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Tumor Necrosis Factor-α-induced Proteolytic Activation of Pro-matrix Metalloproteinase-9 by Human Skin Is Controlled by Down-regulating Tissue Inhibitor of Metalloproteinase-1 and Mediated by Tissue-associated Chymotrypsin-like Proteinase*
* This work was supported in part by the Plastic Surgery Education Society and the Wound Healing Foundation (to Y. P. H.) and National Institutes of Health Grant GM 50967 (to W. L. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The proteolytic activation of pro-matrix metalloproteinase (MMP)-9 by conversion of the 92-kDa precursor into an 82-kDa active form has been observed in chronic wounds, tumor metastasis, and many inflammation-associated diseases, yet the mechanistic pathway to control this process has not been identified. In this report, we show that the massive expression and activation of MMP-9 in skin tissue from patients with chronically unhealed wounds could be reconstituted in vitro with cultured normal human skin by stimulation with transforming growth factor-β and tumor necrosis factor (TNF)-α. We dissected the mechanistic pathway for TNF-α induced activation of pro-MMP-9 in human skin. We found that proteolytic activation of pro-MMP-9 was mediated by a tissue-associated chymotrypsin-like proteinase, designated here as pro-MMP-9 activator (pM9A). This unidentified activator specifically converted pro-MMP-9 but not pro-MMP-2, another member of the gelatinase family. The tissue-bound pM9A was steadily expressed and not regulated by TNF-α, which indicated that the cytokine-mediated activation of pro-MMP-9 might be regulated at the inhibitor level. Indeed, the skin constantly secreted tissue inhibitor of metalloproteinase-1 at the basal state. TNF-α, but not transforming growth factor-β, down-regulated this inhibitor. The TNF-α-mediated activation of pro-MMP-9 was tightly associated with down-regulation of tissue inhibitor of metalloproteinase-1 in a dose-dependent manner. To establish this linkage, we demonstrate that the recombinant tissue inhibitor of metalloproteinase-1 could block the activation of pro-MMP-9 by either the intact skin or skin fractions. Thus, these studies suggest a novel regulation for the proteolytic activation of MMP-9 in human tissue, which is mediated by tissue-bound activator and controlled by down-regulation of a specific inhibitor.
). Within the MMP family, MMP-9 (gelatinase B; EC184.108.40.206) is particularly important, because it has a documented role in many human diseases. Like most MMPs, MMP-9 is secreted as a latent zymogen that is maintained by the interaction between a cysteine in the N-terminal pro-domain and the active-site zinc atom. Proteolytic cleavage of the pro-domain is a common control mechanism for MMP activation, which triggers a conformational change of the enzyme and is termed the “cysteine switch” (
Metastasis is a multistep process, including detachment of cancer cells from a primary site, invasion into surrounding tissue through breakdown matrix, spreading through circulation, and proliferation in distant organs. Breakdown of the basement membrane zone (BMZ) is necessary to allow malignant cells to migrate from the primary location. MMP-9 is not normally expressed in developed tissues, yet it is highly expressed in many cancers (
). An interesting pattern of MMP-9 expression has also been documented in wound healing. In the normal repair of acute trauma or burn wound, pro-MMP-9 is transiently expressed and declines with the progress of healing (
). Given the proteolytic function of MMP-9 in the digestion of BMZ components such as type IV collagen, the pathogenesis of these diseases could be due to the activity of MMP-9.
A critical unanswered question regarding MMP-associated disease pathogenesis is the identification of specific factors controlling the expression and activation of the proteinase. MMP activities are regulated at multiple levels from their expression to activation by cleavage of the inhibitory domain as well as blockage by tissue inhibitors. Although the proteolytic activation of MMP-9 has been well documented in many diseases, the mechanism for the proteolytic conversion is not established. In this report, we show for the first time that the increased expression and activation of MMP-9 found in patients with chronic wounds could be reconstituted in cultured normal human skin by treatment with specific cytokines. Furthermore, we found that activation of pro-MMP-9 was mediated by a tissue-associated chymotrypsin-like proteinase. The TNF-α-mediated activation of pro-MMP-9 is controlled by down-regulation of the MMP inhibitor, TIMP-1. These findings provide a novel model to explain the MMP-9 activation in the pathogenesis of chronic wounds and tumor metastasis through the action of specific proinflammatory cytokines.
In this paper we present for the first time a novel model for the proteolytic activation of pro-MMP-9 in human skin. As shown schematically in Fig. 10, multiple factors, including specific cytokines, a tissue-secreted inhibitor, and a tissue-bound proteinase participate in the regulation of pro-MMP-9 activation in human skin. In quiescent tissue, very little MMP-9 is expressed, and the proteinase inhibitor, TIMP-1, is constantly expressed. During any inflammatory process, TGF-β induces the expression of pro-MMP-9. The MMP retains its latency by forming a complex with TIMP-1, which prevents its access to the tissue-associated activator, pM9A. When TNF-α is also present, it down-regulates TIMP-1. Then pro-MMP-9 can be converted by the tissue-associated chymotrypsin-like pM9A. We believe that this model can be extended to the pathogenesis of many inflammation-associated human diseases. Because these cytokines are present in significant concentrations in diseases such as chronic wounds (
), we believe that these cytokines may be the causal factors for MMP-9 expression and activation in these conditions and may ultimately lead to basement membrane remodeling.
MMP-9 has been implicated in the pathogenesis of several different types of diseases. A major biological function for MMP-9 has been suggested in the remodeling of the BMZ of the skin, which is based on its substrate preference for type IV collagen, the major component in BMZ and type VII collagen, which anchors the BMZ to the dermis (
). The association between nonhealing wounds and MMP-9 is thought to be related to a breakdown of the matrix upon which keratinocytes migrate to reepithelialize the tissue. The best evidence for a role for MMP-9 in BMZ remodeling comes from the study of a skin disease called bullous pemphigoid (BP), which is characterized by the separation of the epidermis from the dermis at the BMZ and is caused by autoantibodies and complements (
Our model proposes two distinct components in the biochemical control of MMP-9 activation. One is the tissue-associated chymotrypsin-like proteinase, the unidentified pM9A. We have characterized much about this enzyme, although its specific identity is not yet determined. Based on the inhibition by TPCK and other inhibitors for chymotrypsin,
Y.-P. Han, Y.-D. Nien, and W. L. Garner, unpublished data.
pM9A is a chymotrypsin-like proteinase. The tight tissue/cell association of pM9A suggests interesting possibilities about its nature. We found that ionized salt such as NaCl can easily dissociate pM9A from tissue, while the nonionized components such as urea and Triton X-100 failed to do so. This suggests that pM9A may bind to a charged component in skin, which is likely to be an extracellular matrix. The inhibition of aprotinin on pM9A also suggests a potential way to purify this protein through the inhibitor-conjugated chromatography. We anticipate that the initial biochemical characterization of pM9A presented here will provide the basis for the purification of this enzyme in the future. Under “Results,” we provide compelling evidence showing that candidate proteinases for the pM9A, MMP-3 and mast cell chymase, are not likely to be pM9A. We considered mast cell α-chymase as a candidate for pM9A because mast cells are strongly associated with skin inflammation. At the basal state, this chymase is located intracellularly in granules, and it is released by IgE-mediated stimulation. The best evidence to rule out this chymase as pM9A comes from the extraction experiment; NaCl can extract pM9A but not the chymase, and conversely, urea can extract the chymase but not pM9A. Two other groups have studied dog mast cell chymase on MMP-9 activation. These studies gave contradictory conclusions (
). We believe that the difference may be due to the nature of substrates used in those experiments. Taken together, the work presented in this paper indicates that the mast cell α-chymase is not likely to be the pM9A. Based on in vitro reconstitution experiments, MMP-3 was shown to convert pro-MMP-9 (
). In this study, we tested the role of MMP-3 in pro-MMP-9 activation in human skin and provide a clear finding that expression of MMP-3 is not sufficient for MMP-9 activation. In addition, the inhibitor for MMP-3 does not affect the skin-mediated pro-MMP-9 activation. Finally, MMP-3 and pM9A are distributed differently; MMP-3 is secreted, and pM9A is tissue-bound. Although we believe that we have excluded these two candidates as pM9A, the final conclusive evidence must wait until the molecular identification of pM9A, which will be the subject of our future work.
Initially, we assumed that TNF-α-mediated activation of pro-MMP-9 by human skin was through induction of its activator. To our surprise, the pM9A activities are steadily expressed and not regulated by the cytokines. This led us to a notion that TNF-α-mediated activation of pro-MMP-9 is probably governed at the inhibitor level. We believe that we have identified this inhibitor as TIMP-1. The most compelling single piece of evidence for a pM9A inhibitor comes from the effects of TGF-β. In the organ explant experiments, TGF-β potently induces pro-MMP-9 but fails to promote its activation. However, once the conditioned medium was removed from the explant tissue, the remaining skin tissue could activate pro-MMP-9. This indicates that the skin secretes an inhibitor, which in turn prevents the tissue-associated pM9A from processing pro-MMP-9. This putative pM9A inhibitor should satisfy the following criteria. First, it should be expressed at basal state and not down-regulated by TGF-β. Second, it must be down-regulated or sequestrated by TNF-α. Third, it must be secreted as a soluble factor. Fourth, down-regulation of the pM9A inhibitor should be linked to the activation of pro-MMP-9, and finally, the accumulation of the inhibitor should block the activation of pro-MMP-9. We provide evidence in this report that TIMP-1 meets all of these demands: 1) TIMP-1 is steadily expressed by skin; 2) most of TIMP-1 is secreted as a soluble factor; 3) TNF-α down-regulates TIMP-1, and this down-regulation correlates with activation of pro-MMP-9 in a dose-dependent manner; and 4) when exogenous TIMP-1 is added to skin explant culture, it blocks the activation of pro-MMP-9. However, whether TIMP-1 is the only inhibitor regulated by TNF-α is currently unknown to us. This possibility will be examined by depletion of TIMP-1 from the conditioned medium and testing whether the pM9A inhibitor function is also eliminated. An additional question we have not yet addressed is the nature of TNF-α-induced down-regulation of TIMP-1; it could be achieved at the level of transcriptional suppression or at the post-transcriptional level such as protein degradation. The cytokine-induced down-regulation of TIMP-1 is unlikely to occur through the sequestration or translocation of the protein, because we measured both the secreted and tissue-associated fractions, and the total amount of the inhibitor is down-regulated.
The findings we have reported here show that multiple factors, including specific cytokine, tissue-bound proteinase, and soluble factor, together participate in the activation of pro-MMP-9 in human skin. This complexity, with the simultaneous involvement of multiple factors, may explain why others failed to observe MMP-9 activation when utilizing the homogenous cell culture. In fact, we also failed to measure the proteolytic activation by culturing the keratinocytes and dermal fibroblasts (
). A comparison of the primary sequences between pro-MMP-9 and pro-MMP-2 shows that they are different at their N-terminal regions, where the presumed activation cleavage sites are located. This also indicates that there is a different mechanism for the activation of these two gelatinases, allowing for differences in control and regulation.
Finally, we believe that the model we have presented here may be extended to other human tissues besides skin. This model may also provide targets for pathophysiological responses in many inflammation-associated diseases. Blocking the potential linkage between inflammation and tumor metastasis by preventing the induction and activation of MMP-9 may prevent BMZ breakdown, limiting tumor cell migration. Acute liver failure induced by viral hepatitis, alcohol, or other hepatotoxic drugs has been reconstituted by injection of TNF-α into mice (
). In these TNF-α-treated mice, MMP-9 was massively induced and proteolytically activated in the liver. However, the cytokine-induced liver failure could be prevented by either knockout ofmmp genes or administration of MMP inhibitor. Thus, TNF-α-regulated factors such as TIMP-1 and the pM9A, as we show in this article, may also participate in the liver failure. Taken together, identification of the factors involved in MMP-9 expression and activation in human tissue may provide useful targets for potential therapeutic treatment of the diseases that involve matrix degradation.
We gratefully acknowledge the determination of TNF levels in tissue biopsies by Dr. Dan Remick (University of Michigan). We thank Dr. Ronald A. Kohanski (Mount Sinai School of Medicine) for valuable comments and editing of this manuscript. We thank Dr. Susan Downey (USC) for supplying the human skin.