Hyaluronan regulates PPARγ and inflammatory responses in IL-1β-stimulated human chondrosarcoma cells, a model for osteoarthritis
Highlights
► HMW-HA induced Akt and increased PPARγ in chondrosarcoma cells. ► HMW-HA reduced COX-2 and MMPs expression by suppressing MAPKs and NFκB signaling. ► Oligo-HA inhibited Akt, and reduced PPARγ in the cells. ► Oligo-HA increased COX-2 and MMPs expression via activation of p38 and NFκB.
Introduction
Hyaluronan (also called hyaluronic acid or HA) is a ubiquitous carbohydrate polymer and a major component of the extracellular matrix in multiple tissues (Toole, 2000). It is composed of repeating GlcNAcβ(1→4)-GlcUAβ(1→3) disaccharide units which molecular weight often reaches million Dalton. Studies showed that patients with an arthropathy have a lower concentration and molecular weight of hyaluronan in their joints than healthy humans (Dahl et al., 1985, Wu et al., 1997). Intra-articular injection of hyaluronan has been used in diverse clinical applications, including ECM regeneration, wound healing, and treatment of osteoarthritis (OA) (Altman, 2010, Mei-Dan et al., 2010, Muzzarelli et al., 2012). Injection of hyaluronan into OA-afflicted joints helps relieve pain in some, but not all, patients (Mei-Dan et al., 2010). The underlying mechanisms of hyaluronan in cells are not fully understood.
OA accounts for the major pain and disability of older adults. It is a gradually progressing disorder affecting mammalian joints and is characterized by the destruction of articular cartilage, resulting in discomfort and dysfunction of the affected joint (Cahue et al., 2007, Yelin, 1992). The pathologic changes that occur during OA development include proteoglycan degradation at the early stages, type II collagen degradation, and eventual localized or complete loss of the cartilage matrix (Homandberg, 2001). Cytokines and their downstream targets play major roles in the pathogenesis of OA (Daheshia and Yao, 2008, Kardel et al., 2003). For example, pro-inflammatory cytokine such as interleukin (IL)-1β is produced by activated synoviocytes and articular chondrocytes. IL-1β activates cyclo-oxygenase-2 (COX-2), and increases expression of several matrix metalloproteinases (MMPs), including MMP-1, MMP-3, and MMP-13 from normal articular human chondrocytes (Fan et al., 2006, Hiramitsu et al., 2006). Its signal transduction utilizes three classical mitogen-activated protein kinase (MAPK) pathways: p38, extracellular signal regulated kinase (ERK), and Jun terminal kinase (JNK) (Saklatvala, 2007). Upon activation, MAPKs then regulate the expression of MMPs (Ancha et al., 2007, Im et al., 2007). In OA cartilage, MMP-1, MMP-3, and MMP-13 levels are increased, and participate in the degradation of the extracellular matrix (ECM) under various physiological and pathological conditions (Sternlicht & Werb, 2001).
Chondrocytes may differentially respond to hyaluronan derivatives. Researchers observed that production of MMP-1 was significantly suppressed by a 1900-kDa hyaluronan (high-molecular-weight hyaluronan; HMW-HA) (Tanaka, Masuko-Hongo, Kato, Nishioka, & Nakamura, 2006). In contrast, others found that hyaluronan oligosaccharides (oligo-HA) increased MMP-13 expression which induced the loss of extracellular matrix proteoglycan and collagen of human articular cartilage (Ohno, Im, Knudson, & Knudson, 2006). A recent study also showed better protection of articular cartilage by hyaluronan with higher MW via inhibiting MMP-2, MMP-9 and plasminogen activator pathways (Hsieh, Yang, Lue, Chu, & Lu, 2008). Studies suggested that hyaluronan regulates MMPs expression via MAPK and nuclear factor kappa B (NF-κB) pathways (Campo et al., 2009, Noble et al., 1996, Ohno et al., 2006). HMW-HA may be degraded into hyaluronan oligosaccharides (oligo-HA) in vivo, and activate an NF-κB/IκBα auto-regulatory loop and induce transcription of MMP-9 and MMP-13 (Fieber et al., 2004, Noble et al., 1996). Although many effects of hyaluronan have been reported, the mechanism of the differential effects by its size remains elusive.
Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily and ligand-activated transcription factors. Studies suggest that PPARγ plays an important role in the pathogenesis of OA (Fahmi, Martel-Pelletier, Pelletier, & Kapoor, 2011). Among the two PPARγ isoforms, human cartilage mainly expresses PPARγ1, and its expression is suppressed by IL-1β and significantly lower in OA tissue than in normal tissue (Afif et al., 2007). Since oligo-HA and HMW-HA modulate inflammation in OA and MMPs differentially, we hypothesized that they may exhibit divergent effects on the regulation of PPARγ and inflammatory responses in chondrocytes.
The human chondrosarcoma SW-1353 cells respond to IL-1β stimulation by activation of MAPKs and induction of MMPs (Afif et al., 2007, Chao et al., 2011, Wang et al., 2010). These cells resembled properties of primary chondrocytes from OA subjects. Using this cell model, we examined the effects of HMW-HA and oligo-HA pretreatment in IL-1β-stimulated SW-1353 cells on (1) expression levels of MMP-1, MMP-13, COX-2, and PPARγ; and (2) intracellular signaling pathways (MAPK, NF-κB, and PI3K/Akt). Our data further elucidated the mechanisms of hyaluronan in human chondrocytes.
Section snippets
Reagents
Medical grade high-molecular-weight sodium hyaluronate (HMW-HA, MW = 600–1200 kDa) was purchased from Seikagaku Co (Japan). This hyaluronan product is endotoxin-free. Because the molecular weight of HMW-HA encompassed a wide range, molar solutions were not formulated, and doses were in mg/mL. The endotoxin-free hexamer sodium hyaluronate oligosaccharide (oligo-HA, MW = 1.22 kDa) was purchased from Cosmobio Co., Ltd. Most chemicals were purchased from Sigma (St. Louis, MO), unless indicated otherwise.
Dosage testing for hyaluronan on cell viability
Treatment of SW-1353 cells with HMW-HA up to 1000 μg/mL slightly but not significantly reduced viability or proliferation (Fig. 1A). In the contrary, oligo-HA less than 10 μg/mL reduced SW1353 cell viability although without significance (Fig. 1B). Therefore doses up to 1000 μg/mL HMW-HA and 10 μg/mL oligo-HA were used in the following experiments.
Differential modulation of IL-1β-induced MMPs and COX-2 by HMW- and oligo-HA
OA joints exhibited high MMP1 and MMP13 levels which are induced by IL-1β (Fan et al., 2006, Hiramitsu et al., 2006). To evaluate the effects of HMW-HA
Discussion
In this study, we investigated the role of HMW-HA and oligo-HA on modulation of IL-1β-induced expression of MMPs, COX-2, PPARγ and other inflammation-related genes in chondrosarcoma SW-1353 cells. In agreement with previous reports, HMW-HA suppressed the IL-1β-induced expression of MMP-1, MMP-13, and COX-2 (Mitsui et al., 2008, Tanaka et al., 2006). HMW-HA exerted an anti-inflammatory role through significant activation of Akt and suppression of NF-κB. Additionally we found that HMW-HA
Author's contributions
CCC designed research and wrote paper. MSH performed research and analyzed data. STL performed research. YHC performed research. CWC analyzed data. TKL analyzed data. PTH analyzed data. YFL analyzed data and wrote paper. CHC designed research and wrote paper.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
We thank Dr. Yu-Chih Liang in the School of Medical Laboratory Science and Biotechnology at Taipei Medical University for kindly providing antibodies against COX-2 and PPARγ. This work was supported by Taipei Medical University Wan Fang Hospital (100TMU-WFH-05) and the National Science Council of Taiwan (NSC98-2314-B-038-005-MY3).
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These authors contribute equally to this work.