Nuclear matrix metalloproteinases: functions resemble the evolution from the intracellular to the extracellular compartment.

Nuclear MMPs are found in disease cases. Nuclear localization signal is identified in MMP. Some membrane-bound MMPs contain nuclear localization signal. Nuclear localization signal is conserved among MMPs, and is comparable to transmembrane domain for conservation among membrane-bound MMPs in different animals. Nuclear localization of MMP induces cell death. Nuclear MMPs are found in disease cases. Nuclear localization signal is identified in MMP. Some membrane-bound MMPs contain nuclear localization signal. Nuclear localization signal is conserved among MMPs, and is comparable to transmembrane domain for conservation among membrane-bound MMPs in different animals. Nuclear localization of MMP induces cell death.

MMP proteins are present not only in vertebrates but also in plants and invertebrates, which shows evolutionary history of these proteinase enzymes. 55 Due to enormous functions of membrane MMPs in comparison with nuclear MMP, the nuclear MMP family might have evolved first. This can be confirmed from figures generated using database and software of phylogenetic trees ( Supplementary Data ) that compare NLS and TM of MT-MMPs, 56–58 such as MMP-14 and MMP-15. The resulting similarities between nuclear localization sequences in various species indicate the old evolutionary origin of NLS of this protein family. The comparison of NLS and TM of TM-MMPs can reflect the domain evolution of MMP translocation in different cellular compartment. The surprising results suggest that NLS and TM underwent a parallel evolution and in some species NLS has fewer branches than TM ( Supplementary Data ). Our analysis suggests that TM-MMPs may have undergone accelerated evolution in TM domain to overcome nuclear localization. Thus nuclear functions of MMP may be ancestral events that evade cancer or severe fatal diseases. Mutations could lead to the modification of MMP proteins including NLS sequence. Therefore, nuclear MMPs might have been evolved to translocate to the membrane through death-induced natural selection for survival. At that time, both nuclear and membrane MMPs were common, but eventually, as 'only the fittest survives', the function of nuclear MMP that underwent cell death became very limited and the fittest MMPs, membrane MMPs with plenty of functions that enhance cell survival became the most popular type. Nuclear MMPs, but not extracellular MMPs, in most cases, could induce cell death (apoptosis), 1 , 6 because evolution needs clearance of nuclear MMP to allow MMPs to translocate to the membrane. Therefore, cells with nuclear MMPs will eventually die and those cells without expression of nuclear MMPs are more likely to survive. We mentioned above that MMP-2 may contribute to tumor cell apoptosis. 21 However, it has been shown that MMP-2 co-localizes with TIMP-1 in neonatal rat neurons, while TIMP-1 inhibits MMP-2 function. 21 Thus, TIMP-1 can prevent MMP-2-induced apoptosis and allow cells without expression of nuclear MMP-2 can survive and evolve. Therefore, TIMP-1 might be the promoter for accelerated evolution of MMPs from nucleus to extracellular compartment. In addition, direct evidence suggests that extracellular MMP-9 and MMP-10 can disrupt apoptosis induced by p53 by multiple mechanisms including survival signaling such as IGF1. 59 Moreover, nuclear MMP-1 showed anti-apoptosis function by prevention of lamin A/C degradation and inhibition of caspase activation in glial Müller cells. 60 The counteract of extracellular and nuclear MMPs in inducing and inhibiting apoptosis suggests that the origin of MMPs might be in the nucleus. Apoptosis might be a selective tool to allow MMPs to evolve to localize to extracellular compartment. During the evolution, the nuclear MMP functions might be inhibited by TIMPs or other membrane MMPs for accelerated evolution. Cells that can survive the natural selection by apoptosis induced by nuclear MMPs may have undergone second gene mutation, abnormal signaling elevation or cross-talk signaling complementation. This might lead to cells to be survived, express nuclear MMPs and promote more aggressive diseases. For example, we and our collaborators found that nuclear MMP-7 associates with advanced prostate cancer. 14 Therefore, the natural selection and fitted survival might give the selection pressure for accelerated evolution of MMPs. In summary, based on our hypothesis, the evolution of MMPs can be outlined as shown in Figure 2 .

Why is nuclear localization signal conserved among MMPs, and comparable to transmembrane domain for conservation among membrane-bound MMPs in different animals? Does it mean that nuclear localization of MMP has an old origin and extracellular localization by transmembrane trafficking is later event during evolution? Why does nuclear localization of MMP induce cell death? Does it suggest that trafficking from nucleus to membrane is through Darwin’s natural selection based on cell survival? Why is nuclear localization signal conserved among MMPs, and comparable to transmembrane domain for conservation among membrane-bound MMPs in different animals? Does it mean that nuclear localization of MMP has an old origin and extracellular localization by transmembrane trafficking is later event during evolution? Why does nuclear localization of MMP induce cell death? Does it suggest that trafficking from nucleus to membrane is through Darwin’s natural selection based on cell survival?

It has been shown that extracellular MMPs can regulate transcription through signaling pathways. Particularly, membrane-bound membrane type 1 (MT1)-MMP (also named as MMP-14) can regulate transcription of VEGFA through Src kinase signaling, 47 Smad1 48 and Dickkopf-3. 49 , 50 It is possible that ECM degradation by MMPs can affect the integrin–ECM interactions, which can produce signals that regulate transcription. 47 However, whether nuclear MMP-mediated transcriptional regulation is direct has been unclear until recent studies showed that MT1-MMP or MMP in the nucleus can directly regulate transcription. 47–52 It has been shown that MMP-12 can directly bind to promoter and regulates transcription in immune response. 51 , 52 Interferons are proteins that signal the cell on the presence of bacteria, viruses or tumor cells, and induce immune system of the host cells. 51 After viral infection, macrophages release MMP-12 which is internalized and translocated into the nucleus. 52 Intracellular MMP-12 binds to NFKIBA promoter region and induces transcription of interferon-alpha (IFN- α ), so that it can induce antiviral immunity by destroying viral proteins. 51 , 52 However, extracellular MMP-12 has shown to degrade IFN- α receptor 2 binding site of systemic IFN- α , to reduce systemic toxicity. 51 , 52 Hence, it was suggested that inhibiting extracellular MMP-12 might lead to new avenues in the development of immune system against pathogenic viruses. 51 , 52 In addition, nuclear MMP-3 can bind directly to promoters and regulate transcription of multiple target genes. First, nuclear MMP-3 was identified as a DNA-binding protein in a screening using transcription enhancer dominant in chondrocytes. 53 Further nuclear MMP-3 was found to bind and transactivate promoter of connective tissue growth factor (CCN2/CTGF). 53 CCNs regulate wound healing, fibrotic disease, arthritis and cancer; and CCN2 promotes chondrocytic growth. 53 Further, nuclear MMP-3 can bind heterochromatin protein gamma as cofactors on the promoter of CCN2/CTGF. 53 Moreover, when we were revising the manuscript, one report just published the most recent finding that nuclear MMP-3 target genes by transcriptional regulation include Heat Shock family proteins such as Hsp70B and Hsp40. 54 In more details, nuclear MMP-3 can recruit nuclear heat shock factor 1 as a cofactor and bind together to transactivate Hsp70B enhancer. 54 Mechanistically, hemopexin-like domain is required for nuclear MMP-3 transcriptional activity. 54

By concluding the review of nuclear MMP family proteinases, many studies have shed the light to both cell death and evolutionary functions. Further research of detailed analysis of structure and function of matrixin is needed to screen inhibitors of proteinases to regulate abnormal cell proliferation, apoptosis or any other toxic effects. Understanding roles of nuclear MMP in signaling cascade ( Figure 3 ) and evolution ( Supplementary Data ) will create the development of therapeutic avenues. Moreover, gain of knowledge in evolutionary translocation of nuclear MMP to the membrane by cleavage and loss of NLS function in trafficking might shed the light to unknown properties of intracellular evolutionary functions of secreted proteins.

Among MMP subtypes, MMP-2, MMP-3, MMP-9 and MMP-13 have been found inside the nucleus of a variety of cell types of the heart, brain, breast and epithelium. 1 It has been shown that upon activation, some types of nuclear MMPs are translocated into the nucleus due to the presence of NLS on carboxyl terminus of pro-MMPs. 2 NLS is a peptide sequence that allows the active transport of a protein to the nucleus of cells. 11 The composition of the nuclear matrix is similar to the extracellular matrix, though they maintain various cellular functions such as the degradation of nuclear proteins. 12 Therefore, many MMPs in the nucleus can participate in various nuclear events such as the degradation of nuclear proteins and the regulation of transcription. We previously compared the putative NLS of different types of MMPs and found that various members of MMP family have conserved NLS. 13 Particularly, most of them appeared to have in common amino acids of lysine (K), tryptophan (W) and KW sequences. Furthermore, the comparison of NLS of MMP-7 protein in different species established the old evolutionary origin of NLS of MMP-7, which indicates the evolvement of this sequence through evolution. 13 Other studies showed that deletion of Arg 110 -Lys 111 and their substitution with Asn and Gln (R110N/K111Q) in NLS of MMP-3 lead to a significant decrease in nuclear localization of MMP-3. 6 Thus NLS-mediated nuclear localization is one of the mechanisms of nuclear translocation of MMPs. MMPs that do not contain NLS may translocate to the nucleus by cargoes, including RNA, RNA interaction proteins and NLS partner proteins. 14 In addition, endocytosis may be a mechanism underlying MMPs entering into nucleus. 15 For example, Membrane Type 1-MMP (MT1-MMP) which is elevated in hepatocellular carcinoma and promotes intrahepatic metastasis interacts and co-localizes with caveolin-1 at the perinuclear compartment. 15 This finding suggests that MT1-MMP may enter into the nucleus though caveolae by endocytosis. 15 Moreover, post-translational modifications may affect nuclear localization of MMPs. MMPs may undergo post-translational modifications through Golgi which localizes closely to the nucleus. Recent evidence suggests that MMPs entering into the nucleus might not through accidental invasion via Golgi–ER–nuclear pore. In human endothelial cells, Brefeldin A, an inhibitor that abolishes Golgi endosomal structures did not inhibit MMP-2 nuclear localization, suggesting that nuclear localization of MMP-2 is a stable event independent on the emission of cytosol-nucleus. 4 In addition, the nuclear MMP-2 was found to be an active form, which has undergone cleavage of pro-enzyme. 4 Thus, nuclear MMP-2 may have the similar function to extracellular MMP-2 or has been evolved in the nucleus. However, we found that MMP-7 localizes in the nucleus of prostate cancer cells with pro-enzyme form. 16 These data suggest that MMPs may undergo differential modifications during nuclear translocation. In conclusion, NLS-mediated import may link MMP to Importin proteins 16 and ARF-mediated protein modification may link MMP to SUMOylation, as we, our collaborators and other labs reported. 17

Nuclear MT1-MMP was found in the centrosome and cleaves centrosomal protein and pericentrin, which are required for centrosome function and mitotic spindle formation. Overexpression of MT1-MMP promotes spindle abnormality, aneuploidy, chromosome instability and thereby tumorigenesis. 29 , 30 In details, MT1-MMP can transform human mammary epithelial cells and cleave pericentrin through upregulating oncogenes, such as aurora kinase and β -catenin. 30 Moreover, MT2-MMP (also named as MMP-15), was examined to be localized to chromosome 16q12.1 closing to 16q heterochromatin using FISH in situ hybridization analysis, suggesting the role of chromosomal nuclear MMPs in genomic stability. 31

Matrix metalloproteinases (MMPs) are related to zinc-dependent endopeptidases of Metzicin family. 1 MMP family has various types of proteins such as MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, MMP-13 and MMP-14. MMPs play essential roles in physiological processes such as organogenesis, tissue wound healing, involution of uterus, angiogenesis, apoptosis, ovulation, cell proliferation and motility. 2 MMPs are also involved in some abnormal processes including childhood infections, cardiovascular, immune disorders and tumor formation. 3 In principle, MMPs are essential in intracellular as well as extracellular matrix remodeling. 4 Some types of MMPs degrade extracellular proteins and extracellular matrix. For example, MMP-3 and MMP-10 can break down fibronectin, proteoglycans and laminin while MMP-8, MMP-13 and MMP-1 degrade collagen types I, II and III. 5 MMP family proteins can be divided into five major groups based on their structural differences: (1) matrilysins; (2) collagenases; (3) gelatinases; (4) transmembrane and GPI-linked MT-MMPs; (5) vitronectin-like and other MMPs. 1 According to Figure 1 , all MMP family members contain a pre-domain sequence that is cleaved while entering endoplasmic reticulum; a pro-domain sequence, which maintains a protein in inactive state and needs to be cleaved for activation, and the active site that is bound to Zn 2+ . 1 , 2 Between the pro-domain and the active site there is furin recognition motif, which allows the furin-like proteinases to cleave the pro-domain and activate the zymogen. 1 , 2 Many types of MMPs, like MMP-2 and MMP-3 have also been found in the nucleus of the cell. 4 , 6 There is strong evidence that MMPs contain nuclear localization signaling sequence (NLS) that allows MMPs to enter the nucleus and regulate certain nuclear events. 1 , 6 NLS sequence is mostly found in the catalytic domain and leads to nuclear translocation of these proteins. 6 The simplest structure described above is possessed by matrilysins, more precisely MMP-7 and MMP-26. 1 On the other hand, collagenases (MMP-1, MMP-8, MMP-13, MMP-18) contain hemopexin-like domain that is connected to the active site by hinge region. 1 This domain, which resembles the portion that is made up of four repeated sequences similar to hemopexin and binds to inhibitors of MMPs, is also present in gelatinases (MMP-2, MMP-9), MT-MMPs (MMP-17, MMP-25, MMP-14, MMP-15, MMP-16, MMP-24), vitronectin-like (MMP-21) and other MMPs (MMP-23). 7–9 Transmembrane MT-MMPs such as MMP-14, MMP-15, MMP-16, MMP-24 also contain a transmembrane domain bound to a cytoplasmic domain, whereas GPI-linked MT-MMPs contain glycosylphosphatidylinositol portion, a glycolipid that allows a protein to attach to the plasma membrane. 3 MMP-21 contains a vitronectin-like region, a glycoprotein which is the homolog of vitronectin that contributes to cell adhesion and migration. 3 In the catalytic domain, MMP-23 contains a Ig-like portion bound to hemopexin domain. 2 It has been shown that MMPs can be regulated at different levels: transcription, activation of zymogen and inhibition of activated forms. 7 , 8 In fact, all MMPs are secreted as inactivated enzymes, more precisely in the latent form (zymogens). The activation of MMPs occurs via proteolytic cleavage of their pro-domains, 3 by the dissociation of zinc ion from cysteine residue. 7 , 8 It has been demonstrated that MMPs can be inactivated by tissue inhibitors of metalloproteinase (TIMPs). 9 The unequal rate of the expression of MMPs and TIMPs can contribute to tumor cell invasion, rheumatoid arthritis, fibrosis and endometriosis. 9 It was demonstrated that MMPs act as regulatory proteins that cleave and activate several intracellular peptides, such as growth factors and cytokines. 2 The activation of matrix proteinases causes tissue degradation, fibrosis and matrix destruction. 2 Further tissue degradation leads to the cancer invasion and the development of secondary tumors, whereas severe fibrosis can be transformed into lung diseases as well as liver cirrhosis. 3 In addition, MMPs participate in cardiac and vascular remodeling, and their activation can cause the rupture of the atherosclerotic plaque and dilated cardiomyopathy. 10 MMPs usually localize to the extracellular compartment to cleave the extracellular matrix. However, recent studies suggest that MMPs also localize to the nucleus to play distinct roles. 1 The present review will summarize the recent progress in studies on nuclear MMPs activation or inhibition via cellular signaling pathways and biological functions.

Due to limited literatures for references, the mechanisms underlying the nuclear MMP-induced apoptosis is still unclear. The possibility that nuclear MMPs directly cleave nuclear PARP and caspase is widely accepted because it has been found that the cleaved PARP or caspase associates with nuclear MMPs. 61 Moreover, other potential nuclear targets of nuclear MMPs associated with apoptosis have been identified as Ste20-like kinases, p21-activated protein kinase, initiator nuclear caspases such as caspase2, caspase 8, caspase 9 and caspase 10. 61 In addition, reactive oxygen species (ROS) produced by oxidative stress may damage DNA and cause apoptosis or genomic instability. MMP-3 has been shown to induce mitochondrial production of ROS, genomic instability, associate with decreased cell adhesions and oncogenic cellular stress. 37 The MMP-3-activated ROS may not only damage DNA but also induce apoptosis. However, nuclear MMP-3 can induce apoptosis through the cleavage of nuclear caspase. 61 Moreover, nuclear MMPs may mediate damaged DNA repair on neuronal death associated with neuroinflammation. 62 One pathway is through proapoptotic signaling upstream of caspases, such as IL-1 β signaling. 62