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. 2010;12(5):R195.
doi: 10.1186/ar3166. Epub 2010 Oct 18.

Pomegranate Extract Inhibits the interleukin-1β-induced Activation of MKK-3, p38α-MAPK and Transcription Factor RUNX-2 in Human Osteoarthritis Chondrocytes

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Free PMC article

Pomegranate Extract Inhibits the interleukin-1β-induced Activation of MKK-3, p38α-MAPK and Transcription Factor RUNX-2 in Human Osteoarthritis Chondrocytes

Zafar Rasheed et al. Arthritis Res Ther. .
Free PMC article

Abstract

Introduction: Pomegranate has been revered throughout history for its medicinal properties. p38-MAPK is a major signal-transducing pathway in osteoarthritis (OA) and its activation by interleukin-1β (IL-1β) plays a critical role in the expression and production of several mediators of cartilage catabolism in OA. In this study we determined the effect of a standardized pomegranate extract (PE) on the IL-1β-induced activation of MKK3/6, p38-MAPK isoforms and the activation of transcription factor RUNX-2 in primary human OA chondrocytes.

Methods: Human chondrocytes were derived from OA cartilage by enzymatic digestion, treated with PE and then stimulated with IL-1β. Gene expression of p38-MAPK isoforms was measured by RT-PCR. Western immunoblotting was used to analyze the activation of MAPKs. Immunoprecipitation was used to determine the activation of p38-MAPK isoforms. DNA binding activity of RUNX-2 was determined using a highly sensitive and specific ELISA. Pharmacological studies to elucidate the involved pathways were executed using transfection with siRNAs.

Results: Human OA chondrocytes expressed p38-MAPK isoforms p38α, -γ and -δ, but not p38β. IL-1β enhances the phosphorylation of the p38α-MAPK and p38γ-MAPK isoforms but not of p38δ-MAPK isoform in human OA chondrocytes. Activation of p38-MAPK in human OA chondrocytes was preferentially mediated via activation of MKK3. In addition, we also demonstrate that polyphenol rich PE inhibited the IL-1β-induced activation of MKK3, p38α-MAPK isoform and DNA binding activity of the transcription factor RUNX-2.

Conclusions: Our results provide an important insight into the molecular basis of the reported cartilage protective and arthritis inhibitory effects of pomegranate extract. These novel pharmacological actions of PE on IL-1β stimulated human OA chondrocytes impart a new suggestion that PE or PE-derived compounds may be developed as MKK and p38-MAPK inhibitors for the treatment of OA and other degenerative/inflammatory diseases.

Figures

Figure 1
Figure 1
Differential inhibition of IL-1β-induced activation of MKK3 and MKK6 by PE in human OA chondrocytes. (a) Specificity of anti-MKK3 and anti-MKK6 monoclonal antibodies used in these studies was determined by using in vitro translated MKK3 and MKK6 proteins. No cross reactivity with the non-target protein was observed. (b) Expression of MKK3 and MKK6 in human chondrocytes. Human OA chondrocytes were grown to 70 to 80% confluence, serum starved overnight and then stimulated with IL-1β (5 ng/ml) for 15 minutes while control cultures received only the fresh medium. Unstimulated and stimulated human OA chondrocytes were lyzed and lysates were used for the immunoprecipitation of MKK3 and MKK6 using monoclonal antibodies. Immunoprecipitated protein was resolved on a 4 to 20% gradient gel by SDS-PAGE and Western blots were probed with anti-phosphoMKK3/6 antibodies. (c) Effect of PE on MKK3 and MKK6 phosphorylation in IL-1β-stimulated human OA chondrocytes. After treatment with PE (6.25 to 100 μg/ml) for 2 h at 37°C, primary human OA chondrocytes (70 to 80% confluent) were incubated with IL-1β (10 ng/ml) for 30 minutes, and then the phosphorylation of MKK3 and MKK6 was determined by immunoblotting using anti-phospho-MKK3 (Ser189)/anti-phospho-MKK6 (Ser207) antibody. Band images were digitally captured and the band intensities (pixels/band) were obtained using the Un-Scan-It software and are expressed as average pixels/band. The data shown are cumulative of two experiments. Average pixel values are presented as Mean ± SD; data without a common letter differ, P < 0.05.
Figure 2
Figure 2
Activation profile of p38-MAPK in IL-1β-stimulated primary human OA chondrocytes. (a) Primary human OA chondrocytes (70 to 80% confluent) were incubated with IL-1β (10 ng/ml) for 5 to 120 minutes, and then the phosphorylation of p38-MAPK was determined by Western immunoblot analysis using primary antibodies specific for phospho-p38-MAPK and p38-MAPK. (b) SiRNA-mediated knockdown of MKK3 and p38-MAPK phosphorylation in IL-1β-stimulated human OA chondrocytes. MKK3-siRNA (30 to 300 nM) transfected human OA chondrocytes were incubated with IL-1β (10 ng/ml) for 30 minutes, then the phosphorylation of p38-MAPK was determined by Western immunoblot analysis. (c) SiRNA-mediated knockdown of MKK6- and p38-MAPK phosphorylation in IL-1β-stimulated human OA chondrocytes. MKK6-siRNA (30 to 300 nM) transfected human OA chondrocytes were incubated with IL-1β (10 ng/ml) for 30 minutes, then the phosphorylation of p38-MAPK was determined by Western immunoblotting. Band images were digitally captured and the band intensities were obtained using UN-San-It software and are expressed in average pixels/band. Data shown are cumulative of two experiments. Average pixel values presented as Mean ± SD; data without a common letter differ, P < 0.05.
Figure 3
Figure 3
Gene expression of p38-MAPK isoforms in primary human OA chondrocytes. (a) Total RNA was prepared from the human OA chondrocytes, single stranded cDNA was synthesized and the PCR reaction was carried out using specific primers for p38-MAPKα, β, γ and δ isoforms as described in methods section above. (b) Protein expression of p38-MAPK isoforms in human OA chondrocytes, C28-I2 human chondrocytes and Hela cells. Western blot analysis was performed as described under Figure 1. Band images were digitally captured and the band intensities (pixels/band) were obtained using the Un-Scan-It software. Data shown are cumulative of two experiments. Average pixel values presented as Mean ± SD; data without a common letter differ, P < 0.05.
Figure 4
Figure 4
Inhibition of IL-1β-stimulated phosphorylation of p38-MAPK by PE in primary human OA chondrocytes. (a) Primary human OA chondrocytes were pretreated with PE for 2 h and then stimulated with IL-1β for 30 minutes and total cell lysate proteins were resolved by SDS-PAGE and analyzed by Western immunoblotting using primary antibodies specific for phospho-p38-MAPK and total p38-MAPK. (b) Inhibition of IL-1β-stimulated phosphorylation of p38-MAPK isoforms by PE in primary human OA chondrocytes. Primary human OA chondrocytes were pretreated with PE for 2 h and then stimulated with IL-1β for 30 minutes and total cell lysate were used to immunoprecipitate p38-α, γ and δ -MAPKs using monoclonal antibodies specific for each isoform as described above. Immunoprecipited protein was resolved by SDS-PAGE and Western blots were probed with anti-phospho-p38-MAPKantibody. Band images were digitally captured and the band intensities (pixels/band) were obtained as described under Figure 1. Average pixel values presented as mean ± SD; data without a common letter differ, P < 0.05.
Figure 5
Figure 5
PE inhibited the activation of RUNX-2 in primary human OA chondrocytes. (a) Primary human OA chondrocytes were pretreated with PE for 2 h and stimulated with IL-1β for 30 minutes and the presence of activated RUNX-2 in the nucleus was determined by Immunoblotting. Protein loading was verified by using an antibody against the nuclear protein poly (ADP-ribose) polymerase-1 (PARP-1). Band images were digitally captured and the band intensities (pixels/band) were obtained as described under Figure 1. Average pixel values presented as mean ± SD. (b) PE inhibited the DNA binding activity of RUNX-2 in IL-1β-stimulated human OA chondrocytes. DNA binding activity of activated RUNX-2 present in the nucleus was determined by using a Transcription Factor ELISA kit specific for RUNX-2 (Active Motif). The Saos-2 nuclear extract supplied with the kit was used as a positive control. Data is representative of two experiments and is presented as Mean ± SEM. Values differ without a common letter P < 0.05. (c) PE does not block the interaction of IL-1β with IL-1 receptor. Human T cells HSB.2 (70 to 80% confluent) were stimulated with IL-1β and PHA in the presence or absence of PE and the production of IL-2 was quantified by a sandwich ELISA (R&D Systems) and the values were calculated from a standard curve. Data presented as Mean ± SEM; data without a common letter differ, P < 0.05.
Figure 6
Figure 6
PE inhibits IL-1β-induced signaling cascade in human OA chondrocytes. The MAPKs are a group of signaling molecules that include the p38-MAPKs which are activated by upstream MAPK kinases MKK3 and MKK6. In human OA chondrocytes, IL-1β through IL-1R activates preferentially MKK3 which leads to the activation of p38-MAPK → RUNX2 pathway. Polyphenols rich PE inhibited the IL-1β-induced activation of MKK# and p38-MAPK in human OA chondrocytes. Breaking lines indicate reduced phosphorylation. IL-1R, IL-1β receptor; MAPK, mitogen-activated protein kinase; MKK, MAPK kinases; MMPs, matrix metalloproteinases; p, phosphorylation; PE, pomegranate extract; RUNX2, runt-related transcription factor-2.

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References

    1. Loeser RF, Erickson EA, Long DL. Mitogen-activated protein kinases as therapeutic targets in osteoarthritis. Curr Opin Rheumatol. 2008;20:581–586. doi: 10.1097/BOR.0b013e3283090463. - DOI - PMC - PubMed
    1. Kumar S, Votta BJ, Rieman DJ, Badger AM, Gowen M, Lee JC. IL-1- and TNF-induced bone resorption is mediated by p38 mitogen activated protein kinase. J Cell Physiol. 2001;187:294–303. doi: 10.1002/jcp.1082. - DOI - PubMed
    1. Lee JC, Laydon JT, McDonnell PC, Gallagher TF, Kumar S, Green D, McNulty D, Blumenthal MJ, Heys JR, Landvatter SW, Strickler E, McLaughlin MM, Siemens IR, Fisher SM, Livi GP, White JR, Adams JL, Young PR. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature. 1994;372:739–746. doi: 10.1038/372739a0. - DOI - PubMed
    1. Raingeaud J, Gupta S, Rogers JS, Dickens M, Han J, Ulevitch RJ, Davis RJ. Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine. J Biol Chem. 1995;270:7420–7426. doi: 10.1074/jbc.270.13.7420. - DOI - PubMed
    1. Suzuki M, Tetsuka T, Yoshida S, Watanabe N, Kobayashi M, Matsui N, Okamoto T. The role of p38 mitogen-activated protein kinase in IL-6 and IL-8 production from the TNF-alpha- or IL-1beta-stimulated rheumatoid synovial fibroblasts. FEBS Lett. 2000;465:23–27. doi: 10.1016/S0014-5793(99)01717-2. - DOI - PubMed

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