Hyaluronan regulates PPARγ and inflammatory responses in IL-1β-stimulated human chondrosarcoma cells, a model for osteoarthritis

Abstract

The carbohydrate polymer, hyaluronan, is a major component of the extracellular matrix in animal tissues. Exogenous hyaluronan has been used to treat osteoarthritis (OA), a degenerative joint disease involving inflammatory changes. The underlying mechanisms of hyaluronan in OA are not fully understood. Pro-inflammatory interleukin (IL)-1β downregulates peroxisome proliferator-activated receptor gamma (PPARγ), and increases expression of matrix metalloproteinases (MMPs) which are responsible for the degeneration of articular cartilage. The effects of low- and high-molecular-weight hyaluronan (oligo-HA and HMW-HA) on the inflammatory genes were determined in human SW-1353 chondrosarcoma cells. HMW-HA antagonized the effects of IL-1β by increasing PPARγ and decreasing cyclooxygenase (COX)-2, MMP-1, and MMP-13 levels. It promoted Akt, but suppressed mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NFκB) signaling, indicating anti-inflammatory effects. In contrast, the cells had overall opposite responses to oligo-HA. In conclusion, HMW-HA and oligo-HA exerted differential inflammatory responses via PPARγ in IL-1β-treated chondrosarcoma cells.

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.