Address reprint requests to Snehasikta Swarnakar, Department of Physiology, Indian Institute of Chemical Biology, Jadavpur, Kolkata 700032, India. E-mail: firstname.lastname@example.org
Abstract: Matrix metalloproteinases (MMPs) are part of a superfamily of metal-requiring proteases that play important roles in tissue remodeling by breaking down proteins in the extracellular matrix that provides structural support for cells. The intricate balance in protease/anti-protease stoichiometry is a contributing factor in a number of diseases. Melatonin possesses multifunctional bioactivities including antioxidative, anti-inflammatory, endocrinologic and behavioral effects. As melatonin affects the redox status of tissues, the association of reactive oxygen species (ROS) with tissue injury under different circumstances may be mitigated by melatonin. Redox signaling is expanding into all areas of basic and clinical sciences, and this timely review focuses on the topic of regulation of MMP activities by melatonin. This is a rapidly growing field. Accumulating evidence indicates that oxidative stress plays an important role in regulating the activities of MMPs that are involved in various cellular processes such as cellular proliferation, angiogenesis, apoptosis, invasion and metastasis. This review offers sections on MMPs, melatonin, major physiological and pathophysiological conditions in the context to MMPs, followed by redox signaling mechanisms that are known to influence the cellular processes. Finally, we discuss the emerging molecular mechanisms relevant to regulatory actions of melatonin on the activities of MMPs. The possibility that melatonin might have therapeutic significance via regulation of MMPs may be a novel approach in the treatment of some diseases.
MMPs, a family of highly homologous zinc-dependent endopeptidases, are known for their ability to cleave several extracellular matrix (ECM) constituents as well as nonmatrix proteins . MMPs are secreted or anchored to the cell surface thereby confining their catalytic activities to membrane proteins or proteins within the secretory pathway or extracellular space. They are important regulators of the functions of various biologically active molecules such as proinflammatory cytokines, chemokines, growth factors and serine proteinase inhibitors [2–4]. The activities and expressions of MMPs are regulated at several levels of gene transcription, zymogen activation, enzyme secretion and inhibition by endogenous inhibitors e.g., tissue inhibitor of metalloproteases (TIMPs) . Additional mechanisms by which MMP activities are fine-tuned involve regulation of mRNA stability, translational efficiency, enzyme compartmentalization, cell surface recruitment, substrate targeting, shedding, oligomerization, cellular uptake and autolysis. These mechanisms operate in a coordinated manner to assure that MMP expressions are well balanced at their respective sites. In addition to their role in destruction and remodeling of the ECM, MMPs are involved in many physiological and pathological processes including embryonic development, inflammation, immunity, chronic wounds, arthritis, periodontitis, cardiovascular disease and cancer [3, 4, 6]. There is overexpression of members of MMPs in pathological conditions characterized by connective tissue destruction, as evidenced by diseases such as arthritis, atherosclerosis, periodontitis and cancer .
Melatonin is a naturally occurring indoleamine found not only in mammalian species but also in nonmammalian vertebrates, in invertebrates and in plants [8–11]. In mammals, including the human, it is produced mostly in the pineal gland, although several other organs (e.g., retina, extraorbital lacrimal gland, gastrointestinal tract, Harderian gland, bone marrow cells, blood platelets and possibly other organs as well) also synthesize the hormone . Melatonin regulates circadian cycles and sleep by synchronizing the biological clock and by chemically causing drowsiness and lowering body temperature . Melatonin is the effective chronobiotic, i.e., a chemical substance capable of therapeutically re-entraining short-term dissociated or long-term desynchronized circadian rhythms or prophylactically preventing disruption following environmental insults . It controls seasonal reproduction in photoperiodically sensitive mammals .
Melatonin exerts a powerful antioxidant activity in addition to its other functions [16, 17]. In many lower life forms, it serves exclusively as an antioxidant . Unlike other antioxidants, melatonin does not undergo redox cycling; therefore, it cannot be reduced to its former state because it forms several stable end-products upon reacting with free radicals [19, 20]. Melatonin is beneficial for inhibiting apoptosis and liver damage resulting from oxidative stress caused by malarial infection . Recently, we have shown that melatonin protects against gastric injury by increasing angiogenesis, which acts through MMP-2 activation, and it also protects against endometriosis through apoptotic action via regulation of MMP-3 activity . The protective effect of melatonin against different diseases, in part, is because of its free radical scavenging properties. In this review, we will discuss the regulatory role of melatonin in modulating MMP-mediated physiological processes and major human diseases. The possibility of developing new approaches for melatonin’s use as a therapeutic strategy by reversing MMPs activities is considered.
All MMPs share a basic domain structure consisting of (i) a signal peptide that targets them for secretion, (ii) a propeptide with a cysteine residue that ligates the catalytic zinc ion for the preservation of latency, (iii) a catalytic domain containing the zinc-binding site [5, 23] and (iv) a hinge region and a carboxy-terminal hemopexin-like domain, both of which are lacking in MMP-7, -23 and -26. The propeptide domain (about 80 amino acids) has a conserved unique PRCG(V/N)PD sequence. The Cys within this sequence (the ‘cysteine switch’) ligates the catalytic zinc to maintain the latency of proMMPs. The catalytic domain (about 170 amino acids) contains zinc-binding motif HEXXHXXGXXH and a conserved methionine, which forms a unique ‘Met-turn’ structure. In addition, some domains are restricted to subgroups of MMPs, such as gelatin-binding domains present in the catalytic domain of the gelatinases (MMP-2 and MMP-9). These domains contain repeats of fibronectin motifs, which facilitate enzyme binding to gelatin. MMPs are classified in different subgroups, namely, collagenase, gelatinase, stromelysin, membrane-type MMPs, and others.
MMP-1 (collagenase-1), MMP-8 (collagenase-2), MMP-13 (collagenase-3) and MMP-18 (present in Xenopus) are members of the collagenase subgroup (Table 1). Collagenases have the ability to cleave interstitial collagens I, II and III at a specific site three-fourths of the distance from the N-terminus . Different collagenases differ in their substrate specificities and functional roles. Collagenases mainly upregulate during tissue remodeling, including embryonic development, wound healing and different types of malignant tumors, but is undetectable in resting tissues. MMP-2 (gelatinase A) and MMP-9 (gelatinase B) belong to gelatinase subgroup. They readily digest the denatured collagens and gelatins. MMP-2, but not MMP-9, digests collagen type I, II and III [25, 26]. MMP-2 is constitutively expressed by a wide range of cell types, including endothelial cells, macrophages and many malignant cells . The constitutive expression of MMP-9 is restricted to neutrophils . MMP-2 cleaves several ECM components, growth factors, and also proMMP-1, -2 and -13 .
Table 1. Types of different matrix metalloproteinases and their substrate specificity
Collagen I, II, III, VII, VIII, X, and gelatin
Collagen I, IV, V, VII, X, XI, XIV, and gelatin
Collagen II, IV, IX, X, and gelatin, α-casein, β-casein.
Collagen I, II, III, V, IV, X and casein
Gelatin, fibronectin and laminin
Gelatin, fibronectin and laminin
Gelatin, fibronectin and laminin
Fibrinogen and fibrin
Gelatin, fibronectin and laminin
Collagen IV, elastin and gelatin
Collagen I, IV and gelatin
Collagen I, IV, and gelatin
Collagen IV and gelatin
MMP-2 knockout mice show reduced angiogenesis and tumor growth . MMP-9 participates in the angiogenic switch necessary for tumor development , although other reports suggest anti-angiogenic effects . MMP-3 (stromelysin-1), MMP-10 (stromelysin-2), MMP-11 (stromelysin-3) belong to stromelysin subgroup. The stromelysins have a similar domain structure to those of collagenases but they cannot cleave native fibrillar collagens. MMP-3 can activate various MMPs including proMMP-1, -3, -7, -8, -9 and -13 , and it is itself activated also by plasmin, kallikrein, chymase and tryptase . Stromelysin-2 transcripts are expressed generally by normal or malignant cells of epithelial origin, at lower levels than stromelysin-1 and no expression has been detected in skin fibroblasts in vivo [2, 33, 34]. MMP-10 activates proMMPs-1, -2, -7, -8 and -9  and itself is activated by plasmin, elastase and cathepsin G . The other group, matrilysin, lacks a carboxy domain and MMP-7, -23, -26 are members of this subgroup. There are six membrane-type MMPs (MT-MMPs). Of the six MT-MMPs, four (MMP-14, -15, -16 and -24) have transmembrane and intracellular domains, whereas two (MMP-17 and -25) have glycosylphosphatidylinositol anchors, which target them to the cell surface. With the exception of MT4-MMP, they are all capable of activating proMMP-2. These enzymes can also digest a number of ECM molecules and MT1-MMP has collagenolytic activity on type I, II and III collagens . MT1-MMP is also more active in ECM degradation and promoting cell invasiveness in experimental models than its soluble form or the secretory MMPs, highlighting the importance of the cell surface localization and cellular regulation of these enzymes . MMP-18 is among other MMPs expressed in a wide variety of normal human tissues and has closest identity with MMP-1, -3, -10 and -11 . Enamelysin (MMP-20), MMP-21 and -22 are derived in a tissue-specific manner by alternative splicing  and are expressed in testis, ovary and prostate .
Almost all MMPs are inhibited by their natural inhibitors, TIMPs. TIMPs are specific inhibitors that bind MMPs in a 1:1 stoichiometry. Four TIMPs (TIMP-1, -2, -3 and -4) have been identified in vertebrates and their expression is regulated during development and tissue remodeling [24, 39]. TIMP-1 inhibits almost all MMPs, but it is not capable of properly inhibiting MMP-14, -15, -16, -19 and -24 . MMPs have been recently divided into three groups on the basis of the mechanism regulating their expression . Group 1 contains the TATA box and activator protein (AP)-1-binding site (MMP-1, -3, -7, -9, -12, -13, -19 and -26), group 2 the TATA box without an AP-1-binding site (MMP-8, -11 and -21), and group 3 lacks both AP-1-binding site and the TATA box (MMP-2, -14 and -28) . Several MMPs can activate other MMPs in vitro by cleaving their prodomains. Plasmin and other serine proteases have also been implicated in the activation of proMMPs [42, 43]. ProMMPs can further be subjected to allosteric activation. This is achieved through interactions between proMMPs and other molecules that induce a conformational change in the proMMP disrupting the cysteine–zinc interaction and allowing autolytic cleavage of the prodomain.
Melatonin (N-acetyl-5-methoxy tryptamine) is a derivative of tryptophan and is synthesized mainly in the pineal gland . It has multifunctional activities (Fig. 1).
Melatonin exerts powerful antioxidant actions in addition to its function as a synchronizer of the biological clock and seasonal reproduction . In many lower life forms, it serves exclusively as an antioxidant . Melatonin easily can cross cell membranes and the blood–brain barrier and is a direct scavenger of ˙OH, O2−˙ and nitric oxide (NO) among others. In animal models, melatonin prevents damage to DNA by some carcinogens and protects against brain injury caused by ROS in experimental hypoxic brain damage in newborn and adult rats [46, 47]. Unlike other antioxidants, melatonin does not undergo redox cycling; therefore, it cannot be reduced to its former state because it forms several stable end-products upon reacting with free radicals. Hence, it is referred to as a terminal antioxidant. Recent research indicates that a single molecule of AFMK (N(1)-acetyl-N(2)-formyl-5-methoxykynuramine), a metabolite in melatonin’s antioxidant pathway along with other by-products, can neutralize up to ten ROS/RNS [19, 20]. Melatonin’s antioxidant activity is beneficial in models of Parkinson  and Alzheimer disease  and has been shown to increase the average life span of mice by 20% in some studies [19, 20, 50].
It is known that human peripheral blood mononuclear cells synthesize biologically relevant amounts of melatonin . This indicates potential intracrine and paracrine role of melatonin in immune regulation. It is believed that melatonin influences cell signaling of the immune system via melatonin receptors . Both membrane and nuclear melatonin receptors have been identified in leukocytes . Through these receptors, melatonin modulates proliferative response of stimulated lymphocytes. On the other hand, melatonin induces cytokine production by human peripheral blood mononuclear cells via nuclear melatonin receptors . Some studies demonstrated immunoenhancing activity of melatonin. The activating effect of melatonin on the immune system is also mediated through the regulation of gene expression of cytokines in the spleen, thymus, lymph nodes, and bone marrow . It has been shown that gene expression of macrophage-colony-stimulating factor (M-CSF), tumor necrosis factor-alpha (TNF)α, and transforming growth factor (TGF)β is increased in peritoneal macrophage while interleukin (IL)1β, interferon (IFN), M-CSF and TNFα were increased in spleen cells of mice treated with melatonin . Other studies showed administration of melatonin increases natural killer (NK) cell activity in humans .
While many studies have implicated melatonin as a positive regulator of immune response, a number of other reports have showed that melatonin acts as an anti-inflammatory agent. It is believed that anti-inflammatory function of melatonin is at least partly because of the induction of Th2 lymphocytes that produce IL-4 thereby inhibiting the function of Th1 cells . Inflammation begins when cells (whether they be epithelial or stromal cells, tissue resident mast cells or dendritic cells) within the infected site recognize an inflammatory stimulus. This signals lead to recruitment and activation of effector cells of the immune system. Melatonin also reduces recruitment of neutrophils to the site of inflammation . Previous works suggest that, at the onset of a defense response, the increase in circulating TNFα leads to a transient block of nocturnal melatonin production and promotes a disruption of internal time organization . However, melatonin is a potent anti-inflammatory agent and is known to attenuate the increased expression of inflammatory genes. Melatonin has the ability to inhibit myeloperoxidase activity during wound healing. Melatonin has been shown to regulate NO synthesis [59, 60]. Maestroni et al.  have investigated on pathology of septic shock and found that indeed melatonin-treated mice were protected from LPS-induced shock and reduced the mortality correlated with NO synthesis. It has been recently reported that melatonin inhibits expression of inducible nitric oxide synthetase in murine macrophage via suppression of nuclear factor-kappa B (NFκB) . Furthermore, NFκB-dependent genes are transcribed, which encodes for proinflammatory cytokines and chemokines. Melatonin has been shown to reduce binding of NFκB to DNA, probably by preventing translocation to the nucleus , which in turn reduces production of proinflammatory cytokines and chemokines. Because melatonin has been shown to reduce adhesion of leukocytes to endothelial cells as well as transendothelial migration [64, 65], it may also suppress the expression of NFκB-regulated adhesion molecules.
Programmed cell death or apoptosis occurs naturally under normal physiological conditions and in a variety of diseases while necrosis is caused by external factors such as infection, toxins or trauma. Studies in peripheral tissues have documented that melatonin inhibits apoptotic processes via its antioxidant properties . For example, melatonin protects against cyclosporin A-induced hemolysis in human erythrocytes because of depuration resulting from O2˙− produced by mitochondria [67, 68]. Melatonin is also highly protective against mitochondrial ROS-induced cardiotoxicity, resulting from doxorubicin treatment . Many lines of evidence indicate an anti-apoptotic effect of melatonin on thymic cells . The methoxyindole reduces DNA fragmentation induced by glucocorticoids in cultured thymocytes . A reduction in glucocorticoid receptor mRNA levels in the intact thymus as well as in cultured thymocytes that were treated with melatonin seem to be the most likely mechanism whereby melatonin inhibits glucocorticoid-induced cell death [72, 73]. Other studies reported that melatonin inhibits DNA fragmentation and the release of cytochrome c from mitochondria of mouse thymocytes treated with dexamethasone . Melatonin may act by inhibiting the mitochondrial pathway, presumably through the regulation of Bax protein levels . Interestingly, proapoptotic effects of melatonin have been noted in a number of tumor cell lines . In MCF-7 breast tumor cell studies conducted in the absence of exogenous steroid hormones, treatment with melatonin produced a 64% reduction in the cellular ATP levels through a membrane receptor-modulated pathway . These findings in tumor cells are in contrast to the described actions of melatonin in normal cells and suggest melatonin’s potential use in killing cancer cells while preserving the function of normal cells.
Melatonin plays a neuroprotective role via the inhibition of intrinsic apoptotic pathways and the activation of survival signals . Melatonin has the ability to inhibit the release of cytochrome c from Ca++-stimulated mitochondria [77, 78]. It mediates anti-apoptotic signals in neuronal cells and protects damage by enhancing the activation of Akt and its downstream target Bad . In addition, melatonin inhibits apoptotic signals by preventing the injury-induced decrease in phosphorylation of Raf-1, MEK1/2 and extracellular signal–regulated kinase (ERK1/2) and the downstream targets, including Bad and ribosomal S6 kinase . Melatonin effectively attenuates neural brain injury via the Bcl-2-related survival pathway by increasing the expression of Bcl-2  and Bcl-xL  in the ischemic brain of the rat.
Angiogenesis is the most crucial event for wound healing, which is regulated by several growth factors including vascular endothelial growth factor (VEGF) and endothelial NOS. Induction of angiogenesis is triggered by VEGF leading to endothelial cell proliferation and migration . Our laboratory has described a potential action of melatonin on VEGF expression and its influence on angiogenesis, particularly, in relation to the healing process during gastric injury . Melatonin not only alters the pathologic condition of gastric ulceration by regulation of angiogenesis but also maintains the capillary homeostasis of gastric mucosa under normal conditions, i.e., melatonin regulates both physiological and pathological conditions as a proangiogenic accelerator in gastric mucosa. The angiogenic potential of melatonin is significant in the rat cornea model for angiogenesis . Ma et al.  demonstrated that platelets help in gastric ulcer healing by secreting VEGF in serum.
Melatonin has a positive effect on both angiogenesis and wound healing. Daily application of melatonin (20 mg/kg bw) accelerates the ulcer-healing process by affecting cyclooxygenase-2 (COX-2)-mediated prostaglandin (PG) synthesis, expression of hypoxia inducible factor (HIF) and activation of eNOS-NO system thereby restoring mucous secretion and microcirculation in the ulcer bed . Indomethacin blocks VEGF-mediated angiogenesis through mitogen-activated protein kinase (MAPK) and ERK signaling cascades [86, 87]. An in vivo study has shown the attenuation of VEGF expression in injured tissues while melatonin blocks VEGF suppression during healing. Melatonin accelerates gastric ulcer-healing process by overexpression of VEGF suggesting its strong angiogenic potential. On the contrary, Blask et al.  reported that melatonin blocks angiogenesis by attenuation of VEGF secretion during neoplastic growth in cancer patients, which suggests melatonin’s anti-angiogenic property during prevention of cancer proliferation. This tumor growth inhibitory property of melatonin is mediated by the suppression of epidermal growth factor receptor/MAPK-mediated signaling mechanism . Thus, melatonin accelerates angiogenesis via increased secretion of proangiogenic growth factors during the natural wound-healing process while suppresses the upregulation of proangiogenic growth factors during cancer prevention. Hence, melatonin acts differently in terms of angiogenesis, as with apoptosis, according to tissue microenvironment.
MMPs in health and disease
Embryo implantation is a highly controlled process that is regulated by a series of factors. Successful embryo implantation depends upon the synchronized development of both the invasiveness of embryo and the receptivity of uterine endometrium [90, 91]. During implantation, the endometrium undergoes decidualization and manifests the maximal uterine receptivity that provides a suitable environment for the embryo to implant on uterine mucosa [91, 92]. This event is accompanied by extensive degradation and remodeling of ECM. Three enzyme families, including plasminogen activators, cathepsin, and MMPs are responsible for the degradation of ECM . ECM remodeling is a major requirement during invasion and penetration of uterine walls by the trophoblastic cells. The ECM components including collagen, fibronectin, and laminin may themselves direct the fate of events that the trophoblast undergoes . Direct evidence of a crosstalk between ECM components and MMPs was observed when cultured trophoblasts under stimulation by fibronectin, lamin or vitronectin demonstrated induction of MMP-9 expression [95–99]. Another key phenomenon that regulates the development of trophoblasts is the involvement of a plethora of paracrine and autocrine factors. Thus, MMPs may also be tightly regulated in such a hormonal milieu .
Remodeling of collagen, which includes the degradation of existing collagen fibrils and the synthesis of new ones, is a key part of the resolution phase of wound healing . MMPs have been shown to play a key role in collagen remodeling during wound resolution. MMPs have been implicated in inflammation, and this includes control of chemokine activity, the establishment of chemotactic gradients, and extravasation of leukocytes out of the blood into the injured tissue. Inflammatory cells are well known to express MMPs; however, epithelial and stromal cells in wounded tissue have also been demonstrated to express multiple MMPs including MMP-1, 2, 7, 9, 10 and 28. In anterior keratectomy corneal wounds, MMP-2 and 9, and to a lesser extent, MMP-3 are localized to the epithelial–stromal interface behind the migrating epithelial cells, which suggests that they may be involved in remodeling of the stroma and reformation of the basement membrane .
In culture, it has been demonstrated that keratinocytes express both MMP-2 and 9 while fibroblasts express only MMP-2 . More importantly, a co-culture of both keratinocytes and fibroblasts leads to increased MMP-2 and -9 abundance suggesting that interaction between the keratinocytes and fibroblasts, which occurs during wound healing, regulates MMP-2 and -9 expression by these cells . MMPs are able to cleave components of cell–cell junctions and cell–matrix contacts within the epithelium to promote re-epithelialization. Multiple MMPs have been associated with this aspect of wound repair, and these include MMP-1, 3, 7, 9, 10, 14 and 28 [104–108]. MMP-9 has also been implicated in re-epithelialization after injury. Epidermal growth factor and hepatocyte growth factor both stimulate keratinocyte migration in wound assays in vitro . MMP-14 expression is increased early in lung epithelium within the terminal airways following naphthalene injury, and it appears that MMP-14 is involved in the regulation of epithelial cell proliferation after injury .
Although not a life-threatening disease, endometriosis can cripple a patient and pose a severe risk factor for infertility and ovarian cancer. Endometriosis, defined as the presence of endometrial glands and stroma at an extrauterine site, remains a poorly understood and complex disease afflicting millions of women worldwide [110–112]. The etiology and basic pathophysiology of endometriosis is still controversial. The most widely accepted theory, however, is ectopic implantation of refluxed endometrial tissue at the time of menstruation [113–115].
Although endometrial expression of the MMP gene family is normally tightly regulated during the menstrual cycle, altered patterns of MMPs and TIMP expression have been reported in eutopic and ectopic endometrial tissues obtained from patients with endometriosis [116, 117]. MMP-2 and MT1-MMP proteins were found to be highly expressed in endometriotic tissues, compared with normal endometrium, but expression of TIMP-1 and TIMP-2 proteins was significantly reduced in the diseased tissues [118, 119]. More recently, Chung et al.  demonstrated lower levels of TIMP-3 mRNA expression in both eutopic endometrium and endometriotic lesions, compared with disease-free women, but MMP-9 mRNA was increased only in the lesions. It has been suggested that the presence of iron , macrophages , and/or environmental contaminants such as polychlorinated biphenyls [123, 124] in the peritoneal fluid may induce oxidative stress leading to tissue growth and endometriosis. Both MMP-2 and MMP-9 are activated by ROS, and their expressions seem to be regulated by oxidative stress [125–127]. The promoter region of MMP-9 gene contains NFκB, AP-1, stimulatory protein-1 and phorbol ester-responsive elements [128, 129]. NF-κB and AP-1 are redox-sensitive proteins and offer a potential mechanism by which oxidative stress may regulate MMP-9 transcription and activity during endometriosis .
Melatonin treatment increases apoptotic cells in endometriotic zones that accompany reduced Bcl-2 expression along with increased Bax expression and caspase-9 activation . A significant increase in the activity of MMP-3 with the severity of endometriosis in human is observed. Oxidative stress provides the initial trigger for c-fos expression leading to MMP-3 upregulation during the onset of endometriosis. Melatonin inhibits c-Fos overexpression and downregulates MMP-3 thereby repressing endometriosis via diminished AP-1 activity . Although the precise role of endometrial MMPs in the pathophysiology of endometriosis is not fully understood, several laboratories have independently reported the effect of MMPs in the establishment and progression of endometriosis.
Fibrogenesis or fibrosis
Fibrosis is defined by the overgrowth, hardening, and/or scarring of various tissues and is attributed to excess deposition of ECM components including collagen. Fibrosis is the end result of chronic inflammatory reactions induced by a variety of stimuli including persistent infections, autoimmune reactions, allergic responses, chemical insults, radiation and tissue injury [132, 133]. Major tissues affected by fibrosis are liver, lung and kidney. In some diseases, such as idiopathic pulmonary fibrosis (IPF), liver cirrhosis, cardiovascular fibrosis, systemic sclerosis and nephritis, extensive tissue remodeling and fibrosis can ultimately lead to organ failure and death. In contrast to acute inflammatory reactions, which are characterized by rapidly resolving vascular changes, edema and neutrophilic inflammation, fibrosis typically results from chronic inflammation and under conditions of inflammation, tissue remodeling and repair processes occur simultaneously. Despite having distinct etiological and clinical manifestations, most chronic fibrotic disorders have in common a persistent irritant that sustains the production of growth factors, proteolytic enzymes, angiogenic factors and fibrogenic cytokines, which stimulate the deposition of connective tissue elements that progressively remodel and destroy normal tissue architecture [134, 135]. In addition to resident mesenchymal cells, myofibroblasts are derived from epithelial cells in a process termed epithelial-mesenchymal transition (EMT) . EMT is, therefore, an area that merits more investigation because it is involved in tissue remodeling, metastasis as well as fibrosis.
The hypothesis that progression of liver fibrosis is associated with inhibition of matrix degradation in liver is strongly suggested by studies of the relevant MMPs and TIMPs in cultured hepatic stellate cells (HSCs) . The three most relevant MMPs are MMP-2, -9 and -3, all of which have been studied in liver . HSC-mediated activation of progelatinase A is significantly induced in the presence of collagen type I (the principal matrix protein found in fibrotic liver) . Increased MMP-9 activity has been reported in the livers of bile duct-ligated rats , suggesting that this enzyme may also play a role in liver fibrosis. ProMMP-3 is also secreted by HSCs in primary culture, but in contrast to MMP-2, its expression is transient .
In lung fibrosis, a temporal difference is observed in expression and localization of MMPs and TIMPs [141, 142]. In the early stage of this disease, MMP-9 activity is predominant and enhances fibroblast invasion to alveolar spaces . In vivo, it was shown that MMP-9 expression is increased in bronchoalveolar lavage fluid from patients suffering from IPF, the most common type of interstitial lung disease . In vitro, MMP-9 expression in cultured alveolar macrophages is decreased by a treatment with steroids and immunosuppressive agents . In lung fibrosis, MMP-2 could play a role in the regeneration of alveolar epithelial cells . Moreover, MMP-7 is identified as a potential target for the therapy of patients with IPF as MMP-7 gene is detected to be overexpressed by microarray gene analysis . A decreased expression of MMP-8 and -13 has been described in a rat model of lung fibrosis . In support of this, there is an overexpression of TIMPs leading to an imbalance in protease/anti-protease stoichiometry resulting in a microenvironment unfavorable to collagenolytic activity .
MMPs, through their capacity to degrade ECM proteins, are important components of oncologic disease processes . The human genome sequence has revealed more than 500 genes that encode proteases or protease-like proteins, with a large number being associated with tumor processes . Among these, the MMPs have been the focus of a large amount of anti-cancer research and clinical trials. MMPs mediate ECM and basement membrane degradation during the early stages of tumorigenesis, contributing to the formation of a microenvironment that promotes tumor growth . An often fatal characteristic of malignant tumors is their ability for tissue invasion and the generation of metastases [1, 150]. MMPs are also active in the later stages of cancer development in that they promote metastasis, as well as other aspects of tumor growth . MMP-9 is detected in malignant transformation of various cells and is associated with tumor metastasis [1, 151]. Inflammatory processes induce MMP-9 expression in several cells, including endothelial cells, macrophages, fibroblasts, and mast cells . MMPs promote the initiation and sustained growth of both primary tumors and metastatic foci by activating growth factors, by inactivating growth factor-binding proteins or by releasing mitogenic molecules from matrix proteins that are sequestered in the peri-tumor ECM . These MMP-activated growth factors directly induce tumor cell proliferation or indirectly regulate the behavior of fibroblast or endothelial cells that support tumor growth. MMPs also process cell adhesion molecules , and by cleavage of the proapoptotic FAS ligand (FASL), MMP-7 allows tumor cells to become resistant to apoptotic signals . Tumor-derived MMPs assist in circumventing the host anti-tumor defence system by destroying chemokine gradients . MMPs also promote tumor angiogenesis by mobilizing or activating proangiogenic factors, such as basic fibroblast growth factor, TGF-β or VEGF . They also negatively regulate angiogenesis by cleaving precursors of angiostatin and endostatin to generate active angiogenesis inhibitors .
MMPs and oxidative stress
Oxidative stress affects the function of proteins through multiple mechanisms, including regulation of protein expression, post-translational modifications, and alterations in protein stability . As proteins exist in an oxygen-rich environment reactions with ROS are unavoidable. Common ROS in biological systems include O2˙−, NO and ˙OH. Nonradical ROS include hydrogen peroxide, ozone, peroxynitrite and hydroxide . A slight increase in the level of ROS may result in transient cellular alterations, whereas an excessive rises in ROS in cells may cause irreversible oxidative damage, leading to cell death. ROS regulate many signal transduction pathways by directly reacting with and modifying the structure of proteins, transcription factors, and genes to modulate their functions . To prevent the harmful effects of ROS, cells control ROS levels by maintaining the balance between ROS generation and elimination. In mammalian cells, the enzymatic defense system consists mainly of superoxide dismutase, catalase (CAT), glutathione peroxides, and glutathione reductase .
In addition to oxidizing proteins, ROS are responsible for deaminating, racemizing, and isomerizing amino acid residues of proteins. These chemical modifications result in protein cleavage, aggregation and loss of catalytic and structural function by distorting the protein’s secondary and tertiary structure . Carbonyl and carbonyl adducts are the result of ROS reacting with lipids, sugars, and amino acids . Redox signaling, a widespread and essential process governing essential cellular activities, warrants an urgent need for detailed studies. The first direct suggestion that redox processes are involved in cell signaling is the adrenochrome hypothesis of Hoffer and Osmond . This holds that psychoactive oxidation products of catecholamines (adrenochromes) are involved in the etiology of schizophrenia and other neuropsychiatric diseases.
The transcription factor, HIF-1, has a central role in regulating the body’s response to changing oxygen levels. The expression of HIF-1-induced gene occurs when oxygen tension drops below a safe level . Thus, oxygen functions as a negative regulator of transcription. Most MMPs have both AP-1 and NFκB sites in their promoter region [130, 162]. Both these transcription factors contain redox-sensitive cysteine residues at their DNA-binding site. Oxidation of these Cys residues (SOx) may disrupt the transactivation activity and inhibit the expression of MMPs [163–165]. Newly synthesized MMP can be directly modified by oxidation of their Cys, Tyr and Met amino acids, resulting in alterations of their functions . Oxidants can both activate and inactivate MMPs by modifying critical amino acids via oxidation . Several proMMPs are activated in vitro by ROS but their role has not been confirmed in vivo [125–127, 168]. Post-translational modifications such as phosphorylation can either activate or inhibit the function of MMPs . H2O2, peroxynitrite and oxidants produced by the xanthine/xanthine oxidase system can activate both MMP-2 and MMP-9 . Augmentation of glutathione levels with N-acetylcysteine treatment has been shown to inhibit MMP activation . The cysteine switch of MMP-9 can also be activated by NO . Overall, oxidative and proteolytic processes can amplify each other and affect the protease/anti-protease balance in the tissues.
MMPs and melatonin
Melatonin provides multifaceted regulation of MMP gene expression and activity in addition to its anti-inflammatory and antioxidant properties . Several diseases and conditions that arise as a result of oxidative stress may benefit from melatonin treatment. Melatonin has been shown to control redox-dependent negative regulation of MMP-2 gene expression during gastroprotection . The regulatory role of melatonin on the induction and secretion of MMP-9 and -2 in gastric mucosa during gastroprotection has been demonstrated . The novel anti-ulcer activity of melatonin involves enhancement of MMP-2 and MT1-MMP along with downregulation of TIMP-2 . The indole has also been found to inhibit the activity of secreted proMMP-9 in a dose-dependent manner that is associated with upregulation of TIMP-1 and TIMP-2 while blocking ethanol induced gastric ulceration in mice. Melatonin’s ability to protect against ethanol-induced ulceration involves MMP-9 downregulation via inhibition of TNF-α . Melatonin suppresses MMP-3 activities at both enzymatic and protein levels during prevention of acute gastric injury in mice . It may have a beneficial role as a protective and therapeutic agent against NSAID-induced gastric injury by accelerating angiogenesis and ECM remodeling . More interestingly, immunofluorescence and in vitro collagenase studies revealed that during gastroprotection, melatonin induces the MMP-2 activation in gastric ECM (lamina propia), which assists in alteration of the basement membrane of blood vessels resulting in proper homeostasis in angiogenic processes. Melatonin in the system before or after ulcer development may arrest microcirculatory damage or initiate neovessel formation by upregulation of MMP-2 thereby escalating the angiogenic process. Thus, melatonin acts as preventive and therapeutic modulator of angiogenesis in physiological wound healing by ameliorating MMP-2 expression .
Melatonin treatment inhibits airway collagen accumulation, which was probably mediated by the inhibition of MMP-9 in a murine model of chronic asthma . Melatonin and its metabolites exert beneficial effects in different experimental model of spinal cord injury (SCI) in rodents. Melatonin exerts protective effects reducing SCI-induced MMP-9 and MMP-2 activity and expression. TNF-α is involved in the pathogenesis of SCI, and melatonin attenuates the TNFα production in the SCI in mice . Therefore, the inhibition of the MMP-2 and -9 by melatonin is most likely attributed to the suppressive effect on TNF-α production. Melatonin also decreases MMP-9 activation and expression and attenuates reperfusion-induced hemorrhage following transient focal cerebral ischemia in rats . Melatonin protects peritoneal endometriosis by downregulating proMMP-9 and -3 in a time and dose-dependent manner. The attenuated activity and expression of proMMP-9 were associated with subsequent elevation in the expression of TIMP-1 . Reports have described the protective role of melatonin in arresting peritoneal endometriosis in mice via inhibition of MMP-9 and -3. The expression ratio of proMMP-9 versus TIMP-1 is identified as a novel biomarker for assessing severity and progression of endometriosis that can be reversed by melatonin treatment . The action of melatonin on MMP-dependent pathways may be an alternate approach for better therapeutics that may limit disease progression.
Emerging molecular mechanism of action
Much of the research on melatonin following its discovery by Lerner et al. in 1958  was related to reproductive physiology, redox biology, immunology, and cancer biology. Melatonin’s antioxidant property  is the major discovery as this function of melatonin has implications for disease prevention as well as optimal functions of cells and organs in humans. Given the uncommonly low toxicity of melatonin, clinical trials are ongoing to arrest pathophysiological status by its application. The other strategy for combating disease by melatonin is to administer melatonin or its metabolites in proper pharmacological concentration at proper location using cutting-edge technology. It is not the total intracellular concentration of melatonin but rather the subcellular distribution in the immediate vicinity where radical generation occurs that is the important strategy to prevent oxidative stress-related disorders . Alternatively, the key enzyme of biosynthetic pathway of melatonin, acetyl serotonin-O-methyl transferase could be a target molecule for examining the potential involvement of melatonin in disease prevention. Several publications have studied the direct and indirect action of melatonin in reducing oxidative stress as well as in modulating ECM homeostasis. Melatonin has been found equivalent or more effective than other oxidants in terms of reducing oxidative stress in mitochondria-related disorders [17, 44, 68, 77]. Various experiments have also shown melatonin to possess a potent anti-fibrotic effect [179, 180]. Ethanol-administered rats treated with melatonin had significantly higher hydroxyproline and ascorbic acid levels and show an anti-fibrotic effect . Another study has also speculated that collagen accumulation in the intact skin is under the control of the pineal gland and melatonin markedly reduces collagen accumulation in the skin .
The regulatory influence of melatonin on the collagen accumulation in the scar formed after myocardial infarction has been studied. Exogenous melatonin elevates the collagen content but surgical pinealectomy or pharmacological blockade of melatonin exerts the opposite effect and reduces collagen content in the scar . Melatonin application to the pinealectomized or evening metoprolol-treated rats fully reverses both pinealectomy and metoprolol effects . Melatonin-dependent collagen accumulation in the infarcted heart scar could be considered beneficial as it accelerates healing. This may reduce the number of complications (heart rupture, aneurysm enlargement, and heart failure) . On the other hand, daily application of melatonin or l-tryptophan accelerates ulcer healing by affecting the COX-2/PG system with excessive production of protective PG, especially in later period of ulcer healing .
The enhanced expression of the melatonin receptor (MT2) combined with overexpression of key enzymes involves in biosynthesis of melatonin such as N-acetyltransferase and hydroxyindole-O-methyltransferase contribute to the acceleration of ulcer healing by this indole . Melatonin-induced acceleration of ulcer healing is also mediated by release of gastrin and ghrelin, the most potent stimulants of gastric mucosal cell proliferation and mucosal repair . When combined with antibiotics, melatonin causes a significant inhibition of malondialdehyde production and neutrophil infiltration caused by acute pyelonephritis in an experimental rat model; these are responsible for the protective effect of melatonin against renal damage, preventing renal scarring formation . In individuals with a head injury, melatonin can enhance osteogenesis. Osteoblastic activity rises with the increases in melatonin . Melatonin suppresses pinealectomy-induced elevation of the total and insoluble collagen content in wounds. No influence of the pineal gland on the soluble collagen content is observed. Thus, melatonin is involved in the inhibitory control of the collagen content . This review aims to enrich our understanding of MMP regulation by melatonin and the physiological significance of gene regulation at different tissue microenvironment. Hence, new therapeutic strategies for the use of melatonin are needed to protect deregulated expression of MMPs in treatment of diseases that affect millions of people worldwide.
MMPs are the key regulators of multiple aspects of tissue repair, and further study of these enzymes and their interaction with melatonin will not only advance the level of basic knowledge of different diseases, but will also provide insights into possible therapies using melatonin. While research to date has uncovered important aspects of melatonin’s regulatory role on MMPs, additional discoveries would assist in identifying the true therapeutic benefits of the modulation of MMP activity by antioxidants or anti-inflammatory agents; such information would have important implications for the treatment of devastating human diseases. Because of the increasing importance of redox signaling systems in cellular pathways, this review will serve as a timely and important reference source. New therapeutic strategies will surely emerge with understanding of the biochemical and molecular complexities of melatonin’s action on signaling pathways. Preclinical studies using melatonin are compelling but clinical data are just beginning to accumulate .