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Curcumin Modulates Nuclear Factor κB (NF-κB)-mediated Inflammation in Human Tenocytes in Vitro

ROLE OF THE PHOSPHATIDYLINOSITOL 3-KINASE/Akt PATHWAY
Open AccessPublished:June 13, 2011DOI:https://doi.org/10.1074/jbc.M111.256180
      Inflammatory processes play essential roles in the pathogenesis of tendinitis and tendinopathy. These events are accompanied by catabolic processes initiated by pro-inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). Pharmacological treatments for tendinitis are restricted to the use of non-steroidal anti-inflammatory drugs. Recent studies in various cell models have demonstrated that curcumin targets the NF-κB signaling pathway. However, its potential for the treatment of tendinitis has not been explored. Herein, we used an in vitro model of human tenocytes to study the mechanism of curcumin action on IL-1β-mediated inflammatory signaling. Curcumin at concentrations of 5–20 μm inhibited IL-1β-induced inflammation and apoptosis in cultures of human tenocytes. The anti-inflammatory effects of curcumin included down-regulation of gene products that mediate matrix degradation (matrix metalloproteinase-1, -9, and -13), prostanoid production (cyclooxygenase-2), apoptosis (Bax and activated caspase-3), and stimulation of cell survival (Bcl-2), all known to be regulated by NF-κB. Furthermore, curcumin suppressed IL-1β-induced NF-κB activation via inhibition of phosphorylation and degradation of inhibitor of κBα, inhibition of inhibitor of κB-kinase activity, and inhibition of nuclear translocation of NF-κB. Furthermore, the effects of IL-1β were abrogated by wortmannin, suggesting a role for the phosphatidylinositol 3-kinase (PI-3K) pathway in IL-1β signaling. Curcumin suppressed IL-1β-induced PI-3K p85/Akt activation and its association with IKK. These results demonstrate, for the first time, a potential role for curcumin in treating tendon inflammation through modulation of NF-κB signaling, which involves PI-3K/Akt and the tendon-specific transcription factor scleraxis in tenocytes.

      Introduction

      The global incidence of tendon injuries has increased in conjunction with the rise in aging and inflammatory diseases (
      • Maffulli N.
      • Wong J.
      • Almekinders L.C.
      ). The etiology of tendinopathy is considered to be multifactorial, but mechanical loading, overuse injury (or association with arthritis), adverse effects of quinolone antibiotics or other drugs, degeneration, and inflammation that cause tendon injuries and rupture are the major causative factors (
      • Almekinders L.C.
      ,
      • Riley G.
      ,
      • Sendzik J.
      • Shakibaei M.
      • Schäfer-Korting M.
      • Lode H.
      • Stahlmann R.
      ,
      • Sendzik J.
      • Shakibaei M.
      • Schäfer-Korting M.
      • Stahlmann R.
      ). Tenocytes are embedded in an extensive three-dimensional network of extracellular matrix components consisting predominantly of collagen type I fibrils (>95% of the total collagen in tendon), other types of collagen (type III and type V), proteoglycans, elastin, and fibronectin (
      • Bernard-Beaubois K.
      • Hecquet C.
      • Houcine O.
      • Hayem G.
      • Adolphe M.
      ,
      • Kannus P.
      ,
      • Rees S.G.
      • Flannery C.R.
      • Little C.B.
      • Hughes C.E.
      • Caterson B.
      • Dent C.M.
      ). These specific matrix components give tendon its resilience and biomechanical stability.
      Tendon and ligament are dense fibrous connective tissues with a very limited intrinsic potential for regeneration (
      • Kannus P.
      ,
      • Butler D.L.
      • Juncosa N.
      • Dressler M.R.
      ). Therefore, their repair and regeneration poses a complex clinical challenge. It is important to understand the cellular and molecular mechanisms involved in tendon degeneration during the early stages of disease pathogenesis to develop new and effective treatments for tendinopathy. Subtle changes such as the release of IL-1β or other inflammatory cytokines by infiltrating macrophages/monocytes may occur (
      • Tsuzaki M.
      • Guyton G.
      • Garrett W.
      • Archambault J.M.
      • Herzog W.
      • Almekinders L.
      • Bynum D.
      • Yang X.
      • Banes A.J.
      ). Moreover, like in other connective tissue injuries, tendon inflammation is accompanied by the up-regulation of pro-inflammatory cytokines such as IL-1β. In vitro studies have shown that IL-1β can induce inflammatory mediators such as COX-2, prostaglandin E2, and matrix metalloproteinases (MMP),
      The abbreviations used are: MMP
      matrix metalloproteinase
      IKK
      IκBα kinase
      SCXA
      scleraxis
      TRAF1
      TNF receptor-associated factor 1.
      all known to be involved in tendon matrix degradation (
      • Archambault J.
      • Tsuzaki M.
      • Herzog W.
      • Banes A.J.
      ,
      • Tsuzaki M.
      • Bynum D.
      • Almekinders L.
      • Yang X.
      • Faber J.
      • Banes A.J.
      ).
      IL-1β is a potent pro-inflammatory cytokine that has been reported to be present in significantly increased quantities in the synovium where it enhances inflammatory reactions in injured joints (
      • Gotoh M.
      • Hamada K.
      • Yamakawa H.
      • Nakamura M.
      • Yamazaki H.
      • Ueyama Y.
      • Tamaoki N.
      • Inoue A.
      • Fukuda H.
      ,
      • Gotoh M.
      • Hamada K.
      • Yamakawa H.
      • Yanagisawa K.
      • Nakamura M.
      • Yamazaki H.
      • Inoue A.
      • Fukuda H.
      ). The intracellular signaling pathways activated by IL-1β are responsible for stimulating MMP expression and COX-2 production. However, these pathways have not been explored in detail in tendon cells. Pro-inflammatory cytokines (e.g. IL-1β) induce activation of a central transcription factor known as NF-κB, which is a key regulator of gene expression (
      • Barnes P.J.
      • Karin M.
      ,
      • Largo R.
      • Alvarez-Soria M.A.
      • Díez-Ortego I.
      • Calvo E.
      • Sánchez-Pernaute O.
      • Egido J.
      • Herrero-Beaumont G.
      ). NF-κB is present in the cytoplasm in its resting stage as a heterotrimer complex consisting of two subunits and an additional inhibitory subunit, IκBα (
      • Kumar A.
      • Takada Y.
      • Boriek A.M.
      • Aggarwal B.B.
      ). During the activation process, the inhibitory subunit IκBα is phosphorylated at Ser-32 and Ser-36 residues by IKK kinase (IκBα kinase) and is subsequently degraded. Once released, subunits of activated NF-κB translocate to the nucleus and mediate transcription of various inflammatory and catabolic gene products (
      • Largo R.
      • Alvarez-Soria M.A.
      • Díez-Ortego I.
      • Calvo E.
      • Sánchez-Pernaute O.
      • Egido J.
      • Herrero-Beaumont G.
      ,
      • Ding G.J.
      • Fischer P.A.
      • Boltz R.C.
      • Schmidt J.A.
      • Colaianne J.J.
      • Gough A.
      • Rubin R.A.
      • Miller D.K.
      ). NF-κB activation has been shown to regulate the expression of more than 500 different gene products linked with inflammation, tumor cell transformation, survival, proliferation, invasion, angiogenesis, metastasis, and chemoresistance (
      • Sung B.
      • Pandey M.K.
      • Ahn K.S.
      • Yi T.
      • Chaturvedi M.M.
      • Liu M.
      • Aggarwal B.B.
      ). Thus, inhibitors of NF-κB activation may have therapeutic potential and are actively being researched.
      Non-steroidal anti-inflammatory drugs are commonly prescribed for the treatment of tendinitis (
      • Wang J.H.
      • Iosifidis M.I.
      • Fu F.H.
      ). However, the use of non-steroidal anti-inflammatory drugs is associated with numerous side effects, which can be quite adverse. Therefore, the search is still on for safer and more selective pharmacotherapies for tendinopathy. Curcumin (diferuloylmethane) is a naturally occurring polyphenol derived from the rhizome of Curcuma longa Linn, with the potential for treatment of various diseases acting via NF-κB inhibition (
      • Bharti A.C.
      • Aggarwal B.B.
      ,
      • Bharti A.C.
      • Donato N.
      • Singh S.
      • Aggarwal B.B.
      ,
      • Mukhopadhyay A.
      • Bueso-Ramos C.
      • Chatterjee D.
      • Pantazis P.
      • Aggarwal B.B.
      ). Commercially available preparations of curcumin contain three major components: curcumin (77%), demethoxycurcumin (17%), and bisdemethoxycurcumin (3%), altogether referred to as the “curcuminoids” (
      • Bharti A.C.
      • Donato N.
      • Singh S.
      • Aggarwal B.B.
      ,
      • Aggarwal B.B.
      • Kumar A.
      • Bharti A.C.
      ,
      • Buhrmann C.
      • Mobasheri A.
      • Matis U.
      • Shakibaei M.
      ,
      • Hatcher H.
      • Planalp R.
      • Cho J.
      • Torti F.M.
      • Torti S.V.
      ,
      • Jurenka J.S.
      ,
      • Shakibaei M.
      • John T.
      • Schulze-Tanzil G.
      • Lehmann I.
      • Mobasheri A.
      ). Recent studies have shown that curcumin mediates its effects by modulation of several important molecular targets, including transcription factors (e.g. NF-κB, AP-1, β-catenin, and peroxisome proliferator-activated receptor-γ), enzymes (e.g. COX-2, 5-LOX, and iNOS), pro-inflammatory cytokines (e.g. TNF-α, IL-1β, and IL-6), and cell surface adhesion molecules. Because of its ability to modulate the expression of these targets, the therapeutic potential of curcumin for treating cancer, arthritis, diabetes, Crohn disease, cardiovascular diseases, osteoporosis, Alzheimer disease, psoriasis, and other pathologies is now under investigation (
      • Aggarwal B.B.
      • Kumar A.
      • Bharti A.C.
      ,
      • Shakibaei M.
      • John T.
      • Schulze-Tanzil G.
      • Lehmann I.
      • Mobasheri A.
      ,
      • Csaki C.
      • Mobasheri A.
      • Shakibaei M.
      ). Furthermore, curcumin has been studied in clinical trials for its anti-inflammatory, anti-carcinogenic, and free radical scavenger properties (
      • Bharti A.C.
      • Donato N.
      • Singh S.
      • Aggarwal B.B.
      ). Phase I clinical trials have indicated that human subjects can tolerate curcumin doses as high as 8–12 g/day with no adverse side effects (
      • Cheng A.L.
      • Hsu C.H.
      • Lin J.K.
      • Hsu M.M.
      • Ho Y.F.
      • Shen T.S.
      • Ko J.Y.
      • Lin J.T.
      • Lin B.R.
      • Ming-Shiang W.
      • Yu H.S.
      • Jee S.H.
      • Chen G.S.
      • Chen T.M.
      • Chen C.A.
      • Lai M.K.
      • Pu Y.S.
      • Pan M.H.
      • Wang Y.J.
      • Tsai C.C.
      • Hsieh C.Y.
      ,
      • Sharma R.A.
      • McLelland H.R.
      • Hill K.A.
      • Ireson C.R.
      • Euden S.A.
      • Manson M.M.
      • Pirmohamed M.
      • Marnett L.J.
      • Gescher A.J.
      • Steward W.P.
      ). Moreover, several aspects of the pharmacological properties and the use of curcumin for cancer chemoprevention have been reviewed recently (
      • Shehzad A.
      • Wahid F.
      • Lee Y.S.
      ). Although curcumin is a potent inhibitor of NF-κB, its effects on human tenocytes have not been investigated at the cellular or molecular levels.
      Phosphatidylinositol 3-kinases (PI-3Ks) are a highly conserved family of kinases that catalyze the 3-position of the inositol ring of phosphoinositides to generate phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate (
      • Vanhaesebroeck B.
      • Alessi D.R.
      ). PI-3K is a heterodimeric lipid kinase consisting of an 85-kDa regulatory subunit and a 110-kDa catalytic subunit that plays a pivotal role in cell movement, growth, vesicular trafficking, mitogenesis, and cell survival (
      • Coffer P.J.
      • Geijsen N.
      • M'rabet L.
      • Schweizer R.C.
      • Maikoe T.
      • Raaijmakers J.A.
      • Lammers J.W.
      • Koenderman L.
      ,
      • Heldin C.H.
      • Ostman A.
      • Rönnstrand L.
      ). PI-3K is involved in the IL-1β signaling pathway and mediates activation and translocation of NF-κB through targeting IKK-α or phosphorylation of p65, a process that is inhibited by the PI-3K-specific inhibitor wortmannin (
      • Reddy S.A.
      • Huang J.H.
      • Liao W.S.
      ,
      • Reddy S.A.
      • Huang J.H.
      • Liao W.S.
      ). Several reports suggest that PI-3K activates protein kinase B (Akt), one of the main downstream kinases in cells (
      • Vanhaesebroeck B.
      • Alessi D.R.
      ,
      • Burgering B.M.
      • Coffer P.J.
      ). However, the PI-3K/Akt signaling pathway has not yet been implicated in the activation of NF-κB in tenocytes.
      The aim of this study was to exploit an in vitro model of human tenocytes to study the mechanism of curcumin in IL-1β signaling and investigate whether curcumin might antagonize the catabolic effects of pro-inflammatory cytokines by suppressing NF-κB-activation and NF-κB-induced gene expression. We also explored the molecular mechanisms by which curcumin suppresses NF-κB activation in tenocytes, a process that was partly mediated by the PI-3K/Akt signaling pathway.

      DISCUSSION

      The purpose of this study was to investigate the effects of curcumin on the IL-1β-induced NF-κB activation pathway and NF-κB-regulated gene products that influence inflammation in tendon. Using monolayer-cultured human tenocytes, we found that curcumin inhibited IL-1β-induced NF-κB activation through suppression of IκBα phosphorylation, IκBα degradation, IκBα kinase activity, and NF-κB-dependent gene products involved in inflammation (COX-2), in extracellular matrix degradation (MMPs), apoptosis (Bcl-2, Bcl-xL, and TRFA-1), and activation of apoptosis (i.e. activation of caspase-3). This inhibition was correlated with suppression of p65 phosphorylation, p65 nuclear translocation, and p65 acetylation. We also demonstrated that the PI-3K/Akt signaling pathway is activated in response to IL-1β and suppression of IL-1β-induced NF-κB activation by curcumin appears to involve the PI-3K/Akt pathway and its association with IKK.
      Tendinopathy is accompanied by inflammation and degradation of the tendon extracellular matrix. At a tendon injury site, pro-inflammatory cytokines such as IL-1β may initiate a cascade of events leading to tendon destruction and loss of biomechanical structural integrity. Furthermore, besides the up-regulation of inflammatory mediators, we found that IL-1β significantly down-regulates the expression of collagen types I and III, decorin, and tenomodulin in tenocytes. Thus, IL-1β-mediated suppression of collagen type I and other tendon-specific extracellular matrix compound expression may lead to the reduced deposition of extracellular matrix and consequently, it might affect normal tissue remodeling and lead to the development of tendinopathy. The control of IL-1β secretion may be critical for protecting tendons from pathological processes. In fact, human tenocytes express IL-1β receptors so that the ligand-receptor signal is transduced via the specific and functional IL-1β receptor. Moreover, IL-1β activates numerous signal transduction systems through protein kinases and this causes induction of genes by activation and suppression of specific transcription factors such as NF-κB (
      • Williams D.H.
      • Jeffery L.J.
      • Murray E.J.
      ).
      We found that curcumin suppresses the activation of NF-κB in human tenocytes in vitro and inhibits the expression of NF-κB-regulated gene products, including COX-2, MMPs, Bax, and caspase-3. Curcumin can both stimulate and inhibit apoptotic signaling, and the treatment time as well as the concentration may determine the effects of curcumin on various cell types (
      • Chan W.H.
      • Wu H.Y.
      • Chang W.H.
      ). Although curcumin has been shown to inhibit cytokine-induced NF-κB activation in many different primary cells and cell lines of various origins (
      • Bharti A.C.
      • Donato N.
      • Singh S.
      • Aggarwal B.B.
      ,
      • Buhrmann C.
      • Mobasheri A.
      • Matis U.
      • Shakibaei M.
      ,
      • Shakibaei M.
      • John T.
      • Schulze-Tanzil G.
      • Lehmann I.
      • Mobasheri A.
      ,
      • Csaki C.
      • Mobasheri A.
      • Shakibaei M.
      ,
      • Jang M.K.
      • Sohn D.H.
      • Ryu J.H.
      ,
      • Kumar A.
      • Dhawan S.
      • Hardegen N.J.
      • Aggarwal B.B.
      ,
      • Plummer S.M.
      • Holloway K.A.
      • Manson M.M.
      • Munks R.J.
      • Kaptein A.
      • Farrow S.
      • Howells L.
      ,
      • Sandur S.K.
      • Deorukhkar A.
      • Pandey M.K.
      • Pabón A.M.
      • Shentu S.
      • Guha S.
      • Aggarwal B.B.
      • Krishnan S.
      ), to the best of our knowledge, this is the first such report in human tenocytes.
      A possible mechanism underlying the inhibition of inducible NF-κB by curcumin could be its capacity to inhibit the PI-3K and Akt signaling pathways. Previous studies using other cells have shown that curcumin inhibits the DNA binding function of NF-κB through suppression of IκBα phosphorylation (
      • Buhrmann C.
      • Mobasheri A.
      • Matis U.
      • Shakibaei M.
      ,
      • Csaki C.
      • Mobasheri A.
      • Shakibaei M.
      ,
      • Zhang C.
      • Li B.
      • Zhang X.
      • Hazarika P.
      • Aggarwal B.B.
      • Duvic M.
      ). Thus, down-regulation of upstream signaling proteins, such as PI-3K/Akt, may be involved in curcumin-mediated activation of IL-1β-induced NF-κB inhibition in tenocytes. In fact it has been reported that the PI-3K pathway is required for activation of NF-κB by cytokines such as IL-1β (
      • Reddy S.A.
      • Huang J.H.
      • Liao W.S.
      ). Our results demonstrate that wortmannin, a specific inhibitor of the PI-3K pathway, inhibits NF-κB activation and its translocation in the nucleus in tenocytes. These observations suggest that the PI-3K pathway may be involved in IL-1β signaling. Furthermore, IL-1β-induced activation of PI-3K/p85 and Akt could be inhibited clearly by curcumin in human tenocytes. We found that inhibition of PI-3K/p85 by curcumin, a process required for Akt activation, inhibits IKK and phosphorylation of both IκBα and p65. We have also shown that curcumin stimulates the expression of several anti-apoptotic proteins that are regulated by NF-κB, including Bcl-2, Bcl-xL, and TRAF1. Curcumin also inhibits the pro-apoptotic protein caspase-3, the matrix degrading MMPs, as well as the inflammatory enzyme COX-2. This is consistent with previous reports that have shown that NF-κB activation requires the PI-3K/Akt signaling pathway (
      • Ozes O.N.
      • Mayo L.D.
      • Gustin J.A.
      • Pfeffer S.R.
      • Pfeffer L.M.
      • Donner D.B.
      ,
      • Kane L.P.
      • Shapiro V.S.
      • Stokoe D.
      • Weiss A.
      ,
      • Romashkova J.A.
      • Makarov S.S.
      ) and that curcumin suppresses the expression of pro-apoptotic proteins (Bax and caspase-3) and matrix degrading enzymes (MMPs) and mediators of inflammation (COX-2) (
      • Pahl H.L.
      ,
      • Shishodia S.
      • Aggarwal B.B.
      ). Several groups have demonstrated that curcumin can also inhibit the two subunits of PI-3K (p110 and p85) and phosphorylation of the Akt signaling pathway (
      • Hussain A.R.
      • Al-Rasheed M.
      • Manogaran P.S.
      • Al-Hussein K.A.
      • Platanias L.C.
      • Al Kuraya K.
      • Uddin S.
      ,
      • Shankar S.
      • Srivastava R.K.
      ). These findings might explain the anti-inflammatory and anti-apoptotic effects of curcumin in tenocytes.
      It is well known that the tendon-specific transcription factor SCXA is required for expression of tendon-specific extracellular matrix genes (
      • Schweitzer R.
      • Chyung J.H.
      • Murtaugh L.C.
      • Brent A.E.
      • Rosen V.
      • Olson E.N.
      • Lassar A.
      • Tabin C.J.
      ). We also observed a reduction in collagen type I and SCXA expression in tenocytes after treatment with IL-1β. However, curcumin pre-treatment inhibited the IL-1β-induced down-regulation of collagen type I and SCXA expression. Thus, curcumin stimulated tenocytes, at least in part, through activation of the tenogenic transcription factor scleraxis, enhancing transcription of tendon-associated collagens in a SCXA-dependent fashion.
      Moreover, curcumin did not exert any toxicity on the cells. Studies on the phase I clinical trials suggest that curcumin can be orally administered safely at doses of 0.2–12 g/day with no dose-limiting toxicity, reaching peak serum concentration at 1–2 h (0.51 ± 0.11 μm at 4000 mg, 0.63 ± 0.06 μm at 6000 mg, and 1.77 ± 1.87 μm at 8000 mg) and is eliminated within 12 h (
      • Cheng A.L.
      • Hsu C.H.
      • Lin J.K.
      • Hsu M.M.
      • Ho Y.F.
      • Shen T.S.
      • Ko J.Y.
      • Lin J.T.
      • Lin B.R.
      • Ming-Shiang W.
      • Yu H.S.
      • Jee S.H.
      • Chen G.S.
      • Chen T.M.
      • Chen C.A.
      • Lai M.K.
      • Pu Y.S.
      • Pan M.H.
      • Wang Y.J.
      • Tsai C.C.
      • Hsieh C.Y.
      ,
      • Sharma R.A.
      • McLelland H.R.
      • Hill K.A.
      • Ireson C.R.
      • Euden S.A.
      • Manson M.M.
      • Pirmohamed M.
      • Marnett L.J.
      • Gescher A.J.
      • Steward W.P.
      ,
      • Goel A.
      • Kunnumakkara A.B.
      • Aggarwal B.B.
      ,
      • Sharma R.A.
      • Euden S.A.
      • Platton S.L.
      • Cooke D.N.
      • Shafayat A.
      • Hewitt H.R.
      • Marczylo T.H.
      • Morgan B.
      • Hemingway D.
      • Plummer S.M.
      • Pirmohamed M.
      • Gescher A.J.
      • Steward W.P.
      ). Recently, a phase II study of this agent has shown that this compound has biological activity in patients with pancreatic cancer (
      • Dhillon N.
      • Aggarwal B.B.
      • Newman R.A.
      • Wolff R.A.
      • Kunnumakkara A.B.
      • Abbruzzese J.L.
      • Ng C.S.
      • Badmaev V.
      • Kurzrock R.
      ).
      Overall, our data suggest that curcumin down-regulates NF-κB and NF-κB-regulated gene products involved in apoptosis, matrix degradation, and inflammation in human tenocytes in vitro. These effects are mediated, at least in part, through down-regulation of PI-3K/Akt signaling. This study provides additional support for designing anti-inflammatory compounds based on curcumin for diseases mediated through NF-κB activation. Therefore, curcumin might have prophylactic potential for the treatment of tendinitis.

      Acknowledgments

      Katharina Sperling and Ursula Schwikowski are gratefully acknowledged for excellent technical assistance.

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