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A new player in cartilage homeostasis: adiponectin induces nitric oxide synthase type II and pro-inflammatory cytokines in chondrocytes  R. Lago, B.S.,

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Presentation on theme: "A new player in cartilage homeostasis: adiponectin induces nitric oxide synthase type II and pro-inflammatory cytokines in chondrocytes  R. Lago, B.S.,"— Presentation transcript:

1 A new player in cartilage homeostasis: adiponectin induces nitric oxide synthase type II and pro-inflammatory cytokines in chondrocytes  R. Lago, B.S., R. Gomez, B.S., M. Otero, Ph.D., F. Lago, Ph.D., R. Gallego, Ph.D., C. Dieguez, M.D., Ph.D., J.J. Gomez-Reino, M.D., Ph.D., O. Gualillo, Ph.D., Pharm.D.  Osteoarthritis and Cartilage  Volume 16, Issue 9, Pages (September 2008) DOI: /j.joca Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

2 Fig. 1 Adiponectin receptors expression in chondrocytes. Left panel: AdipoR1 and AdipoR2 are expressed in undifferentiated ATDC5 murine cell line (a and b, respectively), in differentiated ATDC5 murine cell line (c and d, respectively), and in C28/I2 human immortalized chondrocytes (e and f, respectively). Right panel: AdipoR1 is expressed in human normal cultured chondrocytes (a) and in human cultured chondrocytes from osteoarthritic patients (g). AdipoR2 is expressed in human normal cultured chondrocytes (d) and in human cultured chondrocytes from osteoarthritic patients (j). All cultured chondrocytes showed positive immunofluorescence for collagen type II as chondrocyte specific marker (b, e, h and k). Co-localization of AdipoRs and collagen type II is shown in c, f, i and l. Osteoarthritis and Cartilage  , DOI: ( /j.joca ) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

3 Fig. 2 AdipoRs expression and functionality in chondrocytes. Panel A: AdipoR1 and AdipoR2 mRNA expression in ATDC5 (AT) cells determined by RT-PCR. SM and Liv were used as positive controls. Panel B: AdipoR1 and AdipoR2 mRNA expression in C20A4 (C20), C28/I2 (C28), TC28a2 (TC) and human cultured chondrocytes (hC), determined by RT-PCR. SM and Liv were used as positive controls. Panel C: WB for phosphorylated AMPK. Phosphorylated AMPK was identified by phospho-AMPKα Ab Thr172 (Cell Signalling catalog number 2531, 62kDa) in lysates coming from ATDC5 cells stimulated with adiponectin (1μg/ml). Phosphorylation levels were evaluated, in time-dependent experiments, and compared to total AMPK (AMPKα Ab, Cell Signalling catalog number 2532, 62kDa). Blot is representative of at least four independent experiments (C1). Densitometric analysis of WB experiments is reported in panel C2. Data are represented as percent±s.e.m. over basal AMPK phosphorylation in unstimulated cells. Osteoarthritis and Cartilage  , DOI: ( /j.joca ) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

4 Fig. 3 NOS2 induction by adiponectin. Panel A: Adiponectin (0.1 and 1μg/ml) induces nitrite accumulation in ATDC5 cells in a dose-dependent manner. LPS (500ng/ml) was used as a positive control. Nitrite has been evaluated at 24 (black bars) and 48h (gray bars). This result was confirmed by WB. Adiponectin-induced NOS2 synthesis in a dose-dependent manner and LPS was used as a positive control at 48h (panel inside). Panel B: Adiponectin-induced (1μg/ml) nitrite accumulation in ATDC5 cells was modulated by different specific pharmacological inhibitors [aminoguanidine (1mM), LY (10μM) and dexamethasone (10μM)]. All inhibitors used 1h before adiponectin treatment, reduced significantly nitrite accumulation whereas NOS2 synthesis is decreased by LY (B2). PI3-K is phosphorylated upon adiponectin treatment. Adiponectin-induced phosphorylation of PI3-K is prevented by LY (B1). Panel C: Nitrite accumulation in human cultured chondrocytes induced by adiponectin (1μg/ml) in presence or not of different specific pharmacological inhibitors [aminoguanidine (1mM), LY (10μM)] at 24 (black bars) and 48h (gray bars). NOS2 protein expression is selectively inhibited by LY (C1). Osteoarthritis and Cartilage  , DOI: ( /j.joca ) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

5 Fig. 4 Adiponectin induces pro-inflammatory cytokines. Adiponectin (0.1 and 1μg/ml) induces a significant increase in IL-6, MCP-1, MMP-3, and MMP-9 production by murine ATDC5 cells in a dose-dependent manner (panels A–F). This effect is strongly decreased by the PI3-K inhibitor LY (10μM) for IL-6, and MMP-9 (panels A and F). LY partially attenuated adiponectin-induced MCP-1 (B). Adiponectin is unable to modulate IL-1β, MMP-2 levels (panels C and D). Osteoarthritis and Cartilage  , DOI: ( /j.joca ) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

6 Fig. 5 Adiponectin does not modulate prostanoids, TIMP-1 or TNF-α. Adiponectin (0.1 and 1μg/ml) does not induce any significant modulation in TIMP-1 (A), LTB4 (B), PGE2 (C) and TNF-α (D). Osteoarthritis and Cartilage  , DOI: ( /j.joca ) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

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