Chronic low back pain affects >70% of people at some point in their lives (1), with ∼10% being chronically disabled. The causes of low back pain are multifactorial, although ∼40% of all cases involve degeneration of the intervertebral discs (2). During degeneration, the matrix of the intervertebral disc undergoes structural, mechanical, and molecular changes resulting in a loss of demarcation between the outer annulus fibrosus and the inner nucleus pulposus. Additionally, alterations in collagen type and a decrease in proteoglycan content result in loss of tissue integrity, decreased hydration, and inability to withstand load (3). Importantly, the loss of proteoglycan, predominantly aggrecan, is considered to be an early indicator of intervertebral disc degeneration (4). Aggrecan molecules possess long core proteins with many chondroitin sulfate (CS) and keratan sulfate (KS) glycosaminoglycan (GAG) side chains (3). These GAG side chains are polyanionic due to the high content of carboxyl and sulfate groups, and thus they attract and bind water molecules, hydrating the tissue. Degradation of the proteoglycans, especially aggrecan, in disc degeneration results in decreased hydration and therefore in a reduced ability to resist compressive load.
As with articular cartilage, the extracellular matrix (ECM) in normal discs undergoes a process of remodeling and relies upon a delicate balance between matrix synthesis and degradation. In intervertebral disc degeneration there is a net increase in matrix-degrading proteinase activity compared with proteinase inhibitors, which disrupts the normal balance and leads to breakdown of ECM (5). Although the matrix metalloproteinases (MMPs) are thought to play a role in this process (6), it has been suggested that aggrecanases may also be involved, particularly since they participate in the degradation of aggrecan in articular cartilage and osteoarthritis (OA) (7, 8).
Aggrecanases are proteinases that cleave a particular glutamyl bond in the interglobular domain (IGD) of aggrecan, thereby releasing the bulk of the aggrecan molecule from the tissue (9). The first aggrecanase to be identified was termed aggrecanase 1 by Tortorella et al in 1999 (10) and is now known as ADAMTS-4. Later that year, the same research team reported the identification of a second enzyme, aggrecanase 2 (11), now known to be ADAMTS-5. The ADAMTS belong to a branch of the M12B adamalysin subfamily of metalloendopeptidases (12, 13) (http://merops.sanger.ac.uk/), and there are 19 representatives in the human genome. They are related to the MMP (M10) family but have different ancillary domains and are differentially expressed and regulated in model culture systems (14, 15). The common components of all ADAMTS family members are a signal peptide domain, a prodomain, a metalloproteinase domain, a disintegrin domain, a thrombospondin type 1 motif, a spacer domain, and a second thrombospondin module of a variable number of repeats at the C-terminal region (16). A phylogenetic subgroup of the ADAMTS enzymes (ADAMTS 1, 4, 5, 8, 9, and 15) possess aggrecanolytic properties (i.e., they are capable of cleaving aggrecan at the unique “aggrecanase” cleavage site in the IGD) (10, 11, 17–19). These ADAMTS are inhibited by tissue inhibitor of metalloproteinases 3 (TIMP-3) (20), which, like the ADAMTS, is bound in the ECM via interactions with sulfated GAGs.
To date, few studies have investigated the expression or activity of the aggrecanolytic ADAMTS in intervertebral discs and the role they may play in matrix degradation. Roberts et al (6) reported that aggrecanase-generated fragments increased with disease, their levels correlating with a higher degenerative grade of intervertebral discs, and Sztrolovics et al suggested a link with age (21). Hatano et al (22) showed expression of ADAMTS-4 messenger RNA (mRNA) and protein in herniated discs, while Le Maitre et al (23) demonstrated that native disc cells express ADAMTS-4 which increases with intervertebral disc degeneration. Recently, Patel et al (24) reported the presence of typical aggrecanase-generated aggrecan fragments in intervertebral discs, with ADAMTS-4 (but not ADAMTS-5) levels correlating with degeneration, particularly the p68 form of the enzyme. The aim of the present study was to investigate the mRNA and protein expression of the aggrecanolytic ADAMTS (ADAMTS 1, 4, 5, 8, 9, and 15) and their endogenous inhibitor TIMP-3 in nondegenerated human intervertebral discs and to investigate whether gene and protein expression and aggrecanase activity were altered during intervertebral disc degeneration.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
The ECMs of articular cartilage and the nucleus pulposus of the intervertebral disc share many similarities, being rich in type II collagen and proteoglycans, and these matrices both undergo a continuous process of homeostatic turnover driven by their resident populations of chondrocytic cells. Although nucleus pulposus cells and chondrocytes show a similar phenotype (25), these cells also differ in several aspects, including expression of certain key genes such as type IIA procollagen (32) and regulation of intracellular pH (33). One key feature that distinguishes the 2 tissues is the relative proportions of proteoglycan and collagen in their matrices, with nucleus pulposus tissue having a much higher ratio of proteoglycan:collagen (∼27:1) than that in cartilage (∼2:1) (34). During diseases such as OA and intervertebral disc degeneration, an imbalance occurs between anabolic matrix production and catabolic matrix degradation, resulting in an overall loss of both proteoglycans and collagens and eventual failure of the tissues. The role of MMPs in both homeostatic turnover and pathologic breakdown of articular cartilage and intervertebral disc ECMs has been studied for a number of years and is relatively well established. However, while the importance of members of the aggrecanase enzyme family in ECM breakdown is still under debate, the high proportion of proteoglycans in nucleus pulposus tissue suggests that aggrecanases may play a fundamental role in the initiation and progression of intervertebral disc degeneration.
A range of studies have demonstrated the importance of both ADAMTS-4 and ADAMTS-5 in the breakdown of articular cartilage in arthritis diseases (7, 35–37), but at present it is not known whether this is matched in degeneration of the intervertebral disc. ADAMTS-4 expression has been demonstrated in herniated intervertebral discs (38), and increased expression of the same enzyme was seen in disc degeneration (23). The same enzyme, particularly the processed p68 form, was found to be associated with degenerated intervertebral disc tissue and with increased levels of aggrecanase-generated aggrecan fragments (24). However, the present study is the first to investigate the gene and protein expression of all of the recognized aggrecanolytic ADAMTS (1, 4, 5, 8, 9, and 15) in a large group of nondegenerated and degenerated human intervertebral disc samples.
The results demonstrate for the first time that ADAMTS-1, -4, -5, -9, and -15 and TIMP-3 mRNA and protein are present in both nondegenerated and degenerated human intervertebral discs. The fact that expression is seen in nondegenerated discs could indicate a possible role for the ADAMTS enzymes in the normal turnover of aggrecan and other matrix molecules in the healthy disc matrix.
Interestingly, ADAMTS-8 was the only “aggrecanolytic” ADAMTS not expressed in either nondegenerated or degenerated discs despite exhibiting expression in both normal and OA articular cartilage (19). However, ADAMTS-8 is thought to be only weakly aggrecanolytic compared with other ADAMTS enzymes (37), and a study by Demircan and coworkers using a chondrosarcoma cell line and human chondrocytes failed to find expression of ADAMTS-8 and demonstrated that expression was not inducible by interleukin-1β (IL-1β) or tumor necrosis factor α (38). Since these 2 cytokines are thought to be key regulators of matrix enzyme–mediated catabolism in human intervertebral disc degeneration (39, 40), these findings may help explain the lack of ADAMTS-8 expression in either the nondegenerated or degenerated samples examined in the current study. The study by Demircan et al (38) also demonstrated that IL-1β was capable of increasing expression of ADAMTS 4, 5, and 9, and in our current study there was an increase in the number of cells expressing these genes and a significant increase in the mRNA and protein levels of ADAMTS 4, 5, and 9 in degenerated nucleus pulposus compared with nondegenerated samples. Since IL-1 is known to be increased in intervertebral disc degeneration (39), this may also explain the increased levels of these aggrecanases.
When we compared nucleus pulposus and annulus fibrosus regions, no differences were observed between the levels of mRNA expression, except for ADAMTS-4 expression in degeneration, where mRNA expression was higher in the annulus fibrosus than in the nucleus pulposus. Since the annulus fibrosus samples used for RNA analysis incorporated both inner annulus fibrosus and outer annulus fibrosus and since our data showed increased protein expression in the inner annulus fibrosus, this would suggest that the majority of aggrecanase mRNA expression detected in the annulus fibrosus may be derived from the inner annulus fibrosus. At the protein level, significantly lower expression of all targets was seen in the outer annulus fibrosus compared with the nucleus pulposus and inner annulus fibrosus regions. Thus, these data suggest that the ADAMTS play a more dominant role in the central regions of the disc, which consist predominantly of proteoglycans that are substrates for the aggrecanolytic ADAMTS.
The ECM of the annulus fibrosus region of the intervertebral disc is predominantly composed of type I collagen, and the amount of proteoglycan in the annulus fibrosus, particularly in the outer annulus fibrosus region, is minimal compared with that in the nucleus pulposus. Degeneration has been shown to start within the nucleus pulposus, and as the degradation becomes more severe, the inner annulus fibrosus and outer annulus fibrosus regions become affected. In the present study, increased mRNA and protein levels of ADAMTS expression were observed in the nucleus pulposus and inner annulus fibrosus as degeneration progressed, suggesting that the ADAMTS enzymes are likely to play a prominent role in the degradation process and loss of aggrecan.
In addition to increases in ADAMTS mRNA and protein expression, the present study has demonstrated, although in a small number of samples, that there is aggrecanase and MMP activity in vivo in the disc, and indeed these findings appear to suggest an up-regulation in degeneration. While the antibody used in the present study was directed toward a neoepitope in the IGD of aggrecan, this is not the only site susceptible to cleavage by the aggrecanases (41, 42). However, this site is arguably the most damaging to aggrecan function, since cleavage in this region removes the entire CS- and KS-rich regions, completely removing all GAG side chains, which would be detrimental to the hydration of the intervertebral disc. There is mounting evidence demonstrating that in degraded cartilage, aggrecan is primarily cleaved by aggrecanases and then later by MMPs (7, 9, 43, 44). This theory is supported by the report by Mercuri et al (45) that aggrecanase-generated G1 fragments are substrates for MMPs, but the MMP-generated 342FFGVG fragments are resistant to cleavage by aggrecanases. Further investigation would be necessary to establish whether MMPs or aggrecanases play the dominant role in intervertebral disc degeneration; however, the data presented here strongly suggest an important role for aggrecanases in the breakdown of aggrecan in the nucleus pulposus during intervertebral disc degeneration.
TIMP-3 mRNA expression was substantially higher in nondegenerated nucleus pulposus and annulus fibrosus samples than was mRNA expression of any of the ADAMTS enzymes studied, although levels of expression did not increase significantly with degeneration. While TIMP-3 is known to inhibit the ADAMTS, it also inhibits many other matrix proteinases such as the MMPs and ADAMs (46, 47) by binding noncovalently to the active sites of the target enzymes in a 1:1 stoichiometry (48). It can also be sequestered into the matrix via interaction with GAGs, and the high levels of TIMP-3 in healthy tissues may help to maintain the homeostatic balance of matrix turnover by inhibiting a wide range of catabolic enzymes. However, it is not known whether the static level of TIMP-3 in the degenerated state would be able to cope with the substantial increases in the ADAMTS, combined with the known increases of MMPs reported in intervertebral disc degeneration (23, 49). An increased number of degenerated samples (compared with nondegenerated samples) exhibiting expression of ADAMTS 4, 5, and 15 at the mRNA level, and increased mRNA copy numbers of all ADAMTS target genes in degeneration combined with no corresponding increase in TIMP-3 gene expression, could indicate a role for the ADAMTS aggrecanases in degeneration of intervertebral discs. This theory is supported by the fact that (although in a limited number of samples) we also demonstrated aggrecanase and MMP activity, suggesting that both these groups of enzymes are indeed active in the intervertebral disc, particularly in degenerated tissue.
While there has been much debate over the last decade about the relative importance of MMPs and aggrecanases in the degradation of aggrecan in cartilage, several studies have highlighted the importance of a number of aggrecanases, predominantly ADAMTS-4 and ADAMTS-5, in both the initiation and progression of OA (8, 9, 35, 36). Furthermore, inhibition of ADAMTS-4 and ADAMTS-5 in OA cartilage explants prevented the breakdown of aggrecan (8). While the results presented here do not give a definitive answer as to which enzyme or group of enzymes are the most important for intervertebral disc degeneration, they demonstrate that a wide range of aggrecanases (ADAMTS 1, 4, 5, and 15) show significant increases in degenerated tissue and therefore suggest a role for the ADAMTS enzymes in intervertebral disc tissue breakdown and degradation of aggrecan. Results of Western blotting using aggrecan neoepitopes also suggest that MMPs and aggrecanases may be working simultaneously to break down aggrecan, and the combined increase in their expression levels, without a coordinate increase in TIMP-3, may be sufficient to cause an imbalance in the normal homeostatic mechanism, leading to tissue breakdown. Further studies are therefore required to establish whether any one particular ADAMTS is vital for matrix degradation, or whether matrix degradation is a combined effect of multiple enzymes.
Evidence from imaging studies suggests that loss of aggrecan from the nucleus pulposus is an early and reversible process in degeneration which precedes the irreversible breakdown of the collagen network and eventual loss of disc height and function. Identification of a key enzyme, or enzymes, could lead to the development of a therapy aimed at preventing aggrecan loss in the early stages of degeneration, which would remove the need for surgical intervention.
- Top of page
- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
Prof. Hoyland had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study design. Buttle, Freemont, Hoyland.
Acquisition of data. Pockert, Lyon, Deakin.
Analysis and interpretation of data. Pockert, Richardson, Le Maitre, Lyon, Deakin, Buttle, Freemont, Hoyland.
Manuscript preparation. Pockert, Richardson, Buttle, Freemont, Hoyland.
Statistical analysis. Pockert, Le Maitre, Hoyland.