Curcumin synergizes with resveratrol to stimulate the MAPK signaling pathway in human articular chondrocytes in vitro
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The mitogen-activated protein kinase (MAPK) pathway is stimulated in differentiated chondrocytes and is an important signaling cascade for chondrocyte differentiation and survival. Pro-inflammatory cytokines such as interleukin 1β (IL-1β) play important roles in the pathogenesis of osteoarthritis (OA) and rheumatoid arthritis (RA). In this study, we investigated whether curcumin and resveratrol can synergistically inhibit the catabolic effects of IL-1β, specifically the inhibition of the MAPK and subsequent apoptosis in human articular chondrocytes. Chondrocytes were either left untreated or treated with 10 ng/ml IL-1β or 1 μM U0126, a specific inhibitor of MAPK pathway alone for the indicated time periods or pre-treated with 10 μM curcumin, 10 μM resveratrol or 10 μM resveratrol and 10 μM curcumin for 4 h followed by co-treatment with 10 ng/ml IL-1β or 1 μM U0126 and 10 μM resveratrol, 10 μM curcumin or 10 μM resveratrol and 10 μM curcumin for the indicated time periods. Cultures were evaluated by immunoblotting and transmission electron microscopy. Incubation of chondrocytes with IL-1β resulted in induction of apoptosis, downregulation of β1-integrins and the extracellular signal-regulated kinase 1/2 (Erk1/2). Interestingly, U0126 induced apoptosis and blocked the above-mentioned proteins in a similar way to IL-1β. Furthermore, curcumin and resveratrol inhibited IL-1β- or U0126-induced apoptosis and downregulation of β1-integrins and Erk1/2 in human articular chondrocytes. These results suggest that combining these two natural compounds activates MEK/Erk signaling, a pathway that is involved in the maintenance of chondrocyte differentiation and survival.
KeywordsChondrocyte MAPK pathway Integrins OsteoArthritis IL-1β Curcumin Resveratrol
Pro-inflammatory cytokines produced from activated synoviocytes and articular chondrocytes promote degradation of extracellular matrix components [21, 22]. Several lines of evidence suggest that cytokines, such as IL-1β and TNF-α, are produced in high quantities in OA and RA in cartilage. Once released, they stimulate the synthesis of more pro-inflammatory cytokines, catabolic factors such as matrix metalloproteinases (MMPs) [22, 39, 40] and mediators of inflammation such as prostaglandins produced by cyclooxygenase-2 (COX-2) . Pro-inflammatory cytokines also induce chondrocyte apoptosis , a process that is thought to play a pivotal role in joint diseases (OA and RA) in humans and animals [3, 24, 25]. It has been reported that cytokine-mediated chondrocyte apoptosis can be induced by different signals such as caspase-3, cytochrome-c release, protein kinases and reactive oxygen species (ROS) [38, 50]. Activation of certain caspases such as caspase-3 plays a pivotal role in initiating apoptosis. However, the mechanism of chondrocyte apoptosis in joint diseases has not been fully elucidated.
Cell–matrix interactions in cartilage are essential for the differentiation and survival of chondrocytes and are mediated by specific receptors e.g. integrins [18, 19, 35, 45, 46]. Integrins play a crucial role in early cartilage differentiation, since the presence of anti-integrin antibodies can inhibit differentiation of blastemal cells to chondroblasts . This interaction mediates the activation of the MAPK pathways . Studies from our laboratory have shown that loss of chondrogenic potential is accompanied by reduced expression in key signaling proteins of the mitogen-activated proteins (MAP) kinase (MAPK) pathway and apoptosis [41, 42]. Specific inhibition of the MAPK pathway leads to apoptosis of human chondrocytes in vitro . Furthermore, activation of Sox-9, the chondrogenic transcription factor, seems to be mediated by the MAPK pathway [16, 37]. It has been shown that cytokines partially reduce Sox-9 protein levels through an NF-κB-dependent pathway in mouse chondrocytes [37, 52] demonstrating how active transcription factors, sharing common co-factors, regulate gene expression. Interestingly, naturally occurring polyphenolic compounds, such as curcumin and resveratrol, are potent agents for modulating inflammation mediating their effects in part by targeting the NF-κB signaling pathway [10, 44].
Resveratrol (trans-3, 4′-trihydroxystilbene) is a natural phytoalexin that is found in the skin of red grapes, cranberries, peanuts and root extracts of the weed Polygonum cuspidatum . Several lines of evidence indicate that Resveratrol is also capable of immunomodulatory, anti-oxidative, anti-apoptotic and anti-inflammatory functions [14, 20, 29, 44]. Curcumin (diferuloylmethane), a dietary non-toxic pigment in curry, acts as a potent inhibitor of nuclear transcription factor κB (NF-κB)-activation in several cell types [10, 47, 53]. Curcumin inhibits the constitutive IκBα phosphorylation through the inhibition of IκB-kinase (IKK) [9, 10, 15, 31, 47]. It has been shown that dietary supplements and herbal remedies are capable of suppressing the NF-κB pathway and seem to have positive effects in arthritis therapy; furthermore, it has been reported that curcumin is a potent anti-inflammatory and anti-cancer compound .
Evidence is accumulating to support the idea that signaling pathways malfunction in chondrocytes and synovial cells in aging and well as joint diseases such as OA and RA. Treatment of OA with novel agents that can simultaneously target multiple cellular signaling pathways in chondrocytes will benefit from effectively downregulating inflammation without adverse systemic effects. We and others have shown that phytochemicals such as curcumin and resveratrol target the catabolic pathways mediated by the NF-κB signal transduction pathway in cartilage and might be used as clinically safe nutritional factors for the treatment of OA [13, 15, 26, 34, 36, 47, 50, 54]. We have also shown that resveratrol and/or curcumin stimulate Sox-9 expression and inhibit the IL-1β-induced decreased Sox-9 expression that is necessary for the expression of cartilage matrix genes . Therefore, the aim of the present study was to focus on the MAPK signaling pathway in human chondrocytes and examine the effects of resveratrol and curcumin in combination and in isolation on IL-1β-mediated cellular responses.
Materials and methods
Monoclonal anti-β1-integrin, alkaline phosphatase–linked sheep anti-mouse and sheep anti-rabbit secondary antibodies were obtained from Chemicon International (Temecula, CA, USA). Antibody to β-actin was from Sigma (Munich, Germany). Antibodies to phospho-p42/p44 Erk1/2 and U0126 were purchased from Promega (Mannheim, Germany). Anti-pan Erk1/2 antibodies were purchased from Transduction Laboratories (Heidelberg, Germany). Polyclonal antibody to active caspase-3 was from R&D System (Heidelberg, Germany). All antibodies were used at concentrations and dilutions recommended by the manufacturer (dilutions ranged from 1:100 to 1:10,000 for western blot analysis).
Growth medium and chemicals
Growth medium (Ham’s F-12/Dulbecco’s modified Eagle’s medium (50/50) containing 10% fetal calf serum (FCS), 25 μg/ml ascorbic acid, 50 IU/ml streptomycin, 50 IU/ml penicillin, 2.5 μg/ml amphotericin B, essential amino acids and l-glutamine) was obtained from Seromed (Munich, Germany). Trypsin/EDTA (EC 18.104.22.168) was purchased from Sigma (Munich, Germany). Epon was obtained from Plano (Marburg, Germany). Resveratrol was purchased from Sigma. Curcumin was purchased from Indsaff (Punjab, India). Resveratrol was prepared as a 100 mg/ml solution in ethanol and then further diluted in cell culture medium. Curcumin was diluted in DMSO as a 5,000 μM concentration and then further diluted in cell culture medium. IL-1β was obtained from Strathman Biotech GmbH (Hannover, Germany).
Chondrocyte isolation and culture
Cartilage samples from healthy femoral head articular cartilage obtained during joint replacement surgery for femoral neck fractures were used to isolate primary human articular chondrocytes . Cartilage slices were digested primarily with 1% pronase for 2 h at 37°C and subsequently with 0.2% (v/v) collagenase for 4 h at 37°C. Primary chondrocytes were cultured at a density of 200,000 cells per 60-mm petri dish in monolayer culture for a period of 24 h at 37°C with 5% CO2. Cartilage samples were derived from human patients with full informed consent and local ethics committee’s approval.
To see the effects of pro-inflammatory cytokines in the absence of any other stimulation caused by serum growth factor, primary human articular chondrocytes were washed three times with serum-starved medium (0.5% FCS) and incubated for 1 h with serum-starved medium. Serum-starved human articular chondrocytes were either left untreated or treated with 10 ng/ml IL-1β, or 1 μM U0126 alone for the indicated time periods or pre-treated with 10 μM resveratrol, 10 μM curcumin or 10 μM resveratrol and 10 μM curcumin for 4 h followed by co-treatment with 10 ng/ml IL-1β and 10 μM resveratrol, 10 μM curcumin or 10 μM resveratrol and 10 μM curcumin for 24 h or for the indicated time periods.
Transmission electron microscopy
Chondrocytes were fixed 1 h with Karnovsky’s fixative followed by post-fixation in 1% OsO4 solution (0.1 M phosphate buffer). Monolayer cell pellets were rinsed and dehydrated in an ascending alcohol series before being embedded in Epon and cut on a Reichert-Jung Ultracut E (Heidelberg, Germany). Ultrathin sections were contrasted with 2% uranyl acetate/lead citrate. A transmission electron microscope (TEM 10, Zeiss, Jena, Germany) was used to examine the cultures.
Electron microscopic evaluation of apoptotic cell death
Serum-starved human chondrocytes were exposed to 10 ng/ml IL-1β or 1 μM U0126 alone for 1, 12, 24 and 48 h or pre-stimulated with 10 μM resveratrol, 10 μM curcumin alone or 10 μM resveratrol and 10 μM curcumin for 4 h and then co-treated with IL-1β (10 ng/ml) or U0126 (1 μM) for 1, 12, 24 and 48 h. Ultra-thin sections of the samples were prepared and evaluated with an electron microscope (TEM 10, Zeiss, Jena, Germany). For statistical analysis, the number of cells with morphological features of apoptotic cell death was determined by scoring 100 cells from 20 different microscopic fields.
Western blot analysis
To determine the effect of resveratrol/curcumin on IL-1β- or U0126-induced apoptosis and inhibition of MAPK signaling pathway, whole cell lysates of chondrocyte monolayers were prepared [44, 46, 49] and equal quantities (500 ng of protein per lane) of total proteins were separated by SDS-PAGE (5, 7.5, 12% gels) under reducing conditions. The separated proteins were transferred onto nitrocellulose membranes. Membranes were pre-incubated in blocking buffer (5% (w/v) skimmed milk powder in PBS/0.1% Tween 20) for 1 h and incubated with primary antibodies against β1-integrin, pan-Erk1/2, phospho-Erk1/2, caspase-3 and β-actin (overnight, 4°C). Membranes were washed three times with blocking buffer and incubated with alkaline phosphatase–conjugated secondary antibodies for 30 min. They were finally washed three times in 0.1 M Tris pH 9.5 containing 0.05 M MgCl2 and 0.1 M NaCl. Nitro blue tetrazolium and 5-bromo-4-chloro-3-indoylphosphate (p-toluidine salt; Pierce, Rockford, IL, USA) were used as substrates to reveal alkaline phosphatase–conjugated specific antigen–antibody complexes. The density (specific binding) of each band was measured by densitometry using “Quantity One” (Bio-Rad Laboratories Inc. CA, USA).
The results are expressed as the means ± SD of a representative experiment performed in triplicate. The means were compared using student’s t-test assuming equal variances. P < 0.05 was considered statistically significant.
Resveratrol and curcumin suppress IL-1β-induced apoptosis in chondrocytes
Work from our group has previously demonstrated that U0126 suppresses the MAPK pathway and leads to apoptosis . To determine whether curcumin and resveratrol can act synergistically to modulate the cytotoxic effects of U0126 in human chondrocytes, serum-starved chondrocytes were treated with 1 μM U0126 alone for 1, 12, 24 and 48 h or were pre-stimulated with 10 μM resveratrol, 10 μM curcumin or a combination of phytochemicals (each 10 μM) for 4 h and followed by co-treatment with U0126 1 μM for the indicated time periods. After 12-h treatment with U0126 alone, chondrocytes exhibited morphological features typical of apoptosis, with nuclear changes including chromatin condensation into dense areas along the nuclear membrane and nuclear fragmentation. The cellular membrane was irregular (blebbing), and cytoplasmic vacuoles (dilation of mitochondria and the endoplasmic reticulum) were observed (Figs. 1a, F–I).
Interestingly, pre-treatment of the chondrocytes with resveratrol (Figs. 1a, J–M) or curcumin (Figs. 1a, N–Q) alone or in combination (Figs. 1a, R–U) followed by co-treatment with U0126 or IL-1β inhibited the adverse effects of IL-1β (data not shown) or U0126 (Fig. 1a, J–U). After 48 h, chondrocytes demonstrated less cellular degeneration at the ultrastructural level; they regained a flattened shape and exhibited numerous microvilli-like cytoplasmic processes (Figs. 1a, J–U).
Statistical evaluation of apoptotic cell death was carried out by counting the number of apoptotic cells in selected fields from the transmission electron micrographs. This clearly highlighted changes in the number of cells with apoptotic features before and after IL-1β or U0126 treatment. Pre-treatment with resveratrol and curcumin clearly decreased the number of cells with apoptotic features compared to cells treated with IL-1β or U0126 alone (Fig. 1b). However, pre-treatment of the chondrocytes with a combination of the two phytochemicals decreased the total number of apoptotic cells compared to experiments where each chemical was used in isolation. These results demonstrate that curcumin and resveratrol inhibit the cytotoxic and apoptotic effects induced by IL-1β or U0126 in human chondrocytes.
Resveratrol and curcumin block U0126-induced stimulation of activated caspase-3 and inhibition of β1-integrins in chondrocytes
It is well known that cell–matrix interactions are primarily mediated via multi-functional β1-integrins . β1-integrins organize cell surface mechanoreceptor complexes and function as signal transduction molecules [6, 35] capable of stimulating the MAP kinase pathway [33, 46, 49]. To determine whether curcumin and resveratrol can act synergistically to modulate the inhibitory effects of U0126 on β1-integrin expression, serum-starved human chondrocytes were pre-stimulated with a combination of resveratrol and curcumin for 4 h and co-treated with U0126 for the indicated time periods and then examined by western blotting. As shown in Fig. 2b, U0126 inhibited the expression of β1-integrins in a time-dependent manner. In contrast, combination of resveratrol and curcumin stimulated β1-integrin expression and inhibited the U0126-induced decreased β1-integrin expression.
Resveratrol and curcumin prevent U0126-induced inhibition of Erk 1/2 in a dose- and time-dependent manner in chondrocytes
Resveratrol and curcumin block IL-1β- or U0126-induced inhibition of Erk1/2 in chondrocytes
Taken together, these results suggest that resveratrol and curcumin inhibit the cytotoxic and apoptotic effects induced by IL-1β or U0126, at least in part by activation of MAPK pathway in human chondrocytes.
In this paper, we investigated the synergistic effects of resveratrol and curcumin on apoptotic signaling in IL-1β-stimulated human chondrocytes. The present study leads to the following findings: (1) treatment of chondrocytes with 10 ng/ml IL-1β or 1 μM U0126, a specific inhibitor of the MAPK signaling pathway, which can inhibit the phosphorylation and activation of Erk1/Erk2, results in morphological alterations and apoptosis, which were abolished through pre-treatment with resveratrol and curcumin. (2) U0126 induced an increase in activated caspase-3 and decrease in β1-integrin signal receptor synthesis, and these were relieved by resveratrol and curcumin in combination in a time-dependent manner. (3) Resveratrol potentiates the anti-apoptotic effects of curcumin on IL-1β or U0126-stimulated chondrocytes, and this correlates with up-regulation of the MAPK pathway. (4) Additionally, both resveratrol and/or curcumin suppressed U0126-induced downregulation of the p-Erk1/2 in a dose- and time-dependent manner. (5) Finally, IL-1β or U0126 inhibition of activation and phosphorylation of Erk1/2 could be clearly blocked by resveratrol and/or curcumin.
In the present study, we demonstrated that IL-1β and the specific inhibition of the MAPK pathway led to apoptosis and downregulation of β1-integrin receptor and Erk1/2 expression in chondrocytes. Our data show for the first time that this process is inhibited when chondrocytes are treated with the natural phytochemicals resveratrol and curcumin.
Chondrocytes synthesize a cartilage-specific pericellular matrix, which consists primarily of type-II collagen and cartilage-specific proteoglycans . This specific extracellular matrix and several additional substances are required for chondrocyte differentiation and survival . Interaction of chondrocytes and their surrounding matrix plays an essential role in maintaining their differentiated phenotype. This is partly mediated by β1-integrins, a specific surface receptor and transmembrane signal transduction molecule [18, 32, 43, 46, 48, 49, 51]. Furthermore, several lines of evidence suggest that β1-integrins stimulate different signal transduction pathways, such as the mitogen-activated protein (MAP) kinase pathway, and thus control proliferation, differentiation and survival of cells [23, 33, 55].
Erk1/2 regulates the activities of several nuclear transcription factors , such as cartilage-specific transcription factor Sox-9, which is an important transcription factor for the expression of chondrocyte-specific marker genes, including collagen types II and XI and cartilage-specific proteoglycans [4, 5]. Indeed, a specific inhibitor (U0126) of the MAP kinase signaling pathway, which can block the activation of Erk1/2, suppresses the FGF-2-stimulated increase in Sox-9 levels and hence also the transcription of cartilage-specific extracellular matrix genes . Moreover, we have previously shown that the U0126-induced inhibition of the MAP kinase signaling pathway suppresses the activation of Erk1/2 and promotes apoptosis in cultured human chondrocytes, as revealed by the activation of caspase-3 and the cleavage of poly(ADP-ribose)polymerase (PARP) .
Ms. Christina Pfaff, Ms. Stefanie Schreiber and Ms. Ursula Schwikowski are gratefully acknowledged for their excellent technical assistance.
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