Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that play a key role in tissue-remodeling events under both normal and pathologic conditions (1). Their expression is regulated by growth factors, cytokines, and hormones, as well as by interactions with extracellular matrix (ECM) proteins (1). Endogenous inhibitors, such as tissue inhibitors of matrix metalloproteinases (TIMPs), also exist to counterbalance MMP activity (2).
To date, 4 TIMP family members have been described. TIMPs 1, 2, and 4 are secreted as soluble proteins, whereas TIMP-3 is associated with matrix components as an insoluble protein (3). Noncovalent, 1:1 stoichiometric binding of each TIMP to the catalytic site of the activated forms of secreted MMPs leads to the MMPs' inhibition (3). All TIMPs inhibit active MMPs with relatively low selectivity. However, it has been reported that TIMP-1 binds to and delays the activation of latent MMP-9 and MMP-3 pro forms, while TIMP-2 binds to and regulates activation of proMMP-2 (4). MMP activity is tightly controlled by TIMPs, and the resulting equilibrium regulates integrity of the ECM and, in so doing, plays a key role in a wide range of physiologic processes (1).
As a consequence, imbalances in MMP/TIMP expression ratios have been implicated in various pathologic conditions, including cardiovascular diseases, cancer, and autoimmune diseases (5–7). In this context, it is interesting to note that patients with severe and active Sjögren's syndrome (SS), a chronic autoimmune disorder that causes alterations in the structure and function of exocrine glands, express elevated levels of MMP-3 and MMP-9 in acinar and ductal cells from labial salivary glands (LSGs). These changes are closely associated with destruction of the basal lamina of acini and ducts, as well as degradation of the major interstitial structural proteins of the ECM (8, 9). Additionally, high MMP-9 activity correlates with structural and functional changes of the gland in these patients, whereas MMP-2 activity does not differ from that in controls (8, 9). In other structures of LSGs that lack both MMP-3 and MMP-9, such as blood vessels, the integrity of the basal lamina is maintained (8, 9). In contrast, in acini and ducts, where expression and activity of these MMPs is high, the basal lamina is completely disorganized, suggesting a localized role of MMPs in the cells that produce them (10). Furthermore, studies have shown that the activity of MMP-9 is elevated in saliva of SS patients (11).
When transformed cell lines derived from salivary glands are treated with cytokines (tumor necrosis factor α, interleukin-1β, interferon-γ), MMP messenger RNA (mRNA) and protein levels, as well as gelatinolytic activity, are highly stimulated (12, 13). Levels of the same cytokines are strongly increased locally in salivary glands of patients with SS (14).
Expression of MMPs is mainly transcriptionally regulated. Hence, MMP protein levels correlate well with mRNA expression (15). In the present study, the molecular mechanisms underlying salivary gland remodeling processes in the LSGs of patients with primary SS were characterized. For this purpose, levels of mRNA for both MMPs and TIMPs were quantified, and correlations between the MMP/TIMP ratios obtained and both the quantity of inflammatory cells present in LSG and the morphologic integrity of the residual gland parenchyma were calculated.
We found that in LSGs from patients with SS, both MMP-9/TIMP-1 and MMP-3/TIMP-1 ratios exceeded 1, suggesting elevated levels of proteolytic activity. The latter was independent of the focus score of inflammatory cells but correlated well with dramatic changes in the morphology of acini, manifested mainly as a loss in nuclear polarity. Since these endopeptidases are reportedly produced by acinar and ductal cells in LSGs, our findings suggest that these cells play a crucial role in ECM remodeling processes and in the pathophysiology of SS.
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- PATIENTS AND METHODS
MMPs are secreted as latent pro forms that require N-terminal truncation for activation. Members of the TIMP family inhibit MMPs. Several studies analyzing their expression, protein levels, and gelatinolytic activity in salivary glands, saliva, and tears (13, 20, 21) suggest that MMPs, particularly MMP-9, are involved in the development of SS. Given that TIMPs represent the major regulators of MMP activity, we conducted the current study to investigate alterations in MMPs in relation to their respective TIMPs in LSGs of SS patients, by estimating MMP/TIMP ratios. These ratios were compared with morphologic findings of acinar damage and inflammatory infiltration of the gland.
A ratio of MMP/TIMP expression favoring proteolytic activity was observed in many of the SS patients, with R values greatly exceeding 1 for MMP-9/TIMP-1 and exceeding 1 for MMP-3/TIMP-1 (Figure 2), while R values for MMP-2/TIMP-2 remained close to 1. R values did not correlate with the focus score and, interestingly, among SS patients with similar quantities of inflammatory cells infiltrating the gland tissue, very different R values were determined (Figure 2). In contrast, a strong correlation between high R values and marked histologic alterations of the glandular acini of SS patients was observed. Notably, patients with high R values for MMP-3/TIMP-1 and MMP-9/TIMP-1 exhibited the most severe morphologic changes in LSG acini (Figure 3). MMP protein levels also correlated well with mRNA expression (Figure 4), whereas, although both TIMP-1 mRNA and TIMP-1 protein levels appeared to be low in SS patients, protein levels analyzed by Western blotting were not significantly different from those in controls. The tendency toward low TIMP-1 levels was observed in 13 of the 16 SS patients studied; however, only 5 of them had clearly low levels, while 8 showed slightly reduced levels of TIMP-1 protein and 3 had values similar to those in control subjects (Figure 4). The latter could explain the lack of statistical significance of these results. The trend toward low TIMP-1 protein levels was corroborated by the results of immunohistochemical analysis, in which low TIMP-1 reactivity was also seen in SS patients. Therefore, the low protein expression correlated with TIMP-1 mRNA levels.
The balance between the levels of activated enzymes and free TIMPs determines overall MMP activity. Thus, maintenance of this equilibrium is essential, and any disturbance in this balance likely results in tissue damage by increased proteolysis, as observed in this study. This is an important finding, since the reasons underlying the loss of structural integrity of acini from salivary glands of SS patients have been poorly understood.
The formation of a ternary proMMP-9–TIMP-1–MMP-3 complex in vitro has been described as an intermediate stage in the activation of proMMP-9. Excess MMP-3 weakens the interaction between proMMP-9 and TIMP-1, leading to proMMP-9 processing and further activation (22). The authors of that report also evaluated the action of MMP-3 on proMMP-9 by SDS–polyacrylamide gel electrophoresis under reducing conditions and found 3 bands of 92, 84, and 82 kd, with the first corresponding to the MMP-9 latent form and the others to active forms (22). In the present study, the gland extracts from controls and SS patients showed 3 bands of 98, 86, and 83 kd, suggesting that proMMP-9 might be activated through formation of the above-mentioned ternary complex.
A number of purified proteases, including trypsin, chymase, MMP-2, trypsin 2, plasmin, and MMP-3, have been reported to activate proMMP-9 in vitro (22). However, based on in vitro kinetic and catalytic parameters, MMP-3 appears to be the most efficient activator of proMMP-9 (22). Nevertheless, MMP-3 is also produced as a zymogen and requires activation to convert the latent form of MMP-9 into an active enzyme. Our previous studies have demonstrated high levels of expression of MMP-9 and MMP-3 in LSGs of SS patients, with high MMP-9 activity in patients with the most severe pathologic manifestations (9). Relative protein levels of MMP-3 could not be evaluated here with the techniques available. Thus, we were not able to determine whether this might represent a plausible mechanism by which MMP-9 is activated at different stages of the disease.
An alternative proposed mechanism for activation of proMMP-9 is by human trypsin 2, a serine protease present in acinar cells (21, 23). We postulate that this enzyme may be important when the level of active MMP-3 in the LSG of SS patients is low. However, digestion of proMMP-9 with human trypsin 2 produced a unique peptide of 77 kd (23). Since no such band was detected in our experiments (Figure 4), it is unlikely that trypsin 2 activates MMP-9. As discussed in previous reports, other enzymes with serine-arginine protease activity have been detected, but predominantly in patients in whom MMP activity is reduced, perhaps due to advanced progression of the disease (9). Thus, the exact mechanism by which proMMP-9 is activated remains to be defined. Future experiments will explore the nature of the proteases involved.
Recent studies demonstrated increased MMP-9/TIMP-1 ratios in whole mixed saliva from patients with primary SS (24), similar to our findings in individual acini. Given that whole saliva extracts were used, the cellular origin of MMP-9 and TIMP-1 was unclear, and since mucositis occurs frequently in SS, such chronic inflammation may be expected to favor the appearance of enzymes secreted from a variety of cells. Our data provide evidence in support of the notion that the imbalanced enzyme/inhibitor ratio detected in saliva reflected events occurring in the salivary gland. However, to demonstrate this directly, other possible sources of these proteins in saliva would have to be evaluated.
Finally, the altered ratio of MMP/TIMP mRNA expression described herein suggests that regulation of the genes occurs either at the transcriptional level or by changes in mRNA stability. The regulation of MMP genes in normal tissues has yet to be thoroughly examined, but available evidence suggests that MMP family members are expressed as a complex in a highly individualized tissue-specific manner. Inducible MMPs (MMP-1, MMP-3, MMP-7, MMP-9, MMP-10, MMP-12, and MMP-13) contain 1 or more activator protein 1 (AP-1)–binding site(s) in their promoter regions whereas the promoter regions of constitutively expressed MMPs (MMP-2 and MMP-11) do not contain AP-1–binding sites, suggesting that AP-1 may be important in controlling the expression of inducible MMPs while different transcription factors regulate the expression of MMP-2 and MMP-11 (25–28). Additionally, several factors, such as cytokines, oxidative stress, and growth factors, modulate MMP expression at the transcriptional level (27, 29), thereby contributing to the complexity of the issue (27, 29). These factors, in particular, cytokines, are present in salivary glands of SS patients, and are likely to modulate MMP transcription (14).
To date, few reports have focused on the induction of MMPs in SS (12, 13, 20), although it has been suggested that a better understanding of the molecular mechanism regulating induction and repression of specific MMPs or TIMPs may provide valuable information for developing novel therapeutic approaches (27). Inducers seem to also have a strong effect on the stability of MMP and TIMP mRNA, with a few authors reporting modifications of the half-life of these mRNA (30, 31). However, there is a lack of evidence for changes in the stability of MMP and TIMP mRNA in acinar or ductal cells.
The data reported here, together with our previous observations (8, 9), are the first to indicate that increased MMP-3 and MMP-9 expression, as well as activity derived from exocrine epithelial cells of LSGs, is one of the causes of acinar destruction in SS. The current results suggest that substantial increases in MMP expression in the diseased LSG may be potentiated by moderate decreases in TIMPs. This observed imbalance correlates strongly with the morphologic integrity of acini and the extent of ECM remodeling in LSGs of patients with SS.