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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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.
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.
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.
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.
KeywordsWestern Immunoblotting Fibroblast Like Synoviocytes Mankin Score Powder Extract Pomegranate Fruit Extract
fibroblast like synoviocytes
Institutional Review Board
mitogen activated protein kinase
pomegranate fruit extract
runt-related transcription factor-2
Osteoarthritis (OA), the most common forms of arthritis, is a progressive degenerative joint disease that has a major impact on joint function and the patient's quality of life. This disorder is regulated by proinflammatory cytokines such as IL-1 and TNF-α that can activate a broad array of intracellular signal transduction mechanisms . Of the cytokine- activated pathways, MAP kinases are especially important because they regulate the production of several mediators of inflammation and cartilage damage . Members of the MAP kinase family phosphorylate a number of transcription factors including runt-related transcription factor-2 (RUNX-2), with subsequent activation of matrix metalloproteinases (MMPs) and inflammatory cytokine gene expressions . p38-MAPK, in particular, can regulate cytokine production through a variety of transcriptional and translational mechanisms . Furthermore, p38-MAPK participates in other inflammation-related events, such as neutrophil activation , apoptosis  and nitric oxide synthase induction . Inhibition of p38-MAPK with the commonly used pharmacological agent SB203580 reduces proinflammatory cytokine production in monocytes/macrophages, neutrophils, and T lymphocytes . In a rodent model of inflammatory arthritis, p38-MAPK inhibition suppressed the inflammation and bone destruction . TNF-α for instance, activates preferentially the MAPK-p38 isoform p38α in synovial macrophages and synovial fibroblasts (RASFs) in rheumatoid arthritis (RA) . In addition, p38α-MAPK is activated in human osteoclast precursor cells upon binding of RANKL, which indicates a possible role for p38α-MAPK isoform in bone destruction. Inhibition of p38-MAPK with small-molecule compounds has been successful for the treatment of experimental arthritis in different animal models [11, 12]. Of note, inhibition of the p38α-MAPK isoform seems to be particularly effective with regard to cartilage and bone destruction, since a selective p38α-MAPK inhibitor reduced bone loss, numbers of osteoclasts and serum levels of cartilage breakdown metabolites in mice with CIA . The upstream kinases, MKK3 and MKK6 are important regulators of p38-MAPK and represent potential therapeutic targets to modulate cytokine production . Studies in MKK3 or MKK6 knockout mice demonstrate that both are essential for full p38-MAPK activation in vivo . MKK3 selectively phosphorylates p38α-, γ-, and δ-MAPKs whereas MKK6 activates all four p38-MAPK isoforms (α, β, γ, and δ) . This suggests that substrate selectivity might contribute to the distinct functional profiles of MKK activation. It is well documented that activation of the runt-related transcription factor RUNX-2 is mediated by activated p38-MAPK as inhibition of p38-MAPK abrogates its activity and the expression of cartilage degrading enzymes in chondrocytes . These and other studies  clearly show that inhibition of specific MAPKs or transcription factor may be an effective approach for the inhibition of joint destruction in arthritis.
Pomegranate fruit (Punica granatum L) is revered through the ages for its medicinal properties. Pomegranate fruit extract (PFE) is a rich source of highly potent antioxidants and is widely used in several traditional medicinal systems for the treatment of inflammation and pain in arthritis and other diseases. Consistent with this notion, our published study is noteworthy where we showed for the first time that a standardized PFE preparation was non-toxic to human OA chondrocytes and prevented the IL-1β-induced cartilage breakdown by inhibiting the IL-1β-induced production of MMPs by blocking the activation of p38-MAPK and the transcription factor NF-κB in human OA chondrocytes . In other studies, we have shown that bioavailable metabolites of PFE inhibited COX-2 activity in human OA chondrocytes , and that consumption of PFE suppressed inflammation and joint destruction in an animal model of inflammatory arthritis .
Therefore, based on our published data and the studies showing that IL-1β activates the human chondrocytes and induces the expression of mediators that play a major role in cartilage degradation in OA, we determined whether PE inhibits the IL-1β-induced cartilage degradation by modulating the activation of relevant signal transduction pathways and transcription factors in human OA chondrocytes. Our results show that a standardized pomegranate extract (PE) selectively inhibited the IL-1β-induced activation of p38α-MAPK isoform and its upstream regulator MKK3 in primary human OA chondrocytes. In addition, IL-1β-induced DNA binding activity of RUNX-2 was also inhibited by PE in primary human OA chondrocytes and correlated with the inhibition of p38α-MAPK isoform.
Materials and methods
Specimen selection and articular chondrocytes preparation
With Institutional Review Board (IRB) approval, discarded human cartilage samples were obtained from the knee joints of 19 OA patients aged 58 to 77 years (mean age, 62 ± 7.7 years; 14 female, 5 male Caucasians) who underwent joint replacement surgery at MetroHealth Medical Center, Cleveland, Ohio, USA. The macroscopic cartilage degeneration was determined by staining of femoral head samples with India ink  and the cartilage with smooth articular surface ("unaffected cartilage") was resected and used to prepare chondrocytes by enzymatic digestion as previously described [22, 23, 24, 25, 26, 27]. Histological analysis of some of the "unaffected cartilage" samples was performed on 5 μM thick sections stained with H&E and Safranin O and graded using Mankin score . Grading of the histology slides (not shown) revealed that all of the cartilage pieces taken from the unaffected area had a Mankin score of <3 for structure and Mankin score of 2 for cellularity. Isolated chondrocytes were plated at a density of 1 × 106/ml in 35-mm tissue culture dishes (Corning, Corning, NY, USA) in complete DMEM medium as previously described .
Chemical composition of PE
PE is produced in a two-step process: 1) extraction of fruit residue after pressing for juice and 2) solid-phase extraction to produce a powder with a high concentration of polyphenols. Extraction is performed during fruit harvest using pressed pomegranate fruit and arils. Composition of the powder extract (PE) used in this study has been described previously [18, 19, 20].
Treatment of chondrocytes with IL-1β and PE
Human OA chondrocytes (1 × 106/ml) were plated in 35 mm culture dishes (Becton-Dickinson, Franklin Lakes, NJ, USA) in complete DMEM with 10% fetal calf serum and allowed to grow for 72 h at 37°C and 5% CO2 in a tissue culture incubator. PE was dissolved in water and filter sterilized. Primary OA chondrocytes (>80% confluent) were serum starved for 12 hours/overnight and then were pre-treated with different doses of PE (6.25 to 100 μg/ml) for one or two hour and then stimulated with human recombinant IL-1β (# 201LB, R&D Systems, St Paul, MN, USA, 10 ng/ml) for 5 to 120 minutes. OA chondrocytes cultured without IL-1β or PE served as controls. Cell viability was determined using Trypan Blue and the cells were counted using a hemocytometer. All experiments were performed within four days of the initiation of primary culture to avoid dedifferentiation of human OA chondrocytes.
Immunoprecipitation and Western immunoblotting
Stimulated and control human OA chondrocytes were washed with cold PBS and lysed using the cell lysis RIPA buffer (50 mM Tris:HCl, pH 7.5; 150 mM NaCl; 1% IGEPAL, 4 mM EDTA, 0.1% sodium deoxycholate; 10 mM Na4P2O7, 10 mM NaF, 2 mM Na3VO4, 1 mM PMSF, 1 μg/ml leupeptin, 1 μg/ml aprotinin). Cytoplasmic and nuclear fractions were prepared as previously described . Anti-MKK-3 antibody (# 32-6900), anti-MKK-6 (#32-7100) and anti-p38β antibody (#33-8700) were purchased from Zymed (San Francisco, CA, USA); anti-phospho-MKK3/6 antibody (sc-7994-R) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and the anti-RUNX2 antibody (#D130-3 ) was from MBL, Naka-ku Nagoya, Japan. Anti-p38γ antibody (#MAB1347) was from R&D Systems (Minneapolis, MN, USA); anti-phospho-p38MAPK antibody (#9215S), anti-p38α antibody (#9218), and anti-p38δ antibody (#9214) were from Cell Signaling Technologies (Amherst, Beverly, MA, USA). Proteins of interest were immunoprecipitated using 200 μg total cell lysate protein following standard protocols (Cell Signaling Technology). Briefly, cell lysate was pre-cleared with protein- A/G agarose (Pierce, Rockford, IL, USA) at 4°C and then incubated with the appropriate antibody overnight at 4°C, mixed with Protein A/G agarose, centrifuged and the complexes were washed with cold RIPA buffer prior to SDS-PAGE. Total cell lysate or nuclear/cytoplasmic fraction protein (35 μg/lane) or immunoprecipitated proteins were resolved by SDS-PAGE (10% resolving gel with 4% stacking) and transferred to PVDF immobilon-P transfer membranes (Cat. # IPVH00010, Millipore Corporation, Bedford, MA, USA). Membranes were blocked with non-fat dry milk powder in Tris buffered saline and 0.1% Tween-20 (TBS-T). Blots were probed with 1:200 to 1:1000 diluted primary antibodies specific for the target protein. Immunoreactive proteins were visualized by using 1:1000 diluted HRP-linked secondary antibodies and enhanced chemiluminescence (GE Healthcare, Milwaukee, WI, USA) . Images were captured using AFP-Imaging System (Minimedical Series, Elms Ford, NY, USA).
Reverse transcription PCR (RT-PCR)
To analyze the gene expression of the p38-MAPK isoforms α, β, γ and δ, total cellular RNA was prepared using a commercially available kit (Qiagen, Valencia, CA, USA). First-strand cDNA was synthesized using 1 μg total RNA and the SuperScript First Strand cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA). Primers used for PCR assisted amplification were: p38α- MAPK (NM_139012, F 5'-GATCAGTTGAAGCTCATTTTAA-3'; R 5'-CACTTGAATAATATTTGGAGAGT-3', expected size of the DNA fragment 479 bp), p38β- MAPK (NM_002751, F 5'-AGCCATATCTGG CAA GAA GCT GGA-3'; R 5'-AAG TGT CCG AGT CCA AGT CCA CAT-3', expected size of the DNA fragment 262 bp); p38γ- MAPK (NM_002969, F 5'-TTG AAT TGG ATG CGC TAC ACG CAGB-3'; R 5'-AGG GCT TGC ATT GGT CAG GAT AGA-3', expected size of the DNA fragment 255 bp) and p38δ- MAPK (NM_002754, F 5'-TGT GCA GAA GCT GAA CGA CAA AGC-3'; R 5'-TGC CAT GCA AGA TGA GTC CCT ACA-3', expected size of the DNA fragment 380 bp). PCR reaction was carried out using the PTC-100 Thermal Cycler as follows: two minutes at 95°C, followed by 30 cycles of one minutes at 95°C, 30 seconds at 62°C, and one minute at 72°C. The amplified products were electrophoresed on 1.5% agarose gels in TAE buffer and visualized by ethidium bromide staining. Specificity of amplified fragments was verified by cloning and sequencing.
KK3 or MKK6 knockdown by transfection with small interfering RNAs (siRNA)
Human OA chondrocytes were transfected with human MKK3 or MKK6-specific siRNAs using On-Target SMART Pools (# L-00350900, L-00396700, Dharmacon RNA Technologies, Lafayette, CO, USA). Human GAPDH control On-target SMART pool siRNA (# D-0018301020, Dharmacon) was used as negative control. Chondrocytes were transfected using Amaxa Human Chondrocytes Nucleofector Kit (# VPF-1001, Lonza, Walkersville, MD, USA) according to the manufacturer's instructions. In brief, human OA chondrocytes 1.4 × 106 were transfected with 30 nM-300 nM siRNA and with 100 μl nucleofector solution using electroporation program U-024 and the transfected OA chondrocytes were seeded in six-well plates. After 24 hours, the culture medium was changed to serum-free medium for the experiments using IL-1β. Transfection efficiency was monitored with red fluorescent siRNA oligonucleotides (siGLO red indicator, # D0016300220, Dharmacon). Approximately 70 to 80% of the chondrocytes emitted red fluorescence signal when transfected with siGLO.
RUNX-2 DNA binding activity assay
Activation and DNA binding activity of RUNX-2 in human OA chondrocytes pretreated with PE (2 h) and then stimulated with IL-1β (30 minutes) was determined using a highly sensitive and specific Transcription Factor ELISA Kit according to the instructions of the manufacturer (#44496/44996, Active Motif North America, Carlsbad, CA, USA). Color intensity was read at 450 nm using Synergy HT ELISA plate reader (Biotek Instruments, Winooski, VT, USA).
Treatment of human T cells HSB.2 with IL-1β, PHA and PE
To determine whether the inhibition of IL-1β-induced effects in human OA chondrocytes by PE was not due to an interference by PE in the receptor binding of exogenously added IL-1β, we performed a bioassay using the IL-1-dependent human T cell line HSB.2 known to produce high levels of IL-2 in an IL-1-dependent manner . These cells were obtained from ATCC (CCL 120.1) and were cultured essentially as described . Cells were stimulated with IL-1β, PHA (phytohemagglutinin) and PE and the production of IL-2 was quantified using the IL-2-specific ELISA according to the instructions of the manufacturer (R&D Systems). Plates were read at 450 nm using Synergy HT microplate reader (Biotek Instruments).
Measurements of the scanned bands were performed using UN-SCAN-IT software (Silk Scientific Corporation, Orem, UT, USA). Each band was scanned five times, and the mean band intensity (pixels per band) was obtained. Data were normalized to suitable loading controls and expressed as Mean ± SD followed by appropriate statistical analysis.
All measurements were performed in duplicates and repeated at least three times using different age- and sex-matched OA samples. All statistical analyses were performed using Origin 6.1 software package (Northampton, MA, USA) (one paired two tailed t-test with one way ANOVA and Tukey's post-hoc analysis) and P < 0.05 was considered significant. Values shown are mean ± SD unless stated otherwise.
PE was non-toxic to human OA chondrocytes in vitro
In these studies pre-treatment of human OA chondrocytes for up to 24 hr with varying concentrations of PE (up to 200 μg/ml) was found to be non-toxic and showed no effect on the cell viability (data not shown) as reported by us previously (18). Based on this data the maximum concentration of PE used in these studies was 100 μg/ml.
Expression and activation of MKK3 and MKK6 in human OA chondrocytes
Effect of PE on IL-1β-induced activation of MKK3 and MKK6 in human OA chondrocytes
To determine the effect of PE on IL-1β-induced activation of MKK3 and MKK6 in human OA chondrocytes, cells were pretreated with PE (6.25 to 100 μg/ml) for two hours and then stimulated with IL-1β for 30 minutes and cell lysate was analyzed by Western immunoblotting. Treatment of primary human OA chondrocytes with PE attenuated the IL-1β-induced phosphorylation of both MKK3 and MKK6 in a dose dependent manner with the maximum inhibitory effect of PE being at 50 μg/ml concentration (Figure 1c). Higher concentrations produced no additional suppression of phosphorylation of either MKK3 or MKK6 in human OA chondrocytes.
Effect of MKK3 or MKK6 knock down on IL-1β-induced activation of p38-MAPK in human OA chondrocytes
Expression of p38-MAPK isoforms in human OA chondrocytes
Effect of PE and IL-1β-induced activation of p38-MAPK isoforms in human OA chondrocytes
Effect of PE on IL-1β-induced activation of transcription factor RUNX-2 in human OA chondrocytes
To determine whether PE also inhibits the IL-1β-induced DNA binding activity of the transcription factor RUNX-2 we used a sandwich ELISA assay kit. Exposure of OA chondrocytes to IL-1β significantly enhanced the DNA binding activity of RUNX-2 compared to controls (Figure 5b; P < 0.001). This IL-1β-induced increase in the DNA binding activity of RUNX-2 was inhibited significantly by PE in a dose-dependent manner (12.5 to 100 μg/ml) with the maximum inhibitory effect of PE being at 50 μg/ml concentration (Figure 5b). Higher concentrations produced no additional suppression of IL-1β-induced DNA binding activity of RUNX-2 in human OA chondrocytes.
PE does not block the interaction of IL-1β with IL-1 Receptor
To determine whether the observed inhibition of IL-1β-induced activation of p38α-MAPK and RUNX-2 in human OA chondrocytes by PE was not due to an interference by endogenously added PE in the culture medium, we performed a bioassay using the IL-1-dependent human T cell line HSB.2 shown to produce high levels of IL-2 in an IL-1-dependent manner . Human HSB.2 cells were stimulated with IL-1β + PHA and PE and the production of IL-2 was quantified by a sandwich ELISA (R&D Systems). Results presented in Figure 5c clearly demonstrate that PE did not interfere with the binding of IL-1β with IL-1R in these cells.
Progression of OA is characterized by destruction of the articular cartilage and other soft tissues in the joint. Tissue destruction is accompanied by remodeling and hypertrophy of neighboring bone and varying degrees of synovial inflammation . The destruction, remodeling, and inflammation of joint tissues lead to joint failure manifested as joint pain and loss of function. Chondrocytes are the only cellular components of cartilage, and in pathologic conditions such as OA, chondrocytes increase the production of proteolytic enzymes such as aggrecanases and MMPs, resulting in aberrant cartilage destruction .
Intracellular signaling pathways provide the essential link between receptor signals and nuclear gene transcription. Key to these pathways are the evolutionarily conserved mitogen-activated protein kinases (MAPKs); of these, p38-MAPK (also known as MAPK14) has received considerable attention as potential therapeutic target for inflammatory and degenerative diseases such as OA . p38-MAPK is thought to be crucial because selective p38-MAPK inhibitors block joint inflammation and destruction in several animal models of arthritis . Four p38-MAPK isoforms (p38-α, -β, -γ, and -δ) have been characterized . Although the relative expression of p38-MAPK isoforms in osteoarthritis has not been fully explored, but the best-studied is p38α-MAPK, which can be phosphorylated in many inflammatory cell lineages as well and regulates cytokine production by macrophages  and is known to be especially prevalent at sites of joint destruction .
MAPKs and transcription factor NF-κB are important targets of polyphenols such as ellagic acid and quercetin found in PE [18, 39]. Whereas the free radical scavenging and antioxidant properties of PE are well established , emerging literature reports suggest that their anti-arthritic effects may also be ascribed to their ability to modulate many signal transduction pathways including p38-MAPK and NF-κB in a manner that favors inhibition of OA and other inflammatory diseases [18, 19, 20, 27]. It is well established that in chondrocytes activation of RUNX-2 is mediated by activated p38-MAPK . IL-1 is a potent stimulator of many inflammatory genes in chondrocytes, and its effects are diminished in cells expressing a dominant-negative mutant of RUNX-2 . Since PE suppressed the IL-1β-induced activation of p38-MAPK, we determined whether PE also inhibits IL-1β-induced activation of RUNX-2 in human OA chondrocytes. Pretreatment of human OA chondrocytes with PE inhibited the IL-1β- induced activation and DNA binding activity of RUNX-2 in a dose dependent manner. These data indicate that PE attenuates the inflammatory stimuli-induced activation and DNA binding activity of RUNX-2 in human OA chondrocytes.
In summary, this study shows that human OA chondrocytes expressed p38-MAPK isoforms p38α, γ and δ, but not p38β. Treatment of human OA chondrocytes with IL-1β activates p38α-MAPK and p38γ-MAPK but not p38δ-MAPK isoform. In addition, our results also showed that IL-1β activates the p38-MAPK preferentially via the upstream kinase MKK3 compared to MKK6 in human OA chondrocytes. In addition, we also investigated the effect of PE on IL-1β induced activation and DNA binding activity of the transcription factor RUNX2 in human OA chondrocytes. Our results demonstrate that PE suppresses the IL-1β-induced activation of RUNX-2 and this correlates with the inhibition of p38-MAPK activation. However, it is not known whether the inhibition of p38-MAPK - RUNX-2 pathway is related to the antioxidant activity of PE or one or more of the bioavailable constituents of PE exert this inhibitory effect by interfering with the kinase activity of p38-MAPK or both. This will be pursued in future studies.
The present article is the first report demonstrating that IL-1β preferentially activates MKK3 → p38-MAPK → RUNX-2 pathway in human OA chondrocytes. This study also demonstrated that pretreatment of human OA chondrocytes with PE inhibited the IL-1β-induced activation of the upstream kinase MKK3 resulting in the inhibition of p38α-MAPK isoform and the activation and DNA binding activity of the transcription factor RUNX-2. Thus, our results suggest that PE or PE-derived compounds may be useful in blocking the activation of MKK3 and might provide the benefit of p38-MAPK inhibition and its downstream targets such as RUNX-2 without the direct inhibition of p38-MAPK in OA. This in turn may be of value in the development of MKK inhibitors for the treatment of OA and other degenerative diseases.
This work was supported by the National Institute of Health/National Center for Complimentary and Alternative Medicine grants RO1-AT-003267; RO1-AT-005520 and R21-AT-504615 to TMH. Financial support from the MetroHealth Medical Center, Cleveland, Ohio is also gratefully acknowledged.
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