Toxicology in Vitro 28 (2014) 1242–1248
Contents lists available at ScienceDirect
Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
a-Lipoic acid (a-LA) inhibits the transcriptional activity of interferon regulatory factor 1 (IRF-1) via SUMOylation Tao Sun a,1, Fuyu Gao a,1, Xiaoyan Lin b, Ruixiang Yu c, Yong Zhao a, Jingjie Luan a, Hongyan Li a, Mingzhu Song a,⇑ a b c
Department of Orthopedics, Yantaishan Hospital, Yantai 264025, Shandong Province, China Department of Endocrine, Yantai Zhifu Hospital, Yantai 2960319, Shandong Province, China Department of Endocrine, Yantaishan Hospital, Yantai 264025, Shandong Province, China
a r t i c l e
i n f o
Article history: Received 1 February 2014 Accepted 18 June 2014 Available online 26 June 2014 Keywords: Osteoarthritis Interleukin (IL)-1b Lipoic acid Interferon regulatory factor 1 Chondrocyte SUMOylation
a b s t r a c t Osteoarthritis (OA), one of the most common joint disorders, is one of the leading causes of disability among the elderly. Proinflammatory cytokines, such as interleukin (IL)-1b, which is synthesized locally by synovial cells and chondrocytes, have been shown to play a critical role in sustaining cartilage damage in arthritis by creating an imbalance between cartilage degradation and the repair process. Alpha-lipoic acid (a-LA), which is synthesized in animal and plant tissues, has demonstrated its protective effects in a variety of diseases. However, whether or not LA has a protective effect in OA is still unknown. In this study, we found that a-LA inhibits the IL-1b-induced increase in matrix metallopeptidase 3 (MMP-3) and matrix metallopeptidase 13 (MMP-13) expression and activity. Our data also demonstrate that interferon regulatory factor 1 (IRF-1) participates in the induction of MMP-3 and MMP-13. However, a-LA treatment did not change IRF-1 levels. Importantly, we found that a-LA increases SUMOylation of IRF-1, which attenuates IRF-1’s transcriptional activity. Finally, we found that a-LA treatment leads to an increase in SUMO-1, but not in SUMO-2 or SUMO-3. Taken together, this study shows that a-LA exerts anti-inflammatory effects in an IL-1b-stimulated chondrocyte model, thereby suggesting a potential protective effect of a-LA in OA. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Osteoarthritis (OA), a chronic degenerative disorder, consists of a group of mechanical abnormalities involving the degradation of joints. Chondrocytes are the only cells residing in cartilage. Although the underlying pathophysiological process of cartilage destruction in OA has not been completely elucidated, multiple lines of evidence have demonstrated that cytokines, particularly pro-inflammatory cytokines derived from activated chondrocytes, such as tumor necrosis factor a (TNF-a) and interleukin-1 beta (IL-1b), are essential factors in the pathogenesis of OA (Lotz et al., 1995). IL-1b in particular has been considered a key amplifier and perpetuator of cartilage damage because it suppresses matrix protein synthesis and induces matrix-modifying enzymes (matrix metalloproteinase; MMPs) (Kapoor et al., 2011; Daheshia and Yao, 2008). Exacerbated production of matrix metalloproteinases ⇑ Corresponding author. Address: No. 167 Airport Road, Yantai 264025, Shandong Province, China. Tel./fax: +86 0535 6012685. E-mail address: [email protected] (M. Song). 1 Co-first author. http://dx.doi.org/10.1016/j.tiv.2014.06.003 0887-2333/Ó 2014 Elsevier Ltd. All rights reserved.
(MMPs), especially MMP-3 and MMP-13, which are produced primarily by chondrocytes, has been considered a key event in the progression of osteoarthritis (OA) Burrage et al., 2006. The activity of these enzymes is regulated by tissue inhibitors of metalloproteinase (TIMPs) Fernandes et al., 2002. Understanding the signal transduction pathways and molecular mechanisms that control MMP gene expression may provide new opportunities for the development of therapeutics that prevent the joint destruction seen in arthritis. Interferon regulatory factor-1 (IRF-1), identified as a transcriptional activator of the interferon family, has been verified to play a critical role in regulating the expression of MMPs (Sancéau et al., 2002). Previous studies have reported that IRF-1 is a substrate of SUMOylation (Nakagawa and Yokosawa, 2002). SUMOylation is a posttranslation modification in which the SUMO moiety (known as SMT3 in yeast) is covalently conjugated with the lysine residues of target proteins. A recent study has displayed that SUMOylation of IRF-1 attenuates its transcriptional activity and also interferes with IRF-1-mediated apoptosis (Park et al., 2007). Lipoic acid (LA, 1,2-dithiolano-3-pentanoic acid; C8H14O2S2) is a naturally occurring dithiol compound endogenously synthesized in animal and plant tissues (Liu et al., 1995). LA can be rapidly
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Fig. 1. Effects of a-lipoic acid (a-LA) on IL-1b-induced expression of MMP-3 in IL-1b stimulated chondrocytes. Human chondrocytes were pretreated for 24 h with a-LA at 0, 10 nM, 100 nM, 1 lM, 10 lM, 50 lM, 100 lM, or 1 mM and then either stimulated with 10 ng/ml IL-1b or left unstimulated for another 24 h. (A). mRNA expression of MMP-3 was determined by real time PCR; (B). Protein levels of MMP-3 in culture supernatants were quantified by sandwich ELISA at 24 h (*P < 0.01 vs. control group; #P
Contents lists available at ScienceDirect
Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
a-Lipoic acid (a-LA) inhibits the transcriptional activity of interferon regulatory factor 1 (IRF-1) via SUMOylation Tao Sun a,1, Fuyu Gao a,1, Xiaoyan Lin b, Ruixiang Yu c, Yong Zhao a, Jingjie Luan a, Hongyan Li a, Mingzhu Song a,⇑ a b c
Department of Orthopedics, Yantaishan Hospital, Yantai 264025, Shandong Province, China Department of Endocrine, Yantai Zhifu Hospital, Yantai 2960319, Shandong Province, China Department of Endocrine, Yantaishan Hospital, Yantai 264025, Shandong Province, China
a r t i c l e
i n f o
Article history: Received 1 February 2014 Accepted 18 June 2014 Available online 26 June 2014 Keywords: Osteoarthritis Interleukin (IL)-1b Lipoic acid Interferon regulatory factor 1 Chondrocyte SUMOylation
a b s t r a c t Osteoarthritis (OA), one of the most common joint disorders, is one of the leading causes of disability among the elderly. Proinflammatory cytokines, such as interleukin (IL)-1b, which is synthesized locally by synovial cells and chondrocytes, have been shown to play a critical role in sustaining cartilage damage in arthritis by creating an imbalance between cartilage degradation and the repair process. Alpha-lipoic acid (a-LA), which is synthesized in animal and plant tissues, has demonstrated its protective effects in a variety of diseases. However, whether or not LA has a protective effect in OA is still unknown. In this study, we found that a-LA inhibits the IL-1b-induced increase in matrix metallopeptidase 3 (MMP-3) and matrix metallopeptidase 13 (MMP-13) expression and activity. Our data also demonstrate that interferon regulatory factor 1 (IRF-1) participates in the induction of MMP-3 and MMP-13. However, a-LA treatment did not change IRF-1 levels. Importantly, we found that a-LA increases SUMOylation of IRF-1, which attenuates IRF-1’s transcriptional activity. Finally, we found that a-LA treatment leads to an increase in SUMO-1, but not in SUMO-2 or SUMO-3. Taken together, this study shows that a-LA exerts anti-inflammatory effects in an IL-1b-stimulated chondrocyte model, thereby suggesting a potential protective effect of a-LA in OA. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Osteoarthritis (OA), a chronic degenerative disorder, consists of a group of mechanical abnormalities involving the degradation of joints. Chondrocytes are the only cells residing in cartilage. Although the underlying pathophysiological process of cartilage destruction in OA has not been completely elucidated, multiple lines of evidence have demonstrated that cytokines, particularly pro-inflammatory cytokines derived from activated chondrocytes, such as tumor necrosis factor a (TNF-a) and interleukin-1 beta (IL-1b), are essential factors in the pathogenesis of OA (Lotz et al., 1995). IL-1b in particular has been considered a key amplifier and perpetuator of cartilage damage because it suppresses matrix protein synthesis and induces matrix-modifying enzymes (matrix metalloproteinase; MMPs) (Kapoor et al., 2011; Daheshia and Yao, 2008). Exacerbated production of matrix metalloproteinases ⇑ Corresponding author. Address: No. 167 Airport Road, Yantai 264025, Shandong Province, China. Tel./fax: +86 0535 6012685. E-mail address: [email protected] (M. Song). 1 Co-first author. http://dx.doi.org/10.1016/j.tiv.2014.06.003 0887-2333/Ó 2014 Elsevier Ltd. All rights reserved.
(MMPs), especially MMP-3 and MMP-13, which are produced primarily by chondrocytes, has been considered a key event in the progression of osteoarthritis (OA) Burrage et al., 2006. The activity of these enzymes is regulated by tissue inhibitors of metalloproteinase (TIMPs) Fernandes et al., 2002. Understanding the signal transduction pathways and molecular mechanisms that control MMP gene expression may provide new opportunities for the development of therapeutics that prevent the joint destruction seen in arthritis. Interferon regulatory factor-1 (IRF-1), identified as a transcriptional activator of the interferon family, has been verified to play a critical role in regulating the expression of MMPs (Sancéau et al., 2002). Previous studies have reported that IRF-1 is a substrate of SUMOylation (Nakagawa and Yokosawa, 2002). SUMOylation is a posttranslation modification in which the SUMO moiety (known as SMT3 in yeast) is covalently conjugated with the lysine residues of target proteins. A recent study has displayed that SUMOylation of IRF-1 attenuates its transcriptional activity and also interferes with IRF-1-mediated apoptosis (Park et al., 2007). Lipoic acid (LA, 1,2-dithiolano-3-pentanoic acid; C8H14O2S2) is a naturally occurring dithiol compound endogenously synthesized in animal and plant tissues (Liu et al., 1995). LA can be rapidly
T. Sun et al. / Toxicology in Vitro 28 (2014) 1242–1248
1243
Fig. 1. Effects of a-lipoic acid (a-LA) on IL-1b-induced expression of MMP-3 in IL-1b stimulated chondrocytes. Human chondrocytes were pretreated for 24 h with a-LA at 0, 10 nM, 100 nM, 1 lM, 10 lM, 50 lM, 100 lM, or 1 mM and then either stimulated with 10 ng/ml IL-1b or left unstimulated for another 24 h. (A). mRNA expression of MMP-3 was determined by real time PCR; (B). Protein levels of MMP-3 in culture supernatants were quantified by sandwich ELISA at 24 h (*P < 0.01 vs. control group; #P
