Cyclic compressive mechanical stimulation induces sequential catabolic and anabolic gene changes in chondrocytes resulting in increased extracellular matrix accumulation

Abstract

Overcoming the limited ability of articular cartilage to self-repair may be possible through tissue engineering. However, bioengineered cartilage formed using current methods does not match the physical properties of native cartilage. In previous studies we demonstrated that mechanical stimulation improved cartilage tissue formation. This study examines the mechanisms by which this occurs. Application of uniaxial, cyclic compression (1 kPa, 1 Hz, 30 min) significantly increased matrix metalloprotease (MMP)-3 and MMP-13 gene expression at 2 h compared to unstimulated cells. These returned to constitutive levels by 6 h. Increased MMP-13 protein levels, both pro- and active forms, were detected at 6 h and these decreased by 24 h. This was associated with tissue degradation as more proteoglycans and collagen had been released into the culture media at 6 h when compared to the unstimulated cells. This catabolic change was followed by a significant increase in type II collagen and aggrecan gene expression at 12 h post-stimulation and increased synthesis and accumulation of these matrix molecules at 24 h. Mechanical stimulation activated the MAP kinase pathway as there was increased phosphorylation of ERK1/2 and JNK as well as increased AP-1 binding. Mechanical stimulation in the presence of the JNK inhibitor, SP600125, blocked AP-1 binding preventing the increased gene expression of MMP-3 and -13 at 2 h and type II collagen and aggrecan at 12 h as well as the increased matrix synthesis and accumulation. Given the sequence of changes, cyclic compressive loading appears to initiate a remodelling effect involving MAPK and AP-1 signalling resulting in improved in vitro formation of cartilage.

Introduction

The articulating ends of joint surfaces are covered by articular cartilage, which acts as a load-bearing surface that transmits applied forces to the underlying bone. Damage to this tissue from disease or trauma is of particular concern due to the limited ability of articular cartilage to self-repair. Attempts to treat damaged cartilage have met with limited success (Darling and Athanasiou, 2003, Hunziker, 2002). Repair of joint defects using in vitro-formed cartilage tissue is an alternative treatment, but the physical properties of this tissue are a fraction of native cartilage, which may affect its ability to successfully resurface a large joint defect in the long term. We and others have developed methods of applying compressive forces that mechanically stimulate chondrocytes and result in increased matrix accumulation and improved tissue formation (Lima et al., 2004, Mauck et al., 2003, Waldman et al., 2003, Waldman et al., 2005). However, the molecular mechanism regulating this physical improvement has not been determined.

One possible explanation is that mechanical stimulation is inhibiting matrix metalloprotease (MMP) expression, resulting in increased matrix accumulation. Transcriptional regulation of most MMPs is predominately controlled by DNA binding activity at the AP-1 consensus sequence. AP-1 sites are found in most MMPs, except for MMP-2 and MMP-10 (Crawford and Matrisian, 1996)and MT-MMPs (Westermarck and Kahari, 1999). The AP-1 transcription factor is a protein composed of Jun family homodimers (cJUN, JunB, JunD) or Jun/Fos (cFOS, FRA-1, FRA-2, FOS-b) heterodimers, that bind to the AP-1 site (Hai and Curran, 1991, Smeal et al., 1989), thus upregulating the expression of MMP. Activation of cJUN is through phosphorylation by SAPK/JNK whereas cFOS is activated by phosphorylation by the ERK1/2 pathway (Monje et al., 2003). In rat bladder smooth muscle cells, mechanical stretch has been shown to upregulate AP-1 binding activity and alter MMP expression which correlates with the development of bladder hypertrophy (Park et al., 1999). Alternatively, mechanical loading may increase chondrocyte gene expression of the matrix molecules, aggrecan and collagen. AP-1 sites have not been identified in the promoter regions of either type II collagen or aggrecan so regulation of these macromolecules is quite different than the MMPs. For example, type II collagen and aggrecan expression is regulated by several transcription factors including zinc finger Sp-1/Sp-3 (Chadjichristos et al., 2003, Tan et al., 2003), sox 5,6 and 9 (Lefebvre et al., 1998) and the Snail family (Seki et al., 2003). In other studies, mechanical stimulation has been shown to alter expression of these different genes and their corresponding protein but the ultimate response appears to be influenced by the type and duration of the mechanical force applied (Benjamin and Hillen, 2003).

The aim of this study was to determine the mechanism(s) by which cyclic compressive loading leads to increased matrix accumulation by chondrocytes. The profiles of metalloproteases (MMP 1, 2, 3, and 13) which have been implicated in cartilage degradation and collagen type II and aggrecan gene expression were examined post-stimulation and the signal transduction pathway activated by this mechanical loading was assessed. Understanding the mechanism(s) resulting in increased matrix accumulation may identify a way to further improve the properties of in vitro-formed cartilage. In addition it may allow a better understanding of the mechanism(s) regulating matrix production by chondrocytes, a poorly understood process.