TIMP-1 binding to proMMP-9/CD44 complex localized at the cell surface promotes erythroid cell survival

Besides its ability to inhibit MMP activity, TIMP-1 exhibits other biological functions. We earlier reported that TIMP-1 induced UT-7 erythroid cell survival through activation of the JAK2/PI 3-kinase/Akt pathway and we now aim to determine whether the TIMP-1 anti-apoptotic effect requires MMP involvement. We first show that proMMP-9 was expressed in UT-7 cells and associated with the cell plasma membrane. Such proMMP-9 localization was crucial for TIMP-1 intracellular signalling since (i) TIMP-1 specifically bound to proMMP-9 and (ii) proMMP-9 silencing abrogated the TIMP-1 effect. We also demonstrated that TIMP-1 anti-apoptotic effect was independent on MMP inhibition since MMP-9 function blocking antibodies as well as a synthetic MMP inhibitor were unable to reproduce TIMP-1 effect. Nevertheless, these compounds prevented TIMP-1 binding to proMMP-9 and subsequently abolished TIMP-1-induced cell survival. We finally demonstrated that CD44 anchored proMMP-9 to the plasma membrane and enabled TIMP-1-mediated signal transduction. Therefore, our results indicate that the anti-apoptotic signalling of TIMP-1 depends on the formation of a ternary complex between TIMP-1, proMMP-9 and CD44 at the UT-7 erythroid cell surface.     

Effects of oxygen tension and dextran-shelled/2H,3H-decafluoropentane-cored oxygen-loaded nanodroplets on secretion of gelatinases and their inhibitors in term human placenta

Matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) need to be finely modulated in physiological processes. However, oxygen tension influences MMP/TIMP balances, potentially leading to pathology. Intriguingly, new 2H,3H-decafluoropentane-based oxygen-loaded nanodroplets (OLNDs) have proven effective in abrogating hypoxia-dependent dysregulation of MMP and TIMP secretion by single cell populations. This work explored the effects of different oxygen tensions and dextran-shelled OLNDs on MMP/TIMP production in an organized and multicellular tissue (term human placenta). Chorionic villous explants from normal third-trimester pregnancies were incubated with/without OLNDs in 3 or 20% O₂. Explants cultured at higher oxygen tension released constitutive proMMP-2, proMMP-9, TIMP-1, and TIMP-2. Hypoxia significantly altered MMP-2/TIMP-2 and MMP-9/TIMP-1 ratios enhancing TIMP-2 and reducing proMMP-2, proMMP-9, and TIMP-1 levels. Intriguingly, OLNDs effectively counteracted the effects of low oxygen tension. Collectively, these data support OLND potential as innovative, nonconventional, and cost-effective tools to counteract hypoxia-dependent dysregulation of MMP/TIMP balances in human tissues.     

Distribution of lactate dehydrogenase in healthy and degenerative canine stifle joint cartilage

In dogs, degenerative joint diseases (DJD) have been shown to be associated with increased lactate dehydrogenase (LDH) activity in the synovial fluid. The goal of this study was to examine healthy and degenerative stifle joints in order to clarify the origin of LDH in synovial fluid. In order to assess the distribution of LDH, cartilage samples from healthy and degenerative knee joints were investigated by means of light and transmission electron microscopy in conjunction with immunolabeling and enzyme cytochemistry. Morphological analysis confirmed DJD. All techniques used corroborated the presence of LDH in chondrocytes and in the interterritorial matrix of healthy and degenerative stifle joints. Although enzymatic activity of LDH was clearly demonstrated in the territorial matrix by means of the tetrazolium–formazan reaction, immunolabeling for LDH was missing in this region. With respect to the distribution of LDH in the interterritorial matrix, a striking decrease from superficial to deeper layers was present in healthy dogs but was missing in affected joints. These results support the contention that LDH in synovial fluid of degenerative joints originates from cartilage. Therefore, we suggest that (1) LDH is transferred from chondrocytes to ECM in both healthy dogs and dogs with degenerative joint disease and that (2) in degenerative joints, LDH is released from chondrocytes and the ECM into synovial fluid through abrasion of cartilage as well as through enhanced diffusion as a result of increased water content and degradation of collagen.     

Temporospatial expression of tissue inhibitors of matrix metalloproteinases-1, -2 and -3 during development, growth and aging of the mouse skeleton

Proteolytic degradation of collagen-rich extracellular matrices is a key feature in the development, growth and aging of skeleton. Matrix metalloproteinases (MMPs) are a family of enzymes capable of performing this function, whereas tissue inhibitors of MMPs (TIMPs) are believed to play an important role in regulating their activity. To better understand the roles of TIMP-1, -2 and -3, we have studied their mRNA levels in several different mouse tissues with special emphasis on the skeleton and the developing eye. A systematic analysis of TIMP-1, -2 and -3 mRNA levels in mouse knee joints during growth and aging demonstrated markedly different expression patterns for each TIMP. Immunohistochemical analysis revealed several time-dependent changes in the distribution of TIMP-1 and -2 in articular and growth cartilages, synovial tissue and bone. The data suggest that upon aging synovial tissue becomes the major source of synovial fluid TIMPs. In articular cartilage these inhibitors were mainly found in the deep layer and in subchondral bone. Compared with epiphyseal growth plate, the amounts of TIMP-1 and -2 in articular cartilage were quite low. These findings suggest that the capacity of articular cartilage chondrocytes to inhibit MMP activities by local production of TIMPs is limited, which may be of consequence during osteoarthritic cartilage degeneration.     

Cell surface activation of progelatinase A （proMMP—2） and cell migration

Gelatinase A (MMP-2) is considered to play a critical role in cell migration and invasion.The proteinase is cerceted from the cell as an inactive zymogen.In vivo it is postulated that activation of progelationase A (proMMP-2) takes place on the cell surface mediated by membrane-type matrix metalloproteinases (MT-MMPs).Recent studies have demonstrated that proMMP-2 is recruited to the cell surface by interacting with tissue inhibitor of metalloproteinases-2 (TIMP-2) bound to MT1-MMP by forming a ternary complex.Free MT1-MMP closely located to the ternary complex then activates proMMP-2 on the cell surface.MT1-MMP is found in cultured invasive cancer cells at the invadopodia.The MT-MMP/TIMP-2/MMP-2 system thus provides localized expression of proteolysis of the extracellular matrix required for cell migration.     

Independent regulation of matrix metalloproteinases and plasminogen activators in human fibrosarcoma cells

Serine proteases and matrix metalloproteinases have been shown to often cooperate in multiple physiological and pathological processes associated with changes in the extracellular matrix (ECM). We have examined the interaction between the plasminogen activator (PA)-plasmin system and matrix metalloproteinases (MMPs) in HT1080 human fibrosarcoma cells treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). While TPA treatment evoked a temporary increased expression of urokinase type PA (uPA), the production of both types of plasminogen activator inhibitors (PAI) was induced and sustained over 12 h by TPA treatment shifting the protease-protease inhibitors balance in favor of the inhibitors. TPA treatment of HT1080 cells induced the expression of interstitial collagenase (MMP-1) and increased the expression of gelatinase B (MMP-9), tissue inhibitor of metalloproteinases-1 (TIMP-1), and MT-MMP, a membrane-bound activator of progelatinase A (proMMP-2), while MMP-2 and TIMP-2 expression were decreased. Increased MT-MMP expression by TPA treatment was associated with increased activation of proMMP-2. These data show that the regulation of PA-plasmin and metalloproteinase and their specific inhibitors is uncoordinated. In addition, inhibition of the PA-plasmin system by PAI-2 or aprotinin did not prevent the activation of proMMP-2 by TPA, suggesting that plasmin is not involved in MT-MMP-mediated activation of proMMP-2. © 1996 Wiley-Liss, Inc.     

Expression and functional properties of antibodies to tissue inhibitors of metalloproteinases (TIMPs) in rheumatoid arthritis

Tissue inhibitors of matrix metalloproteinases (TIMPs) regulate the breakdown of extracellular matrix components and play an important role in tissue remodelling and growth, in both physiological and pathological conditions. We studied the autoimmune response to TIMPs in patients with rheumatoid arthritis (RA). Eighty-nine paired blood and synovial fluid samples from patients with RA were assessed for their reactivity with recombinant tissue inhibitors of metalloproteinases (TIMPs) 1 to 4 by an ELISA and were compared with blood from 62 healthy controls and 21 synovial fluid samples from patients with degenerative joint diseases. Presence of antibodies was established as the absorbance of the sample more than 2 standard deviations above the mean of the controls. In addition, immunoglobulin G (IgG) from blood samples of RA patients possessing TIMP antibodies was isolated on protein A–sepharose and tested for the in vitro ability to neutralize TIMP-2-dependent effects on metalloproteinase 9 (MMP9). Anti-TIMP antibodies were found in 56% of RA samples but in only 5% of the controls (P < 0.005). RA patients had high frequencies of antibodies against all TIMPs except TIMP-3. TIMP-2 antibodies were most frequently found (33%), being significantly more prevalent (P = 0.024) in patients with nonerosive than erosive RA. TIMP-1 antibodies were significantly more often found in synovial fluid samples than in the matched blood samples (P < 0.025). Importantly, the IgG fraction containing TIMP antibodies down-regulated the TIMP-2 inhibitory effect, thereby supporting MMP9 activity in vitro. In the present study, we show that RA patients frequently develop autoimmune response to TIMPs that may act as a functionally significant regulator of MMP activity and thereby of joint destruction.     

Expression of interstitial collagenase and its endogenous regulators in immortalized and transformed by HPV-16 E7 gene fibroblasts

Matrix metalloproteinases (MMPs) play a critical role in tumor development and invasion. The aim of this study was to elucidate peculiarity of expression of interstitial collagenase (MMP-1) and its endogenous regulators during oncogenic transformation of fibroblasts by HPV-16 E7 gene. Papilloma virus types 16 and 18 are etiological factor of cervical cancer. We have studied expression of MT1-MMP, MMP-1, tissue inhibitor of these proteases, TIMP-1, and urokinase-like plasminogen activator (uPA), activating MMP-1 via plasmin. The study was carried out using fibroblasts immortalized by LT gene (IF) and transformed by E7 gene of HPV-16 fibroblasts (TF). Primary culture of Fisher rat embryo fibroblasts was used as a control (PF). mRNA expression, and enzymatic activity were studied by RT-PCR and by hydrolysis of fluorogenic type I collagen, respectively. Cell transformation was accompanied by: (a) 2–3 fold induction of MT1-MMP mRNA expression vs PF; (b) the decrease in mRNA level of TIMP-1 (1,5–2 fold); c) unchanged uPA expression. Cell immortalization is accompanied by: (a) the increase of MT1-MMP expression (1,5–2 fold); (b) unchanged TIMP-1 expression; (c) the increase of uPA expression (2–4 fold) vs PF and TF. MMP secreted activity and activity in lysates of TF increased but level of free endogenous MMP inhibitors decreased vs IF. Data on gene expression are consistent with enzymatic data on the collagenolytic activity. These results suggest changes in enzyme/inhibitor/activator ratio both TF and IF and significant enhancement of the destructive potential of the TF.     

Pharmacokinetics of omega-3-fatty acids during ingestion of fish oil preparations.

An in vivo comparison of three dosages (3 g, 6 g, 12 g) of two different fish oil preparations in terms of plasma concentrations of their major active components eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) was performed. The plasma accumulation was measured during 28 days of ingestion and an equally long wash out period. Data were scrutinized for bioavailability in order to distinguish between the efficiency of the two preparations. Rapid increases in EPA and DHA plasma concentrations can be demonstrated at all dosages during a 28-day ingestion period. EPA accumulated more during ingestion of high than of low dosages of fish oil. DHA revealed almost identical increases and peak values in plasma concentrations in all subgroups. The present data demonstrate dose dependent increases of EPA concentrations whereas DHA plasma concentrations are comparable in all dosages investigated. Measurable EPA and DHA plasma concentration levels are inappropriate means to explain clinical effectiveness. These results were found in both commercially available fish oil preparations. Direct comparison of both preparations revealed no differences in bioavailability.     

Identification of soluble type of membrane-type matrix metalloproteinase-3 formed by alternatively spliced mRNA

Homology screening for human membrane-type MMP (MT–MMP) was carried out, and cDNA encoding a soluble type of MT3–MMP (SM3), which is considered to be an alternatively spliced variant of MT3–MMP, was obtained. SM3 had a novel sequence consisting of 50 amino acids after Lys407 instead of amino acids containing the transmembrane domain of MT3–MMP. When SM3 tagged with a FLAG epitope (SM3–flag) was expressed in COS-7 cells, SM3–flag was present in the conditioned medium in its activated form. The enzymatic activity of SM3 was studied using a recombinant enzyme expressed in E. coli (SM3-e). The fluorogenic peptide substrate hydrolyzing activity of SM3-e was inhibited by EDTA and by the tissue inhibitor of metalloproteinase-2 (TIMP-2), whereas TIMP-1 had only relatively weak inhibitory ability. SM3-e was able to activate proMMP-2, and this activity was also inhibited by TIMP-2 but not by TIMP-1. SM3-e was able to cleave type III collagen, and also digested fibronectin. In view of the homology of the primary structures, MT3–MMP was considered to have the same catalytic activity as SM3. The results of studies of SM3's activity on extracellular matrix (ECM) protein suggests that MT3–MMP plays a role in ECM turnover not only by activating proMMP-2 but also by acting directly on ECM macromolecules.     

Utilisation of fish oil in ruminants: II. Transfer of fish oil fatty acids into goats’ milk

A study was conducted to determine the transfer of eicosapentaenoic acid (EPA, 20:5) and docosahexaenoic acid (DHA, 22:6) from fish oil into goats’ milk. Goats were sequentially offered three diets: control (C) pellets (lucerne hay-oat grain: 60/40 w/w), C plus tuna oil protected against ruminal biohydrogenation (PTO pellets), and C plus unprotected tuna oil (UTO pellets). In supplemented diets, tuna oil constituted 3% of total dry matter (DM), and each supplement was fed for 7 days, with 12 days allowed between the two fish oil feeding periods to minimise carry-over effects. Dry matter intake, milk yield, protein and fat yield were reduced by feeding UTO, but not PTO, pellets. Goats produced ω-3 enriched milk (0.3–0.5% EPA and 1.01–1.12% DHA) when fed either supplement. The rate of transfer of dietary EPA and DHA to milk ranged from 3.5 to 7.6%. Significant transfer of EPA and DHA from tuna oil into goat milk, without deleterious effects on intake or milk yield is possible, provided that the oil supplement is substantially protected against ruminal biohydrogenation.     

Docosahexaenoic acid reduces in vitro invasion of renal cell carcinoma by elevated levels of tissue inhibitor of metalloproteinase-1

We demonstrate in this study that the n-3 polyunsaturated fatty acids derived from fish oil, namely, eicosapentanoic acid (EPA) and docosahexaenoic acid (DHA), can increase levels of tissue inhibitors of metalloproteinase-1 (TIMP-1) in the renal cell carcinoma cell line caki-1 by 26% and 17.42% respectively. The result of this elevation in TIMP-1 levels is a reduction of 48.48% in caki-1 invasion through the basement membrane component matrigel when cells are treated with DHA. By inhibition of 2-series prostaglandin production, a similar increase in TIMP-1 was observed in caki-1 cells. We conclude that the polyunstaurated fatty acid DHA, a component of fish oil, is capable of significantly reducing the invasive profile of renal cell carcinoma, and that this reduction is regulated by levels of 2-series prostaglandin production.     

Disruption of the TIMP-1 gene product is associated with accelerated endometrial gland formation during early postnatal uterine development

Postnatal uterine development is marked by periods of tissue remodeling. The objective of the present study was to examine the role of tissue inhibitor of metalloproteinase-1 (TIMP-1), a regulator of tissue remodeling events, during postnatal uterine development and to assess the phenotypic consequences of disruption of the TIMP-1 gene product during this time period. To accomplish this goal, wild-type and TIMP-1 null mice were sacrificed at Postnatal Days (PNDs) 5, 10, 15, 20, and 25 and uterine morphology, TIMP expression and matrix metalloproteinase (MMP) activity were assessed. In wild-type mice, TIMP-1 mRNA steady-state levels were highest at PND 5, after which expression decreased. TIMP-2 and TIMP-3 expression in wild-type mice showed no significant changes from PND 5 to 25. In TIMP-1 null mice, TIMP-2 and TIMP-3 expression patterns were similar to those in wild-type counterparts with the exception that, at PND 10, TIMP-2 and TIMP-3 expression was significantly lower in the null mice. Endometrial gland number and uterine histology were similar between genotypes at PNDs 5 and 10, but at PNDs 15 and 20, endometrial glands were more abundant in TIMP-1 null mice. Associated with the increased gland density in the null mice was an increase in total MMP activity above the levels expressed in wild-type mice. In summary, disruption of the TIMP-1 gene product is associated with reduced TIMP-2 and TIMP-3 steady-state mRNA levels, elevated MMP activity, and accelerated endometrial gland formation. We conclude that, during early postnatal uterine development, TIMP-1 may be critical for proper endometrial gland development.     

Matrix metalloproteinases (MMP)—MMP-1,-2,-9 and endogenous regulators of their activity in cervical carcinoma cell lines transformed by the E7 oncogene HPV16 and HPV18

Matrix metalloproteinases (MMP) play a key role in development of tumor invasion and metastasis. The aim of the work was to elucidate peculiarities of expression of MMP-1, MMP-2, MMP-9 and regulators of their activity: plasminogen activator uPA and tissue inhibitors of MMPs TIMP-1 and TIMP-2 in human cell lines of squamous cell carcinoma (SCC).     

Differential regulation of membrane type 1-matrix metalloproteinase activity by ERK 1/2- and p38 MAPK-modulated tissue inhibitor of metalloproteinases 2 expression controls transforming growth factor-beta1-induced pericellular collagenolysis

Acquisition of matrix metalloproteinase-2 (MMP-2) activity is temporally associated with increased migration and invasiveness of cancer cells. ProMMP-2 activation requires multimolecular complex assembly involving proMMP-2, membrane type 1-MMP (MT1-MMP, MMP-14), and tissue inhibitor of metalloproteinases-2 (TIMP-2). Because transforming growth factor-beta1 (TGF-beta1) promotes tumor invasion in advanced squamous cell carcinomas, the role of TGF-beta1 in the regulation of MMP activity in a cellular model of invasive oral squamous cell carcinoma was examined. Treatment of oral squamous cell carcinoma cells with TGF-beta1 promoted MMP-dependent cell scattering and collagen invasion, increased expression of MMP-2 and MT1-MMP, and enhanced MMP-2 activation. TGF-beta1 induced concomitant activation of ERK1/2 and p38 MAPK, and kinase inhibition studies revealed a negative regulatory role for ERK1/2 in modulating acquisition of MMP-2 activity. Thus, a reciprocal effect on proMMP-2 activation was observed whereupon blocking ERK1/2 phosphorylation promoted proMMP-2 activation and MT1-MMP activity, whereas inhibiting p38 MAPK activity decreased proteolytic potential. The cellular mechanism for the control of MT1-MMP catalytic activity involved concurrent reciprocal modulation of TIMP-2 expression by ERK1/2 and p38 MAPKs, such that inhibition of ERK1/2 phosphorylation decreased TIMP-2 production, and down-regulation of p38 MAPK activity enhanced TIMP-2 synthesis. Further, p38 MAPK inhibition promoted ERK1/2 phosphorylation, providing additional evidence for cross-talk between MAPK pathways. These observations demonstrate the complex reciprocal effects of ERK1/2 and p38 MAPK in the regulation of MMP activity, which could complicate the use of MAPK-specific inhibitors as therapeutic agents to down-regulate the biologic effects of TGF-beta1 on pericellular collagen degradation and tumor invasion.     

Distinct regulation of plasma LDL cholesterol by eicosapentaenoic acid and docosahexaenoic acid in high fat diet-fed hamsters: Participation of cholesterol ester transfer protein and LDL receptor

Despite established anti-atherogenic action, previous reports have shown that fish oils or n-3 poly-unsaturated fatty acid (PUFA) increase plasma LDL-C in animals and humans. However, which component of n-3 PUFAs and what mechanisms contribute to this increase are unclear. We investigated the effects of the major components of n-3 PUFA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on plasma LDL-C in high fat diet-fed hamsters. While LDL-C increased significantly with n-3 PUFA oil and DHA, EPA had no effect on LDL-C. Interestingly, a positive correlation was found between plasma cholesterol ester transfer protein (CETP) activity and LDL-C. Only DHA increased plasma CETP activity and significantly decreased LDL receptor expression in the liver. Our data suggest that DHA, not EPA, is a major factor in the LDL-C increasing effect of n-3 PUFA oil. These differential effects on LDL-C may arise from differences in plasma CETP activity and LDL receptor expression.     

A comparison of highly unsaturated fatty acid levels in wild and farmed eels (Anguilla anguilla).

1. Absolute and relative amounts of eicosapentaneoic acid (EPA) and docohexaenoic acid (DHA) in muscle of eels from four different fish farms were compared with samples from wild eels from two different areas of northern Italy. 2. Farmed eels were richer in DHA and EPA than wild animals. 3. The addition of cod liver oil to the diet of farmed eels led to a significant accumulation of EPA and DHA, but no change in total lipid content. 4. The calculation of two indices related to highly unsaturated fatty acid (HUFA) content (FLQ = fish lipid quality; AI = aterogenic index), indicated the higher nutritional value of farmed vs wild fish. 5. We conclude that farmed eels are an adequate source of fish products for human nutrition and propose use of the above-mentioned indices as an effective means for assessing fish nutritional quality for populations at high risk of chronic degenerative disease.