measuring the fluorescence vs cycle number over a dilution range of the constructs. PAR-2 standard curve (a representative result) is shown. Left, fluorescence vs cycles for different dilutions of copy number of the PAR-2 construct. Right, the standard curve for determining the arbitrary units of PAR-2. (b) Real-time PCR was performed to evaluate the PAR-2 mRNA level expressed in chondrocytes from three samples of normal (NC) and eight samples of OA (OC) cartilage tissues, as well as in synovial fibroblast-like cells from normal (NFLS) and OA (OFLS) synovial tissues of the same patient. mRNA levels in the cells were normalized to the mRNA level of β-actin and are expressed as the arbitrary units of PAR-2 mRNA per 1000 mRNA copies of β-actin expressed in the same cells. Osteoarthritis and Cartilage  , DOI: ( /j.joca ) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

4 Fig. 3 Comparison of PAR-2 protein levels between normal and OA chondrocytes and FLS by WB. (a) WB was performed to evaluate PAR-2 protein levels in three normal and eight OA samples of cultured chondrocytes at passage 1 as well as in three NFLS and eight OFLS samples from the same patients. Mouse anti-PAR-2 antibody was diluted to 1:200 in PBS containing 0.05% Tween 20 as the primary antibody, while the secondary antibody was horse radish peroxidase-conjugated sheep anti-mouse IgG diluted to 1:10,000. WB results of three normal samples (NC-1 to -3), three OA samples (OC-1 to -3) as well as one NFLS and one OFLS sample are shown as representatives; upper panel, WB of PAR-2 protein and GAPDH; lower panel, the relative levels of PAR-2 protein extrapolated from the band intensity ratio of PAR-2/GAPDH. Asterisk (*) indicates a significant difference in PAR-2 protein levels between normal and OA cells and between chondrocytes and FLS. *P<0.05; ***P< (b) WB was performed with cytokine-stimulated cell lysates to quantify the effect of stimulation by IL-1β, TNF-α and TGF-β for 24h on protein levels. Upper panel, the representative WB results from one healthy (NC) and one OA chondrocyte (OC) sample in the absence (non) or presence of stimulants (IL-1β, TNF-α and TGF-β); lower panel, the relative levels of PAR-2 protein estimated from the band intensity ratio of PAR-2/GAPDH. Osteoarthritis and Cartilage  , DOI: ( /j.joca ) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

5 Fig. 4 Expression of PAR-2 in chondrocytes at different passages. Real-time PCR was performed to evaluate the PAR-2 mRNA level expressed in chondrocytes at different passages from three samples of normal (NC) and three samples of OA chondrocytes (OC). Total RNA was extracted from uncultured fresh chondrocytes (fresh), and from cultured chondrocytes at passages from 0 to 6 (P0 to P6). The differences between mean PAR-2 mRNA levels were compared between OA and normal chondrocytes. *P<0.05; ***P<0.001. Osteoarthritis and Cartilage  , DOI: ( /j.joca ) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

6 Fig. 5 Regulation of PAR-2 expression in chondrocytes by IL-1β. (a and b) Concentration-response. Total RNA was extracted from eight samples of OA chondrocytes and three samples of normal chondrocytes after culture in FCS-free DMEM without or with 1, 10 or 100ng/ml IL-1β for 24h. Results were normalized to the β-actin mRNA level and the normalized PAR-2 expression in IL-1β-stimulated and unstimulated chondrocytes was compared. The changes of PAR-2 mRNA in arbitrary units (a) and the fold-change in PAR-2 mRNA (b) in chondrocytes without and with stimulation by different concentration of IL-1β are shown. (c and d) Time course. Total RNA was extracted from chondrocytes after culture in FCS-free DMEM without or with 10ng/ml IL-1β for 12, 24 and 48h. The changes in PAR-2 mRNA in arbitrary units (c) and the fold change in PAR-2 (d) before and after stimulation by IL-1β for different durations are shown. NC, normal chondrocytes; OC, OA chondrocytes; Non, unstimulated chondrocytes. *P<0.05; **P<0.01. Osteoarthritis and Cartilage  , DOI: ( /j.joca ) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

7 Fig. 6 Regulation of PAR-2 expression in chondrocytes by TNF-α. (a and b) Concentration-response. Total RNA was extracted from eight samples of OA chondrocytes (OC) and three samples of normal chondrocytes (NC) after culture in FCS-free DMEM without or with 1, 10 or 100ng/ml TNF-α for 24h. The β-actin-normalized mRNA expression levels of PAR-2 in TNF-α-stimulated and unstimulated chondrocytes were compared. The changes in PAR-2 mRNA arbitrary units (a) and the fold increase of PAR-2 (b) in chondrocytes without and with stimulation by different concentrations of TNF-α are shown.(c and d) Time course. Total RNA was extracted from chondrocytes after culture in FCS-free DMEM without or with 10ng/ml TNF-α for 12, 24 and 48h. The changes in PAR-2 mRNA arbitrary unit (c) and the fold-change in PAR-2 (d) before and after stimulation by TNF-α for different durations are shown. NC, normal chondrocytes; OC, OA chondrocytes; Non, unstimulated chondrocytes. *P<0.05; **P<0.01. Osteoarthritis and Cartilage  , DOI: ( /j.joca ) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

8 Fig. 7 Regulation of PAR-2 expression in chondrocytes by TGF-β1. (a and b) Concentration-response. Total RNA was extracted from eight samples of OA chondrocytes and three samples of normal chondrocytes after culture in FCS-free DMEM without or with 0.1, 1 or 10ng/ml TGF-β1 for 24h. The β-actin-normalized mRNA expression levels of PAR-2 in TGF-β1-stimulated and unstimulated chondrocytes were compared. The changes in PAR-2 mRNA arbitrary units (a) and the fold change in PAR-2 (b) before and after stimulation by different concentrations of TGF-β1 are shown. (c and d) Time course. Total RNA was extracted from chondrocytes after culture in FCS-free DMEM without or with 1.0ng/ml TGF-β1 for 12, 24 and 48h. The changes in PAR-2 mRNA arbitrary units (c) and the fold change in PAR-2 (d) after stimulation with TGF-β1 for different durations are shown. NC, normal chondrocytes; OC, OA chondrocytes; Non, unstimulated chondrocytes. *P<0.05; **P<0.01. Osteoarthritis and Cartilage  , DOI: ( /j.joca ) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

9 Fig. 8 Comparison of PAR-2 synthesis by IFA in cultured chondrocytes before and after stimulation. Chondrocytes from three normal and three OA samples, cultured in eight-well chamber-slides and incubated in the absence or presence of IL-1β (10ng/ml), TNF-α (10ng/ml) and TGF-β1 (1ng/ml) for 24h, were stained with mouse anti-human PAR-2 IgG (SAM11) and then visualized with FITC-labeled rabbit anti-mouse IgG. Upper panel (I–VI), OA chondrocytes; lower panel (VII–XII), normal chondrocytes. I and VII, hematoxylin-stained unstimulated chondrocytes matching II and VIII, the representative phase contrast; II and VIII, unstimulated chondrocytes; III and IX, cells stimulated with IL-1β; IV and X, cells stimulated with TNF-α; V and XI, cells stimulated with TGF-β; VI and XII, mouse IgG was used in place of anti-PAR-2 antibody as negative controls. Osteoarthritis and Cartilage  , DOI: ( /j.joca ) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

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