Inhibition of bacterial and human zinc-metalloproteases by bisphosphonate- and catechol-containing compounds.

Compounds containg catechol or bisphosphonate were tested as inhibitors of the zinc metalloproteases, thermolysin (TLN), pseudolysin (PLN) and aureolysin (ALN) which are bacterial virulence factors, and the human matrix metalloproteases MMP-9 and -14. Inhibition of virulence is a putative strategy in the development of antibacterial drugs, but the inhibitors should not interfere with human enzymes. Docking indicated that the inhibitors bound MMP-9 and MMP-14 with the phenyl, biphenyl, chlorophenyl, nitrophenyl or methoxyphenyl ringsystem in the S1'-subpocket, while these ringsystems entered the S2'- or S1 -subpockets or a region involving amino acids in the S1'- and S2'-subpockets of the bacterial enzymes. An arginine conserved among the bacterial enzymes seemed to hinder entrance deeply into the S1'-subpocket. Only the bisphosphonate containing compound RC2 bound stronger to PLN and TLN than to MMP-9 and MMP-14. Docking indicated that the reason was that the conserved arginine (R203 in TLN and R198 in PLN) interacts with phosphate groups of RC2.

Zinc-Chelating Compounds as Inhibitors of Human and Bacterial Zinc Metalloproteases.

Inhibition of bacterial virulence is believed to be a new treatment option for bacterial infections. In the present study, we tested dipicolylamine (DPA), tripicolylamine (TPA), tris pyridine ethylene diamine (TPED), pyridine and thiophene derivatives as putative inhibitors of the bacterial virulence factors thermolysin (TLN), pseudolysin (PLN) and aureolysin (ALN) and the human zinc metalloproteases, matrix metalloprotease-9 (MMP-9) and matrix metalloprotease-14 (MMP-14). These compounds have nitrogen or sulfur as putative donor atoms for zinc chelation. In general, the compounds showed stronger inhibition of MMP-14 and PLN than of the other enzymes, with Ki values in the lower µM range. Except for DPA, none of the compounds showed significantly stronger inhibition of the virulence factors than of the human zinc metalloproteases. TPA and Zn230 were the only compounds that inhibited all five zinc metalloproteinases with a Ki value in the lower µM range. The thiophene compounds gave weak or no inhibition. Docking indicated that some of the compounds coordinated zinc by one oxygen atom from a hydroxyl or carbonyl group, or by oxygen atoms both from a hydroxyl group and a carbonyl group, and not by pyridine nitrogen as in DPA and TPA.

Molecular Interactions Stabilizing the Promatrix Metalloprotease-9·Serglycin Heteromer.

Previous studies have shown that THP-1 cells produced an SDS-stable and reduction-sensitive complex between proMMP-9 and a chondroitin sulfate proteoglycan (CSPG) core protein. The complex could be reconstituted in vitro using purified serglycin (SG) and proMMP-9 and contained no inter-disulfide bridges. It was suggested that the complex involved both the FnII module and HPX domain of proMMP-9. The aims of the present study were to resolve the interacting regions of the molecules that form the complex and the types of interactions involved. In order to study this, we expressed and purified full-length and deletion variants of proMMP-9, purified CSPG and SG, and performed in vitro reconstitution assays, peptide arrays, protein modelling, docking, and molecular dynamics (MD) simulations. ProMMP-9 variants lacking both the FnII module and the HPX domain did not form the proMMP-9∙CSPG/SG complex. Deletion variants containing at least the FnII module or the HPX domain formed the proMMP-9∙CSPG/SG complex, as did the SG core protein without CS chains. The interacting parts covered large surface areas of both molecules and implicated dynamic and complementary ionic, hydrophobic, and hydrogen bond interactions. Hence, no short single interacting linear motifs in the two macromolecules could explain the strong SDS-stable and reduction-sensitive binding.

Method for Determining Gelatinolytic Activity in Tissue: In Situ Gelatin Zymography.

To explore the physiological or pathological roles of proteases, it is important to be able to detect and precisely localize them in a tissue, to differentiate between inactive and active forms, as well as to quantify and determine the nature of the enzyme that degrades a given substrate. Here we present an in situ gelatin zymography method that allows for a precise localization of active gelatin-degrading enzymes in a tissue section. In this method, dye-quenched gelatin is put on top of a tissue section. During an incubation period, active gelatinolytic enzymes will degrade the substrate and fluorescent signals are emitted from the locations of these enzymes.

Method for Determining Gelatinolytic Activity in Tissue Extracts: Real-Time Gelatin Zymography.

To explore the physiological or pathological roles of proteases, it is important to be able to detect and precisely localize them in a tissue, to differentiate between inactive and active forms, as well as to quantify and determine the nature of the enzyme that degrades a given substrate. Here we present a protocol for real-time gelatin zymography that is very useful for the detection of gelatin-degrading proteases in tissue extracts. This method uses fluorescence-labeled gelatin and therefore we also present an easy, fast, and cheap method for labeling gelatin with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF).

The selectivity of galardin and an azasugar-based hydroxamate compound for human matrix metalloproteases and bacterial metalloproteases.

Inhibitors targeting bacterial enzymes should not interfere with enzymes of the host, and knowledge about structural determinants for selectivity is important for designing inhibitors with a therapeutic potential. We have determined the binding strengths of two hydroxamate compounds, galardin and compound 1b for the bacterial zinc metalloproteases, thermolysin, pseudolysin and auerolysin, known to be bacterial virulence factors, and the two human zinc metalloproteases MMP-9 and MMP-14. The active sites of the bacterial and human enzymes have huge similarities. In addition, we also studied the enzyme-inhibitor interactions by molecular modelling. The obtained Ki values of galardin for MMP-9 and MMP-14 and compound 1b for MMP-9 are approximately ten times lower than previously reported. Compound 1b binds stronger than galardin to both MMP-9 and MMP-14, and docking studies indicated that the diphenyl ether moiety of compound 1b obtains more favourable interactions within the S´1-subpocket than the 4-methylpentanoyl moiety of galardin. Both compounds bind stronger to MMP-9 than to MMP-14, which appears to be due to a larger S´1-subpocket in the former enzyme. Galardin, but not 1b, inhibits the bacterial enzymes, but the galardin Ki values were much larger than for the MMPs. The docking indicates that the S´1-subpockets of the bacterial proteases are too small to accommodate the diphenyl ether moiety of 1b, while the 4-methylpentanoyl moiety of galardin enters the pocket. The present study indicates that the size and shape of the ligand structural moiety entering the S´1-subpocket is an important determinant for selectivity between the studied MMPs and bacterial MPs.

A Sensitive Assay for Proteases in Bioaerosol Samples: Characterization and Quantification of Airborne Proteases in Salmon Industry Work Environments.

Proteases are probably underestimated exposure agents in bioaerosols. Their roles as barrier disrupters in allergic sensitization and activators of innate inflammation call for more attention in exposure-response studies. The main objectives of this study was (i) to establish a suitable method for detection of small quantities of proteases in filtered air samples and (ii) to utilize the method to characterize exposure to proteases in a salmon industry work environment. Analysis of proteases in filtered air samples was based on zymography, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis with 0.1% gelatin as substrate added in the polyacrylamide gel. Gelatinase activity was evident as cleared (unstained) regions. The area of these regions was quantified using image analysis (UVP Vision Works®). Standard curves with known amounts of active porcine trypsin were added to each gel. Validation of 11 non-linear standard curves showed R2 (range) = 0.8989-0.9882, limit of detection = 0.056 nM, lower limit of quantification = 0.161 nM, and coefficients of variations (range) = 20-28%. Sampling of bioaerosols in salmon industry was performed using polytetrafluoretylene filters with an airflow of 3 l min-1. All samples contained visible bands close to the size of porcine trypsin (23.3 kDa). The bands did not disappear in the presence of EDTA but abolished by Pefabloc, demonstrating that the enzyme is a serine protease, most likely salmon trypsin. Airborne levels of active protease were below the statistical detection limit in the filleting department but quantifiable in extract samples from the slaughter department. Three filtered air samples from the slaughter department showed air concentrations of 6.2, 16.5, and 27.0 ng m-3 air. We conclude that zymography is a sensitive and reliable method for exposure assessment of active proteases in indoor environmental samples. We recommend this assay for use in occupational studies to characterize and quantify exposure to active proteases in bioaerosols.

Inhibition of chemerin/CMKLR1 axis in neuroblastoma cells reduces clonogenicity and cell viability in vitro and impairs tumor growth in vivo.

Pro-inflammatory cells, cytokines, and chemokines are essential in promoting a tumor supporting microenvironment. Chemerin is a chemotactic protein and a natural ligand for the receptors CMKLR1, GPR1, and CCRL2. The chemerin/CMKLR1 axis is involved in immunity and inflammation, and it has also been implicated in obesity and cancer. In neuroblastoma, a childhood tumor of the peripheral nervous system we identified correlations between high CMKLR1 and GPR1 expression and reduced overall survival probability. CMKLR1, GPR1, and chemerin RNA and protein were detected in neuroblastoma cell lines and neuroblastoma primary tumor tissue. Chemerin induced calcium mobilization, increased MMP-2 synthesis as well as MAP-kinase- and Akt-mediated signaling in neuroblastoma cells. Stimulation of neuroblastoma cells with serum, TNFα or IL-1ß increased chemerin secretion. The small molecule CMKLR1 antagonist α-NETA reduced the clonogenicity and viability of neuroblastoma cell lines indicating the chemerin/CMKLR1 axis as a promoting factor in neuroblastoma tumorigenesis. Furthermore, nude mice carrying neuroblastoma SK-N-AS cells as xenografts showed impaired tumor growth when treated daily with α-NETA from day 1 after tumor cell injection. This study demonstrates the potential of the chemerin/CMKLR1 axis as a prognostic factor and possible therapeutic target in neuroblastoma.

Cleavage of the urokinase receptor (uPAR) on oral cancer cells: regulation by transforming growth factor - ß1 (TGF-ß1) and potential effects on migration and invasion.

BACKGROUND: Urokinase plasminogen activator (uPA) receptor (uPAR) is up-regulated at the invasive tumour front of human oral squamous cell carcinoma (OSCC), indicating a role for uPAR in tumour progression. We previously observed elevated expression of uPAR at the tumour-stroma interface in a mouse model for OSCC, which was associated with increased proteolytic activity. The tumour microenvironment regulated uPAR expression, as well as its glycosylation and cleavage. Both full-length- and cleaved uPAR (uPAR (II-III)) are involved in highly regulated processes such as cell signalling, proliferation, migration, stem cell mobilization and invasion. The aim of the current study was to analyse tumour associated factors and their effect on uPAR cleavage, and the potential implications for cell proliferation, migration and invasion. METHODS: Mouse uPAR was stably overexpressed in the mouse OSCC cell line AT84. The ratio of full-length versus cleaved uPAR as analysed by Western blotting and its regulation was assessed by addition of different protease inhibitors and transforming growth factor - ß1 (TGF-ß1). The role of uPAR cleavage in cell proliferation and migration was analysed using real-time cell analysis and invasion was assessed using the myoma invasion model. RESULTS: We found that when uPAR was overexpressed a proportion of the receptor was cleaved, thus the cells presented both full-length uPAR and uPAR (II-III). Cleavage was mainly performed by serine proteases and urokinase plasminogen activator (uPA) in particular. When the OSCC cells were stimulated with TGF-ß1, the production of the uPA inhibitor PAI-1 was increased, resulting in a reduction of uPAR cleavage. By inhibiting cleavage of uPAR, cell migration was reduced, and by inhibiting uPA activity, invasion was reduced. We could also show that medium containing soluble uPAR (suPAR), and cleaved soluble uPAR (suPAR (II-III)), induced migration in OSCC cells with low endogenous levels of uPAR. CONCLUSIONS: These results show that soluble factors in the tumour microenvironment, such as TGF-ß1, PAI-1 and uPA, can influence the ratio of full length and uPAR (II-III) and thereby potentially effect cell migration and invasion. Resolving how uPAR cleavage is controlled is therefore vital for understanding how OSCC progresses and potentially provides new targets for therapy.

Synthesis, experimental evaluation and molecular modelling of hydroxamate derivatives as zinc metalloproteinase inhibitors.

Enzymes of the M4 family of zinc-metalloproteinases are virulence factors secreted from gram-positive or gram-negative bacteria, and putative drug targets in the treatment of bacterial infections. In order to have a therapeutic value such inhibitors should not interfere with endogenous zinc-metalloproteinases. In the present study we have synthesised a series of hydroxamate derivatives and validated the compounds as inhibitors of the M4 enzymes thermolysin and pseudolysin, and the endogenous metalloproteinases ADAM-17, MMP-2 and MMP-9 using experimental binding studies and molecular modelling. In general, the compounds are stronger inhibitors of the MMPs than of the M4 enzymes, however, an interesting exception is LM2. The compounds bound stronger to pseudolysin than to thermolysin, and the molecular modelling studies showed that occupation of the S2(') subpocket by an aromatic group is favourable for strong interactions with pseudolysin.

Intracellular MMP-2 activity in skeletal muscle is associated with type II fibers.

Matrix metalloproteinase 2 (MMP-2) is a proteolytic enzyme implicated in motility, differentiation, and regeneration of skeletal muscle fibers through processing of extracellular substrates. Although MMP-2 has been found to be localized intracellularly in cardiomyocytes where the enzyme is thought to contribute to post-ischemic loss of contractility, little is known about intracellular MMP-2 activity in skeletal muscle fibers. In the present study we demonstrate intracellular MMP-2 in normal skeletal muscle by immunohistochemical staining. Immunogold electron microscopic analyses indicated that the enzyme was concentrated in Z-lines of the sarcomers, in the nuclear membrane, and in mitochondria. By use of in situ zymography, we found that gelatinolytic activity in muscle fibers was co-localized with immunofluorecent staining for MMP-2. Staining for MMP-9, the other member of the gelatinase group of the MMPs, was negative. The broad-spectrum metalloprotease inhibitor EDTA and the selective gelatinase inhibitor CTT2, but not the cysteine inhibitor E64, strongly reduced the gelatinolytic activity. The intracellular gelatinolytic activity was much more prominent in fast twitch type II fibers than in slow twitch type I fibers, and there was a decrease in intracellular gelatinolytic activity and MMP-2 expression in muscles from mice exposed to high intensity interval training. Together our results indicate that MMP-2 is part of the intracellular proteolytic network in normal skeletal muscle, especially in fast twitch type II fibers. Further, the results suggest that intracellular MMP-2 in skeletal muscle fibers is active during normal homeostasis, and affected by the level of physical activity.

Inhibition of pseudolysin and thermolysin by hydroxamate-based MMP inhibitors.

In the present study, we have investigated the inhibition of thermolysin and pseudolysin by a series of compounds previously identified as matrix metalloproteinase (MMP) inhibitors using experimental binding studies and theoretical calculations. The experimental studies showed that some of the compounds were able to inhibit thermolysin and pseudolysin in the low µM range. The studies revealed that, in general, the compounds bound in the order MMPs > pseudolysin > thermolysin, and the strongest pseudolysin and thermolysin binders were compounds 8-12. Furthermore, compounds 8 and 9 were unique in that they bound much stronger to the two bacterial enzymes than to the MMPs. The docking calculations suggested that the phenyl group of the strongest binders (compounds 8 and 9) occupy the S2(')-subpocket, while a second ring system occupy the S1-subpocket in both thermolysin and pseudolysin. When the compounds possess two ring systems, the largest and most electron rich ring system seems to occupy the S1-subpocket.

Tumour microenvironments induce expression of urokinase plasminogen activator receptor (uPAR) and concomitant activation of gelatinolytic enzymes.

BACKGROUND: The urokinase plasminogen activator receptor (uPAR) is associated with poor prognosis in oral squamous cell carcinoma (OSCC), and increased expression of uPAR is often found at the invasive tumour front. The aim of the current study was to elucidate the role of uPAR in invasion and metastasis of OSCC, and the effects of various tumour microenvironments in these processes. Furthermore, we wanted to study whether the cells' expression level of uPAR affected the activity of gelatinolytic enzymes. METHODS: The Plaur gene was both overexpressed and knocked-down in the murine OSCC cell line AT84. Tongue and skin tumours were established in syngeneic mice, and cells were also studied in an ex vivo leiomyoma invasion model. Soluble factors derived from leiomyoma tissue, as well as purified extracellular matrix (ECM) proteins, were assessed for their ability to affect uPAR expression, glycosylation and cleavage. Activity of gelatinolytic enzymes in the tissues were assessed by in situ zymography. RESULTS: We found that increased levels of uPAR did not induce tumour invasion or metastasis. However, cells expressing low endogenous levels of uPAR in vitro up-regulated uPAR expression both in tongue, skin and leiomyoma tissue. Various ECM proteins had no effect on uPAR expression, while soluble factors originating from the leiomyoma tissue increased both the expression and glycosylation of uPAR, and possibly also affected the proteolytic processing of uPAR. Tumours with high levels of uPAR, as well as cells invading leiomyoma tissue with up-regulated uPAR expression, all displayed enhanced activity of gelatinolytic enzymes. CONCLUSIONS: Although high levels of uPAR are not sufficient to induce invasion and metastasis, the activity of gelatinolytic enzymes was increased. Furthermore, several tumour microenvironments have the capacity to induce up-regulation of uPAR expression, and soluble factors in the tumour microenvironment may have an important role in the regulation of posttranslational modification of uPAR.

PAC-1 and isatin derivatives are weak matrix metalloproteinase inhibitors.

BACKGROUND: Dysregulation of apoptotic cell death is observed in a large number of pathological conditions. As caspases are central enzymes in the regulation of apoptosis, a large number of procaspase-activating compounds (PAC-1 derivatives) and inhibitors (isatin derivatives) have been developed. Matrix metalloproteinases (MMPs) have been shown to have a dual role in apoptosis. Hence compounds that either activate or inhibit caspases should ideally not affect MMPs. As many PAC-1 derivatives contain a zinc chelating ortho-hydroxy N-acyl hydrazone moiety and isatin derivatives has two carbonyl groups on the indole core, it was of interest to determine to which extent these compounds can inhibit MMPs. METHODS: Eight PAC-1 and five isatin derivatives were docked into MMP-9 and MMP-14. The same compounds were synthesized, characterized, purified and tested as inhibitors of MMP-9 and MMP-14, using fluorescence quenched peptide and biological substrates. Some of the compounds were also tested for fluorescence quenching. RESULTS: Molecular docking suggested that the different compounds can bind to the MMP active sites. However, kinetic studies showed that neither of these compounds was a strong MMP inhibitor. IC50 values over 100µM were obtained after the enzyme activities were corrected for quenching. These IC50 values are far above the concentrations needed to activate or inhibit the caspases. CONCLUSION: The use of PAC-1 and isatin derivatives against caspases should have little or no effect on the activity of MMPs. GENERAL SIGNIFICANCE: Activators and inhibitors of caspases are important potential therapeutic agents for several diseases such as cancer, diabetes and neurodegenerative disorders.

Salmon and king crab trypsin stimulate interleukin-8 and matrix metalloproteinases via protease-activated receptor-2 in the skin keratinocytic HaCaT cell line.

Occupational skin symptoms are prevalent among the workers of the seafood processing industry. In this study we investigate the role of salmon (Salmo salar) and king crab trypsin (Paralithodes camtschaticus) as inducers of inflammation in skin via secretion of inflammatory mediators. Human skin keratinocytes (HaCaT cells) were exposed to purified salmon and king crab trypsin. We observed that salmon trypsin enhanced the secretion of IL-8 and MMP-2 and crab trypsin enhanced the secretion of IL-8, MMP-2 and MMP-9 in a dose dependent manner. As protease activated receptors (PAR)-2 in skin are known to play an important role in physiology and pathology, we explored the involvement of these receptors in mediating the release of interleukin (IL)-8 and matrix metalloproteinase (MMP)-2 and -9 subsequent to exposure of skin keratinocytes to salmon and crab trypsin. In addition we observed that salmon and crab trypsin exhibit individual differences in stimulating the release of these inflammatory mediators. Finally, using specific small interfering RNA (siRNA) against PAR-2, we confirmed that the increase in secretion of IL-8, MMP-2 and MMP-9 in skin keratinocytes following exposure to salmon and crab trypsin was mediated via activation of PAR-2. These results suggest that exposure to proteases from the seafood may lead to inflammatory reactions in skin.

Comparative molecular dynamic simulations of wild type and Thr114Val mutated Scaptodrosophila lebanonensis alcohol dehydrogenase.

Enzyme kinetics studies have shown that Scaptodrosophila lebanonensis alcohol dehydrogenase (SlADH) and other drosophilid alcohol dehydrogenases function by a compulsory-ordered mechanism where the coenzyme binds to the free enzyme, and that a proton is released upon formation of the binary enzyme-NAD(+) complex. A proton relay mechanism for the proton abstraction has been suggested that includes an eight-membered chain of water molecules connecting the active site with the bulk solvent. Thr114 bridges between two water molecules in the water chain. In a previous structural and enzyme kinetic study of a Thr114 Val mutant of SlADH, we showed that an intact water chain is essential for full enzyme activity. In the present study, comparative molecular dynamic (MD) simulations of the wild type and the SlADH(T114V) were performed. The simulations showed differences in hydrogen bonding properties and dynamics between the wild type and the SlADH(T114V). Differences in molecular dynamical behaviour were seen in the loop of importance for binding the nicotinamide part of NAD(+), in the region important for binding the adenine part of NAD(+), and in the region of the amino acid at position 114. The substrates also had more freedom for conformational changes in active site of the wild type SlADH than of the SlADH(T114V). The differences in hydrogen bonding properties and MDs between the wild type and mutant could not have been observed from the X-ray crystal structures only.

Matrix metalloproteinases in cancer: their value as diagnostic and prognostic markers and therapeutic targets.

Biomarkers are used as tools in cancer diagnostics and in treatment stratification. In most cancers, there are increased levels of one or several members of the matrix metalloproteinases (MMPs). This is a family of proteolytic enzymes that are involved in many phases of cancer progression, including angiogenesis, invasiveness, and metastasis. It has therefore been expected that MMPs could serve as both diagnostic and prognostic markers in cancer patients, but despite a huge number of studies, it has been difficult to establish MMPs as cancer biomarkers. In the present paper, we assess some of the challenges associated with MMP research as well as putative reasons for the conflicting data on the value of these enzymes as diagnostic and prognostic markers in cancer patients. We also review the prognostic value of a number of MMPs in patients with lung, colorectal, breast, and prostate cancers. The review also discusses MMPs as potential target molecules for therapeutic agents and new strategies for development of such drugs.

In vitro reconstitution of complexes between pro-matrix metalloproteinase-9 and the proteoglycans serglycin and versican.

Previously, we have shown that a proportion of the matrix metalloproteinase-9 (MMP-9) synthesized by the macrophage cell line THP-1 binds to a chondroitin sulfate proteoglycan (CSPG) core protein to form a reduction-sensitive heteromer. It was also shown that the hemopexin-like (PEX) domain and the fibronectin-like (FnII) module in the enzyme are involved in heteromer formation. In this paper, we show that reduction-sensitive and SDS-stable heteromers may be reconstituted in vitro by mixing proMMP-9 with either serglycin, versican or CSPGs isolated from various monocytic cell lines. In addition, a strong but SDS-soluble proMMP-9·CSPG heteromer was formed. The two macromolecules in the SDS-stable reduction-sensitive heteromers were not linked together by disulfide bonds. As for the heteromer isolated from THP-1 cells, in vitro reconstituted SDS-stable and SDS-soluble heteromers showed weaker binding to gelatin than the proMMP-9 monomer. Furthermore, gelatin inhibited in vitro reconstitution of the heteromers, showing that the FnII module is involved in the complex formation. Tissue inhibitor of metalloproteinase (TIMP)-1 was not be detected in the proMMP-9·CSPG complexes. However, the presence of TIMP-1 inhibited formation of the SDS-soluble heteromer, but not the SDS-stable reduction-sensitive heteromer. This indicates that different regions in the PEX domain are involved formation of these heteromers.

Tissue- and cell-specific co-localization of intracellular gelatinolytic activity and matrix metalloproteinase 2.

Matrix metalloproteinase 2 (MMP-2) is a proteolytic enzyme that degrades extracellular matrix proteins. Recent studies indicate that MMP-2 also has a role in intracellular proteolysis during various pathological conditions, such as ischemic injuries in heart and brain and in tumor growth. The present study was performed to map the distribution of intracellular MMP-2 activity in various mouse tissues and cells under physiological conditions. Samples from normal brain, heart, lung, liver, spleen, pancreas, kidney, adrenal gland, thyroid gland, gonads, oral mucosa, salivary glands, esophagus, intestines, and skin were subjected to high-resolution in situ gelatin zymography and immunohistochemical staining. In hepatocytes, cardiac myocytes, kidney tubuli cells, epithelial cells in the oral mucosa as well as in excretory ducts of salivary glands, and adrenal cortical cells, we found strong intracellular gelatinolytic activity that was significantly reduced by the metalloprotease inhibitor EDTA but not by the cysteine protease inhibitor E-64. Furthermore, the gelatinolytic activity was co-localized with MMP-2. Western blotting and electron microscopy combined with immunogold labeling revealed the presence of MMP-2 in different intracellular compartments of isolated hepatocytes. Our results indicate that MMP-2 takes part in intracellular proteolysis in specific tissues and cells during physiological conditions.

Expression of TWEAK/Fn14 in neuroblastoma: implications in tumorigenesis.

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor (TNF) family of cytokines, acts on responsive cells via binding to a cell surface receptor called Fn14. TWEAK binding to an Fn14 receptor or constitutive Fn14 overexpression has been shown to activate nuclear factor κB signaling which is important in tumorigenesis and cancer therapy resistance. In the present study, we demonstrate that TWEAK and Fn14 are expressed in neuroblastoma cell lines and primary tumors, and both are observed at increased levels in high-stage tumors. The treatment of neuroblastoma cell lines with recombinant TWEAK in vitro causes increased survival, and this effect is partially due to the activation of NF-κB signaling. Moreover, TWEAK induces the release of matrix metalloprotease-9 (MMP-9) in neuroblastoma cells, suggesting that TWEAK may play a role in the invasive phase of neuroblastoma tumorigenesis. TWEAK-induced cell survival was significantly reduced by silencing the TWEAK and Fn14 gene functions by siRNA. Thus, the expression of TWEAK and Fn14 in neuroblastoma suggests that TWEAK functions as an important regulator of primary neuroblastoma growth, invasion and survival and that the therapeutic intervention of the TWEAK/Fn14 pathway may be an important clinical strategy in neuroblastoma therapy.