MT1-MMP expression in the odontogenic region of rat incisors undergoing interrupted eruption

MT1-MMP (membrane type matrix metalloproteinase-1) has been considered an important membrane-type matrix metalloproteinase involved in the remodeling process in tissue and organ development, including the processes of the tooth and root growth and dental eruption. Therefore, the aims of this study were to evaluate MT1-MMP expression in the odontogenic region, as well as the eruption rate and morphology of the lower-left rat incisor, where the eruption process was interrupted for 14 days by a steel wire attached from the center of the incisor labial face and braced to the first molar. In the interrupted eruption group, the eruption rate was significantly reduced, producing drastic morphological alterations in the tooth germ and socket area. The MT1-MMP expression was widespread in the dental follicle, in both groups studied (normal and interrupted eruption groups); however a significant decrease in immunostaining was observed in the interrupted eruption group. Results indicate that MT1-MMP may have an important role in the process of dental eruption.     

Differential roles of TIMP-4 and TIMP-2 in pro-MMP-2 activation by MT1-MMP

The tissue inhibitors of metalloproteinases (TIMPs) are specific inhibitors of MMP enzymatic activity. However, TIMP-2 can promote the activation of pro-MMP-2 by MT1-MMP. This process is mediated by the formation of a complex between MT1-MMP, TIMP-2, and pro-MMP-2. Binding of TIMP-2 to active MT1-MMP also inhibits the autocatalytic turnover of MT1-MMP on the cell surface. Thus, under certain conditions, TIMP-2 is a positive regulator of MMP activity. TIMP-4, a close homologue of TIMP-2 also binds to pro-MMP-2 and can potentially participate in pro-MMP-2 activation. We coexpressed MT1-MMP with TIMP-4 and investigated its ability to support pro-MMP-2 activation. TIMP-4, unlike TIMP-2, does not promote pro-MMP-2 activation by MT1-MMP. However, TIMP-4 binds to MT1-MMP inhibiting its autocatalytic processing. When coexpressed with TIMP-2, TIMP-4 competitively reduced pro-MMP-2 activation by MT1-MMP. A balance between TIMP-2 and TIMP-4 may be a critical factor in determining the degradative potential of cells in normal and pathological conditions.     

Immunolocalization and activity of the MMP-9 and MMP-2 in odontogenic region of the rat incisor tooth after post shortening procedure

MMP-9 and MMP-2 are metalloproteinases which degrade the denatured collagen fibers. However, there is no report about roles of these MMPs in the odontogenic region of the adult rat incisor tooth under different eruption conditions. Male Wistar rats were divided in a normofunctional group (NF) in which their lower teeth remained in a normal eruption. In a hypofunctional group (HP) rats underwent shortening of their lower left incisor tooth every 2 days during 12 days. The eruption rate as well as the expression and activities of MMP-9 and MMP-2 were evaluated using imunohistochemistry and zymography. Although the shortening increased the eruption rate, no changes in the MMP-9 and MMP-2 were observed. We conclude that in adult rats, in opposite to development of tooth, the MMP-9 and MMP-2 present in the odontogenic region does not seem to play a direct role in the remodeling matrix, even after post-shortening procedures which to lead an acceleration of the eruption process in the incisor.     

TIMP-2 Interaction with MT1-MMP Activates the AKT Pathway and Protects Tumor Cells from Apoptosis

Membrane-type 1 matrix metalloproteinase (MT1-MMP), a transmembrane proteinase with an extracellular catalytic domain and a short cytoplasmic tail, degrades a variety of extracellular matrix (ECM) components. In addition, MT1-MMP activates intracellular signaling through proteolysis-dependent and independent mechanisms. We have previously shown that binding of tissue inhibitor of metalloproteinases-2 (TIMP-2) to MT1-MMP controls cell proliferation and migration, as well as tumor growth in vivo by activating the Ras—extracellular signal regulated kinase-1 and -2 (ERK1/2) pathway through a mechanism that requires the cytoplasmic but not the proteolytic domain of MT1-MMP. Here we show that in MT1-MMP expressing cells TIMP-2 also induces rapid and sustained activation of AKT in a dose- and time-dependent manner and by a mechanism independent of the proteolytic activity of MT1-MMP. Fibroblast growth factor receptor-1 mediates TIMP-2 induction of ERK1/2 but not of AKT activation; however, Ras activation is necessary to transduce the TIMP-2-activated signal to both the ERK1/2 and AKT pathways. ERK1/2 and AKT activation by TIMP-2 binding to MT1-MMP protects tumor cells from apoptosis induced by serum starvation. Conversely, TIMP-2 upregulates apoptosis induced by three-dimensional type I collagen in epithelial cancer cells. Thus, TIMP-2 interaction with MT1-MMP provides tumor cells with either pro- or anti-apoptotic signaling depending on the extracellular environment and apoptotic stimulus.     

TIMP-2 is released as an intact molecule following binding to MT1-MMP on the cell surface

Binding of tissue inhibitor of metalloproteinase-2 (TIMP-2) to pro-MMP-2 and mature membrane type-1 MMP (MT1-MMP) on the cell surface is required for activation of MMP-2. It has been reported that following binding to cell surface receptors, TIMP-2 undergoes endocytosis and extensive degradation in lysosomes. The purpose of this study was to reexamine the fate of TIMP-2 following binding to transfected HT1080 cell surface MT1-MMP at 4 degrees C. Following 37 degrees C incubation, 125I-TIMP-2 release, endocytosis, and degradation were characterized under varying conditions. More than 85% of the total 125I-TIMP-2 bound to cells was released as intact functional molecules; <15% was degraded. Transfection of HT1080 cells with dominant negative mutant dynamin cDNA resulted in delayed endocytosis and release of 125I-TIMP-2 from cells. Pharmacologic agents that induce clustering of cell surface receptors (concanavalin A) and interfere with endosomal/lysosomal function (bafilomycin A(1)) resulted in enhanced binding of 125I-TIMP-2 to cell surface receptors. Abrogation of activation of proMT1-MMP with a furin inhibitor prevented binding and endocytosis of 125I-TIMP-2. Biotinylation of cell surface MT1-MMP followed by Western blotting confirmed the presence of mature MT1-MMP on the cell surface and degraded MT1-MMP in the intracellular compartment. In conclusion, these studies demonstrate that TIMP-2 is released from cells primarily as an intact functional molecule following binding to MT1-MMP on the cell surface.     

Contrasting expression of membrane metalloproteinases, MT1-MMP and MT3-MMP, suggests distinct functions in skeletal development

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is the most ubiquitous and widely studied of the membrane-type metalloproteinases (MT-MMPs). It was thus surprising to find no published data on chicken MT1-MMP. We report here the characterization of the chicken gene. Its low sequence identity with the MT1-MMP genes of other species, high GC content, and divergent catalytic domain explains the absence of data and our difficulties in characterizing the gene. The absence of structural features in the chicken gene that have been suggested to be critical for the activation of MMP-2 by MT1-MMP; for the effect of MT1-MMP on cell migration and for the recycling of MT1-MMP suggest these features are either not essential or that MT1-MMP does not perform these functions in chickens. Comparison of the expression of chicken MT1-MMP with MT3-MMP and with MMP-2 and MMP-13 has confirmed the previously recognized co-expression of MT1-MMP with MMP-2 and MMP-13 in fibrous and vascular tissues, particularly those surrounding the developing long bones in other species. By contrast, MT3-MMP expression differs markedly from that of MT1-MMP and of both MMP-2 and MMP-13. MT3-MMP is expressed by chondrocytes of the developing articular surface. Similar expression patterns of this group of MT-MMPs and MMPs have been observed in mouse embryos and suggest distinct and specific functions for MT1-MMP and MT3-MMP in skeletal development.     

Pro-MMP-9 activation by the MT1-MMP/MMP-2 axis and MMP-3: role of TIMP-2 and plasma membranes

MMP-9 (gelatinase B) is produced in a latent form (pro-MMP-9) that requires activation to achieve catalytic activity. Previously, we showed that MMP-2 (gelatinase A) is an activator of pro-MMP-9 in solution. However, in cultured cells pro-MMP-9 remains in a latent form even in the presence of MMP-2. Since pro-MMP-2 is activated on the cell surface by MT1-MMP in a process that requires TIMP-2, we investigated the role of the MT1-MMP/MMP-2 axis and TIMPs in mediating pro-MMP-9 activation. Full pro-MMP-9 activation was accomplished via a cascade of zymogen activation initiated by MT1-MMP and mediated by MMP-2 in a process that is tightly regulated by TIMPs. We show that TIMP-2 by regulating pro-MMP-2 activation can also act as a positive regulator of pro-MMP-9 activation. Also, activation of pro-MMP-9 by MMP-2 or MMP-3 was more efficient in the presence of purified plasma membrane fractions than activation in a soluble phase or in live cells, suggesting that concentration of pro-MMP-9 in the pericellular space may favor activation and catalytic competence.     

Expression of Ki-67, p53 and p63 proteins in keratocyst odontogenic tumours: an immunohistochemical study

Aim To investigate the immunohistochemical expression of Ki-67, p53 and p63 in Keratocyst Odontogenic Tumours (KOTs) in order to contribute to the biological profile of this tumor. Methods Immunohistochemical technique was performed using the EnVision™ System in 37 cases of KOTs. Results Ki-67- and p53-immunostained cells were mainly located in the suprabasal layers. p63-positive cells were found throughout the lining cystic epithelium. No difference in the immunostaining for these proteins was observed between primary and recurrent KOTs (Ki-67: P = 0.5591; p53: P = 0.9847; p63: P = 0.9127), or between KOTs associated with Nevoid Basal Cell Carcinoma Syndrome (NBCCS) and sporadic KOTs (Ki-67: P = 0.7013; p53: P = 0.3197; p63: P = 0.2427). Conclusions It is possible that biological behavior of KOTs may be related to suprabasal proliferative compartment in the cystic epithelium as observed by high levels of Ki-67, p53 and p63. In addition, p63 immunostaining may represent immaturity of keratinocytes in KOTs, and suggests that this protein may participate in the regulation of epithelial cell differentiation. Taken together, these data may favor tumorigenesis on KOTs.     

miRNA-105 and -128 function as rheostats modulating MMP-2 activities by downregulation of TIMP-2 and upregulation of MT1-MMP

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that remodel and degrade the extracellular matrix. Of various MMPs, MMP-2 plays an important role in tumor metastasis. Recently, microRNAs with pro- or anti-metastatic effects were collectively referred to as metastamiRs. We screened 215 human miRNA mimics for modulators of MMP-2 activities in HT-1080 cells, and found that miR-105 and miR-128 promote MMP-2 activities. Bioinformatics analysis predicted that miR-105 and miR-128 both bind to the 3′ untranslated region (UTR) of TIMP-2, an inhibitor of MMP-2 activities. This prediction was verified by reduced luciferase activity in HT-1080 cells co-transfected with miR-105 or miR-128 mimics and plasmids encoding luciferase fused to 3′ UTR of TIMP2. In addition, Western blotting showed that transfection of HT-1080 cells with miR-105 or miR-128 suppressed TIMP-2 levels and enhanced levels of MT1-MMP, an activator of MMP-2 activities. The mechanism by which miR-128 upregulates MT1-MMP was determined to be downregulation of PRKD1, an inhibitor of MT1-MMP, at least in part. Cell invasion assays using Matrigel demonstrated that HT-1080 cells transfected with miR-105 or miR-128 are more invasive as compared to control cells. Taken together, these findings show that miR-105 and miR-128 are metastamir promoting MMP-2 activities via simultaneously downregulating TIMP-2 and upregulating MT1-MMP, and may provide a platform for the development of therapeutics against metastasis.     

MT1-MMP modulates the mechanosensitivity of osteocytes

Membrane-type matrix metalloproteinase-1 (MT1-MMP) is expressed by mechanosensitive osteocytes and affects bone mass. The extracellular domain of MT1-MMP is connected to extracellular matrix, while its intracellular domain is a strong modulator of cell signaling. In theory MT1-MMP could thus transduce mechanical stimuli into a chemical response. We hypothesized that MT1-MMP plays a role in the osteocyte response to mechanical stimuli. MT1-MMP-positive and knockdown (siRNA) MLO-Y4 osteocytes were mechanically stimulated with a pulsating fluid flow (PFF). Focal adhesions were visualized by paxillin immunostaining. Osteocyte number, number of empty lacunae, and osteocyte morphology were measured in long bones of MT1-MMP(+/+) and MT1-MMP(-/-) mice. PFF decreased MT1-MMP mRNA and protein expression in MLO-Y4 osteocytes, suggesting that mechanical loading may affect pericellular matrix remodeling by osteocytes. MT1-MMP knockdown enhanced NO production and c-jun and c-fos mRNA expression in response to PFF, concomitantly with an increased number and size of focal adhesions, indicating that MT1-MMP knockdown osteocytes have an increased sensitivity to mechanical loading. Osteocytes in MT1-MMP(-/-) bone were more elongated and followed the principle loading direction, suggesting that they might sense mechanical loading. This was supported by a lower number of empty lacunae in MT1-MMP(-/-) bone, as osteocytes lacking mechanical stimuli tend to undergo apoptosis. In conclusion, mechanical stimulation decreased MT1-MMP expression by MLO-Y4 osteocytes, and MT1-MMP knockdown increased the osteocyte response to mechanical stimulation, demonstrating a novel and unexpected role for MT1-MMP in mechanosensing.     

Cellular activation of MMP-2 (gelatinase A) by MT2-MMP occurs via a TIMP-2-independent pathway

The role of membrane-type (MT) 2-matrix metalloproteinase (MMP) in the cellular activation of MMP-2 and the tissue inhibitor of matrix metalloproteinase (TIMP) requirements for this process have not been clearly established. To address these issues a TIMP-2-free cell line derived from a Timp2-/- mouse was transfected for stable cell surface expression of hMT2-MMP. Untransfected cells did not activate endogenous or exogenous TIMP-2-free MMP-2 unless both TIMP-2 and concanavalin A (ConA) were added. Transfected cells expressing hMT2-MMP efficiently activated both endogenous and exogenous MMP-2 (within 4 h) via the 68-kDa intermediate in the absence of TIMP-2 and ConA. In contrast, activation of MMP-2 by Timp2-/- cells expressing recombinant hMT1-MMP occurred more slowly (12 h) and required the addition of 0.3-27 nm TIMP-2. Addition of TIMP-2 or TIMP-4 did not enhance MMP-2 activation by MT2-MMP at any concentration tested; furthermore, activation was inhibited by both TIMPs at concentrations >9 nm, consistent with the similar association rate constants (k(on)) calculated for the binding of TIMP-4 and TIMP-2 to MT2-MMP (3.56 x 10(5) m(-1) s(-1) and 6.52 x 10(5) m(-1) s(-1), respectively). MT2-MMP-mediated activation involved cell surface association of the MMP-2 in a hemopexin carboxyl-terminal domain (C domain)-dependent manner: Exogenous MMP-2 hemopexin C domain blocked activation, and cells expressing hMT2-MMP did not bind or activate a truncated form of MMP-2 lacking the hemopexin C domain. These studies demonstrate the existence of an alternative TIMP-2-independent pathway for MMP-2 activation involving MT2-MMP, which may be important in mediating MMP-2 activation in specific tissues or pathologies where MT2-MMP is expressed.     

The role of MT2-MMP in cancer progression

The role of MT2-MMP in cancer progression remains to be elucidated in spite of many reports on MT1-MMP. Using a human fibrosarcoma cell, HT1080 and a human gastric cancer cell, TMK-1, endogenous expression of MT1-MMP or MT2-MMP was suppressed by siRNA induction to examine the influence of cancer progression in vitro and in vivo. In HT1080 cells, positive both in MT1-MMP and MT2-MMP, the migration as well as the invasion was impaired by MT1-MMP or MT2-MMP suppression. Also cell proliferation in three dimensional (3D) condition was inhibited by MT1-MMP or MT2-MMP suppression and tumor growth in the nude mice transplanted with tumor cells were reduced either MT1-MMP or MT2-MMP suppression with a prolongation of survival time in vivo. MT2-MMP suppression induces more inhibitory effects on 3D proliferation and in vivo tumor growth than MT1-MMP. On the other hand, TMK-1 cells, negative in MT1-MMP and MMP-2 but positive in MT2-MMP, all the migratory, invasive, and 3D proliferative activities in TMK-1 are decreased only by MT2-MMP suppression. These results indicate MT2-MMP might be involved in the cancer progression more than or equal to MT1-MMP independently of MMP-2 and MT1-MMP.     

Co-recycling of MT1-MMP and MT3-MMP through the trans-Golgi network. Identification of DKV582 as a recycling signal

Members of the membrane-type matrix metalloproteinases (MT-MMPs) have been implicated in a wide range of physiological and pathological processes from normal development to tumor growth. Tethered on plasma membrane, these enzymes are potentially regulated by the trafficking machinery of the cells. Here we demonstrate that both MT1-MMP and MT3-MMP are internalized, transported to the trans-Golgi network through early endosomes, and recycled back to cell surface in 60 min in a manner distinct from the one employed by transferrin receptor. Interestingly, co-expressed MT1-MMP and MT3-MMP are localized and routed in the same vesicles throughout the trafficking process. We further demonstrated that the carboxyl-terminal sequence DKV(582) of MT1-MMP is required for its recycling, thus defining a novel recycling motif. These results suggest that MT-MMPs may coordinate their proteolytic activities through the cellular trafficking machinery.     

N-i-Propoxy-N-biphenylsulfonylaminobutylhydroxamic acids as potent and selective inhibitors of MMP-2 and MT1-MMP

Structural manipulation of the pharmacophoric model of type A selective MMP inhibitors (MMPi), obtained by the insertion of some alkyl substituents R2 possessing an appropriate geometry, steric bulkiness and lipophilicity, is able to improve potency, in the subnanomolar range on MMP-2, and to give a good MMP inhibition on MMP-14 (MT1-MMP) in the designed MMPi of type C, while maintaining a good MMP-1/MMP-2 selectivity profile. The simultaneous inhibition of these two enzymes yields type C compounds, which are potent antiangiogenic agents, able to block a chemoinvasion model on HUVEC cells in the micromolar range.     

MT1-MMP hemopexin domain exchange with MT4-MMP blocks enzyme maturation and trafficking to the plasma membrane in MCF7 cells

The hemopexin-like domain of membrane-type matrix metalloproteinase-1 (MT1-MMP) enables MT1-MMP to form oligomers that facilitate the activation of pro-matrix metalloproteinase-2 (pro-MMP-2) at the cell surface. To investigate the role of the MT1-MMP hemopexin domain in the trafficking of MT1-MMP to the cell surface we have examined the activity of two MT1-MT4-MMP chimaeras in which the hemopexin domain of MT1-MMP has been replaced with that of human or mouse MT4-MMP. We show that MT1-MMP bearing the hemopexin domain of MT4-MMP was incapable of activating pro-MMP-2 or degrading gelatin in cell based assays. Furthermore, cell surface biotinylation and indirect immunofluorescence show that transiently expressed MT1-MT4-MMP chimaeras failed to reach the plasma membrane and were retained in the endoplasmic reticulum. Functional activity could be restored by replacing the MT4-MMP hemopexin domain with the wild-type MT1-MMP hemopexin domain. Subsequent analysis with an antibody specifically recognising the propeptide of MT1-MMP revealed that the propeptides of the MT1-MT4-MMP chimaeras failed to undergo proper processing. It has previously been suggested that the hemopexin domain of MT4-MMP could exert a regulatory mechanism that prevents MT4-MMP from activating pro-MMP-2. In this report, we demonstrate unambiguously that MT1-MT4-MMP chimaeras do not undergo normal trafficking and are not correctly processed to their fully active forms and, as a consequence, they are unable to activate pro-MMP-2 at the cell surface.     

Expression of proteins: D1 cyclin and Ki-67 in papillary thyroid carcinomas

The aim of the study was an evaluation of expression of D1 cyclin and Ki-67 proteins in tissue of human papillary thyroid carcinoma (PTC) in a group of papillary microcarcinomas and in a group of PTC with a degree of staging higher than pT1a in TNM classification. We performed immunohistochemical staining and found no statistical differences between groups. These results suggest that changes of expression of D1 cyclin are an early event in tumorigenesis.