Role of the transcription factor Sp1 in regulating the expression of the murine cathepsin E gene.

Cathepsin E (CE) is an intracellular aspartic proteinase that is exclusively expressed in cells of the gastrointestinal tracts, lymphoid tissues, urinary organs and red blood cells. However, the molecular mechanism by which CE is predominantly expressed in these cells remains unknown. Here, we report the identification of several transcription start sites of the CE gene and their regulatory factors in gastric adenosarcoma cells. We first identified several unique transcription start sites in mouse CE genes by an oligo cap method. Their analysis also revealed the existence of a non-coding region ∼24-kb upstream of exon 1 in the CE gene and also the existence of two transcripts for CE. Luciferase analyses in upstream of exon 1 revealed that this site contained putative binding regions for the transcription factors Sp1, AP-1 and cEts-1 essential for the expression of CE gene. Moreover, electrophoretic mobility shift assays revealed that the protein-oligonucleotides complex of the Sp1 site were supershifted by an anti-Sp1 antibody. The chromatin immunoprecipitation assay showed that Sp1 bound to the CE promoter region. In addition, overexpression of the Sp1 protein increased the expression of the CE protein. Altogether, these results suggest that Sp1 binding plays a particularly important role in the regulation of CE gene expression.

Higher concentrations of extracellular ATP suppress proliferation of Caco-2 human colonic cancer cells via an unknown receptor involving PKC inhibition.

BACKGROUND/AIMS: Adenosine 5'-triphosphate (ATP) mediates a variety of signal transductions via ATP receptors such as P2X and P2Y receptors. The present study aimed at understanding the mechanism underlying extracellular ATP-induced suppression of Caco-2 human colonic cancer cell proliferation. METHODS: Caco-2 cells were cultured. To examine cell viability and cell cycling, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, fluorescent cytochemistry, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and flow cytometry were carried out. To see mRNA expression of ATP receptors, reverse transcription-polymerase chain reaction (RT-PCR) was performed. To examine PKC activity and mitogen-activated protein (MAP) kinase activity, in situ PKC assay and Western blotting using an anti-extracellular signal-regulated kinase 1 (ERK1)-antibody and an anti-phospho-ERK antibody were carried out. RESULTS: Extracellular ATP or the unhydrolyzed ATP analogue 5'-adenylyimido-diphosphate (AMP-PNP) reduced Caco-2 cell viability in a concentration (10 microM-10 mM)-dependent manner at 48-h treatment, and the effect was not affected by caspase inhibitors. Caco-2 cells were little reactive to propidium iodide and Hoechst 33342 or little positive to TUNEL after 48-h treatment with ATP (1 mM). In the flow cytometry, 48-h treatment with ATP (1 mM) arrested cell cycling at the S phase in Caco-2 cells. P(2) purinoceptor agonists reduced Caco-2 cell viability with the order of potency: 2-methylthio ATP>UTP>beta, gamma-methylene ATP, and the ATP effect was partially inhibited by suramin, a non-selective inhibitor of P(2) purinoceptors. The PKC inhibitor GF109203X or the MAP kinase kinase inhibitor PD98059 reduced Caco-2 cell viability to an extent similar to that achieved by ATP (1 mM), and no further reduction was obtained with co-treatment with ATP. ATP and its ATP analogues such as AMP-PNP and ATPgammaS, at higher concentrations (1-10 mM), inhibited PKC activation in Caco-2 cells in a fashion that mimics the effect of GF109203X, but PD98059 exhibited no effect on PKC activation. The inhibitory effect of ATP on PKC activation was not found with SK-N-SH cells, a human neuroblastoma cell line, but the cells expressed all the mRNAs for P2X and P2Y receptors that Caco-2 cells did. ATP (10 mM) or GF109203X inhibited activation of ERK, a MAP kinase, in Caco-2 cells. CONCLUSION: Extracellular ATP, at higher concentrations, suppresses Caco-2 cell proliferation at the S phase of cell cycling by inhibiting PKC, possibly as mediated via an unknown ATP receptor, followed by MAP kinase.

Bactericidal effect of Nd:YAG and Er:YAG lasers in experimentally infected curved root canals.

OBJECTIVE: The purpose of this study was to evaluate the bactericidal efficacy of Nd:YAG and Er:YAG laser in the experimentally infected curved root canals. BACKGROUND DATA: Previous studies revealed that laser systems have a significant bactericidal effect in both human and bovine infected straight root canals. MATERIALS AND METHODS: Sixty extracted single-rooted teeth with single root canals were selected and then instrumented with endodontic files to a size 60 (K-type file). The degree of root curvature was determined according to modified Schneider's method. Each of the specimens was incubated in a sterile centrifuge tube with 1 mL of the Enterococcus faecalis suspension at 37°C for 2 weeks under aerobic conditions. After laser irradiation at each of the two settings, 50 mJ, 10 pps (0.5 W) or 100 mJ, 10 pps (1.0 W), the number of E. faecalis in each root canal was examined. RESULTS: In the straight root canals, the Er:YAG laser showed higher bactericidal effects by 6.4-10.8% than did the Nd:YAG laser. Conversely, the bactericidal effect of Er:YAG laser in the curved root canals was higher by 1.5-3.1% than was that with the Nd:YAG laser. The bactericidal effect of the Er:YAG laser in the curved root canal is significantly lower than that in the straight root canal (p < 0.05). CONCLUSION: These results suggest that further development in the endodontic laser tip and technique is required to ensure its success in curved root canals sterilization.

Effect of MTAD on the differentiation of osteoblast-like cells.

INTRODUCTION: The aim of the present study was to investigate the effect of MTAD on the differentiation of osteoblast-like cells. METHODS: The cell viability assay was performed to evaluate the cytotoxicity of MTAD on MC3T3-E1 and periodontal ligament cells at the various concentrations. The bone sialoprotein (BSP) gene expression was also examined by real-time polymerase chain reaction. RESULTS: MTAD exhibited a lower cytotoxicity compared with other intracanal irrigants and medication. The MC3T3-E1 cells treated with H2O2 showed a decrease in the alkaline phosphatase (ALP) activity by 40% on day 14 compared with the control group at the concentration of 50 microg/mlL. No significant difference in the ALP activity was observed between MTAD and control group. Furthermore, MTAD and Ca(OH)2 paste did not change in BSP gene expression in MC3T3-E1 cells on day 21. CONCLUSIONS: These results suggested that MTAD is a less cytotoxic irrigant and does not affect differentiation into osteoblasts compared with other intracanal irrigants, such as H2O2, NaOCl, ethylenediaminetetraacetic acid, and chlorhexidine.

The influence of the antibacterial monomer 12-methacryloyloxydodecylpyridinium bromide on the proliferation, differentiation and mineralization of odontoblast-like cells.

A dentin primer incorporating an antibacterial monomer 12-methacryloyloxydodecylpyridinium bromide (MDPB) shows strong antibacterial effects, and may provide better prognosis for direct capping of infected pulp exposed by caries removal compared with conventional adhesives. However, influences of MDPB on healing of the pulp have not yet been fully elucidated. The purpose of this study was to compare the influences of unpolymerized MDPB on proliferation, differentiation and mineralization of odontoblast-like MDPC-23 cells with those of other resin monomers, Bis-GMA, MDP, TEGDMA and HEMA. The inhibitory effects of MDPB on the proliferation of MDPC-23 were lower than those of Bis-GMA. While MDPB strongly affected the differentiation compared with the other monomers, it was less inhibitory than Bis-GMA and MDP on the mineralization ability of odontoblast-like cells. These findings indicate that MDPB has superior biocompatibility than Bis-GMA in terms of hard tissue formation by odontoblastic cells, suggesting its possible less negative influences on dentinogenesis.

Caspase-4 activation in association with decreased adenosine deaminase activity may be a factor for gastric ulcer.

BACKGROUND/AIMS: Adenosine deaminase (ADA)-deficient disease, a severe combined immunodeficiency, is most commonly associated with gastrointestinal disorders such as ulcer. The present study investigated the role of ADA in the pathogenesis of gastric ulcer. METHODS: ADA activity was measured in a variety of organs and tissues from rats and in human gastric biopsy samples from patients who underwent gastrofiberscope. An MTT assay and TUNEL staining were carried out, and activity of caspase-3 and -4 was enzymatically measured in MKN45 human gastric cancer cells. In the Western blot analysis, caspase-4 activation was identified in gastric biopsy samples. RESULTS: In rat organs and tissues, the epithelium of the gastrointestine exhibited higher ADA activity. The ADA inhibitor EHNA reduced cell viability, increased TUNEL-positive cells, and activated caspase-3 and -4 in MKN45 cells. For gastric biopsy samples, much lower ADA activity was found in gastric ulcer tissues, with a tendency of caspase-4 activation. CONCLUSION: A decline in ADA activity and the ensuing increase in intracellular adenosine concentrations for the stomach could induce gastric epithelial cell apoptosis by activating caspase-4 and the effector caspase-3. This may represent a fresh pathogenetical pathway for gastric ulcer relevant to ADA activity and caspase-4 activation.

Adenosine suppresses CW2 human colonic cancer growth by inducing apoptosis via A(1) adenosine receptors.

Extracellular adenosine-induced apoptosis of CW2 human colonic cancer cells by activating caspase-3, -8 and -9, and the effect was enhanced by adding EHNA, an adenosine deaminase inhibitor. Adenosine-induced CW2 cell death was inhibited by 8-CPT, an A(1) adenosine receptor inhibitor, but otherwise 2-chloroadenosine, an A(1) adenosine receptor agonist, and MDL-12330A, an inhibitor of adenylate cyclase, mimicked the adenosine effect. For mice inoculated with CW2 cells, intraperitoneal injection with adenosine reduced tumor growth and more prominent reduction was obtained with co-injection with EHNA. Adenosine, thus, suppresses CW2 human colonic cancer growth by inducing apoptosis as mediated via A(1) adenosine receptors.

Dual regulation of heat-activated K+ channel in rat DRG neurons via alpha(1) and beta adrenergic receptors.

AIMS: The present study aimed at understanding regulation of heat-activated K+ channels expressed in DRG neurons. MAIN METHODS: Whole-cell patches were made from cultured rat dorsal root ganglion (DRG) neurons and heat-activated currents were monitored. KEY FINDINGS: A temperature rise from 25 to 45 degrees C generated outward currents with the current/voltage relation revealing a reversal potential of approximately -90 mV, that are inhibited by the K+ channel blockers, Ba2+ and tetraethylammonium, indicating that heat activates K+ channels expressed in DRG neurons. Heat-activated K+ channel currents here was depressed by noradrenaline, and a similar effect was obtained with cirazoline, an agonist of alpha1 adrenergic receptors linked to G(q) protein involving PKC activation, and isoproterenol, an agonist of beta adrenergic receptors linked to G(s) protein involving PKA activation, but not UK14304, an agonist of alpha2 adrenergic receptors linked to G(i) protein. The noradrenaline action was prevented by GF109203X, a PKC inhibitor, or H-89, a PKA inhibitor, and a complete inhibition was obtained with co-treatment with GF109203X and H-89. PMA, a PKC activator, or forskolin, an activator of adenylate cyclase, on the other hand, reduced heat-activated outward currents and co-treatment with PMA and forskolin further attenuated the currents to an extent similar to that for noradrenaline. SIGNIFICANCE: The results of the present study show that heat-activated K+ channel currents in DRG neurons are inhibited by both PKC and PKA as mediated via alpha1 and beta adrenergic receptors, respectively. This may represent a new insight into regulation of thermosensation via a noradrenergic pathway.

Extracellular adenosine induces apoptosis in Caco-2 human colonic cancer cells by activating caspase-9/-3 via A(2a) adenosine receptors.

BACKGROUND: Extracellular adenosine has been shown to induce apoptosis in a variety of cells via an intrinsic pathway linked to adenosine uptake into cells and the ensuing signaling cascades and an extrinsic pathway linked to adenosine receptors. The present study was designed to understand the mechanism underlying adenosine-induced apoptosis of Caco-2 human colonic cancer cells. METHODS: To observe cell viability, an MTT assay was carried out in Caco-2 cells untransfected or transfected with the A(2a) adenosine receptor pcDNA3.1. Apoptotic cell death was assessed with flow cytometry using propidium iodide and annexin V and internucleosomal DNA fragmentation analysis. Activities of caspase-3, -8, and -9 were measured using a caspase fluorometric assay kit. Mitochondrial membrane potentials were monitored using a DePsipher kit. Expression of adenosine receptors was examined with the reverse transcription-polymerase chain reaction (RT-PCR) method. RESULTS: Extracellular adenosine induced Caco-2 cell apoptosis in a concentration-dependent (1-20 mM) and treatment time-dependent (24-72 h) manner. The adenosine effect was inhibited by DMPX, an inhibitor of A(2a) adenosine receptors and SQ22536, an inhibitor of adenylate cyclase. CGS21680, an agonist of A(2a) adenosine receptors, and forskolin, an adenylate cyclase activator, mimicked the adenosine action. Caco-2 cell death was still induced by overexpressing A(2a) adenosine receptors, and adenosine further promoted the cell death. Adenosine disrupted mitochondrial membrane potentials and activated caspase-9 and -3, but not caspase-8. CONCLUSIONS: Extracellular adenosine induces apoptosis in Caco-2 cells by activating caspase-9 and the downstream effector caspase caspase-3 in association with mitochondrial damage via A(2a) adenosine receptors.

Impaired chemotaxis and cell adhesion due to decrease in several cell-surface receptors in cathepsin E-deficient macrophages.

Cathepsin E is an endo-lysosomal aspartic proteinase exclusively present in immune system cells. Previous studies have shown that cathepsin E-deficient (CatE(-/-)) mice display aberrant immune responses such as atopic dermatitis and higher susceptibility to bacterial infection. However, the mechanisms underlying abnormal immune responses induced by cathepsin E deficiency are still unclear. In this study, we found that the cell-surface levels of chemotactic receptors, including chemokine receptor (CCR)-2 and N-formyl peptide receptors (FPRs), were clearly diminished in CatE(-/-)macrophages compared with those in wild-type cells. Consistently, chemotaxis of CatE(-/-)macrophages to MCP-1 and N-formyl-methionyl-leucyl-phenylalanine was also decreased. Similar to the chemotactic receptors, the surface expressions of the adhesion receptors CD18 (integrin beta(2)) and CD 29 (integrin beta(1)) in CatE(-/-) macrophages were significantly decreased, thereby reducing cell attachment of CatE(-/-) macrophages. These results indicate that the defects in chemotaxis and cell adhesion are likely to be involved in the imperfect function of CatE(-/-)macrophages.

In vitro evaluation of the antimicrobial activity of a new resin-based endodontic sealer against endodontic pathogens.

The purpose of this study was to compare the antimicrobial activities of a new resin-based SuperBond (SB) Sealer and five other sealers/cements against endodontic pathogens. The antimicrobial activities of SB Sealer, Sealapex, AH plus, Roeko Seal Automix, Canals N, and ProRoot mineral trioxide aggregate (MTA) were examined using a double-layered method. The microorganisms Staphylococcus aureus, Enterococcus faecalis, Candida albicans, Streptococcus mutans, and Streptococcus sanguinis were used. Live microorganisms were stained using triphenyltetrazolium chloride, and the zones of inhibition of microorganism growth were measured. The antimicrobial activity of SB Sealer was significantly lower than that of the other sealers, except for Pro Root MTA, against S. aureus, C. albicans, S. mutans, and S. sanguinis, but no activity against E. faecalis was detected. On the other hand, AH plus exhibited the highest antimicrobial activity. Pro Root MTA showed no antimicrobial activity against any of the microorganisms tested. SB Sealer offered no antimicrobial advantage over the other sealers tested except for Pro Root MTA.

The effect of mineral trioxide aggregate on the mineralization ability of rat dental pulp cells: an in vitro study.

The aim of this study was to investigate the effect of mineral trioxide aggregate (MTA) on cell viability and mineralization ability of rat dental pulp cells. The pulp capping materials, such as MTA, Dycal (Dentsply Caulk, Milford, DE), and Superbond C&B (SB; Sun Medical, Shiga, Japan) were placed on transwell inserts and cultured with rat dental pulp cells. MTA and SB exhibited no cytotoxicity, whereas almost all cells had died after 72 hours of culture with Dycal. MTA significantly stimulated mineralization by 60% compared with the control. MTA and Dycal significantly upregulated by two-fold the level of bone morphogenetic protein (BMP)-2 messenger RNA expression compared with the control. Furthermore, MTA increased BMP-2 protein production by about 40%, whereas Dycal significantly reduced it. Although MTA and Dycal increased the concentration of extracellular calcium by about 0.4 mmol/L, SB had no effect. These results suggest that BMP-2 may play an important role in mineralization stimulated by MTA.

Adiponectin induces dentin sialophosphoprotein in rat dental pulp cells: an in vitro study.

Adiponectin is known to play an important role in the regulation of blood glucose levels through the mediation of adiponectin receptors 1 and 2 (AR1 and AR2, respectively). The purpose of this study was to investigate the role of adiponectin in dental pulp cells. The expressions of both AR1 and AR2 were observed in dental pulp by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. Quantitative analysis of Alizarin Red S staining showed that 10 microg/mL of adiponectin significantly promoted mineralization by 1.6 times compared with control on day 12. However, no significant difference in mineralization was observed between control and 0.1 or 1 microg/mL adiponectin treatment. Moreover, real-time PCR results indicated that adiponectin (10 microg/mL) significantly increased the expression of dentin sialophosphoprotein (DSPP) by 2.3 and 1.8 times compared with control on days 8 and 12, respectively. These results indicated that adiponectin might promote mineralization by inducing DSPP expression in dental pulp cells.

Dentin phosphophoryn promotes cellular migration of human dental pulp cells.

Dentin phosphophoryn (DPP) is a dentin sialophosphoprotein gene product that has an RGD motif and repeat sequences of aspartic acid and phosphoserine. To date, the function of DPP in the early stage of reparative dentin formation still remains unclear. The objective of this study was to evaluate the effects of DPP on pulp cell migration and proliferation. DPP promoted cell migration in a concentration-dependent manner, thus increasing it by about 3-fold at 1000 ng/mL compared with the control, but it had no effect on cell proliferation. Dephosphorylated DPP also promoted cell migration, similarly to DPP. However, cell migration was significantly suppressed by the addition of alphavbeta3 integrin antibody to the medium. Furthermore, porcine DPP-derived RGD peptide, but not its mutant RAD peptide, significantly promoted cell migration. These results indicated that the RGD motif of DPP plays an important role in the migration of human dental pulp cells.

Spontaneous atherothrombosis and medial degradation in Apoe-/-, Npc1-/- mice.

BACKGROUND: The formation of an occluding thrombus on a ruptured or eroded atherosclerotic plaque is the hallmark event leading to acute coronary syndromes, myocardial infarction, and sudden death in humans. However, other species are highly resistant to plaque complications, and the specific processes predisposing to plaque destabilization and thrombosis are poorly understood. METHODS AND RESULTS: Mice carrying a null mutation of a gene regulating intracellular cholesterol transport (the Niemann-Pick C1 [Npc1] gene) were crossed with apolipoprotein E (Apoe) knockout mice to examine the effect of Npc1 on atherosclerotic lesion formation. Double-mutant mice showed greater lesion area compared with Apoe-/- littermates. Remarkably, the double mutants also developed large, protruding thrombi associated with the plaques and prominent medial degradation with inflammatory cell infiltration into the adventitia. Genetic studies suggested that the BALB background was permissive for plaque complications compared with C57BL/6J, and a BALB susceptibility locus was mapped by linkage analysis to chromosome 6. Examination of clotting parameters in double-knockout mice revealed that native clotting times were shortened and thrombin-antithrombin complex and soluble CD40 ligand levels were elevated compared with wild-type controls. In addition, cathepsin K was induced in Npc1-/- macrophages, and cathepsin K immunostaining and elastase activity were increased in proximal aortas of double-mutant mice compared with controls. CONCLUSIONS: A defect in intracellular cholesterol trafficking caused by the Npc1 null mutation predisposes to increased lesion formation, atherothrombosis, and medial degradation. Plaque complications may require a procoagulant state and an increased protease activity, leading to plaque destabilization.

Effects of disease-modifying anti-rheumatic drugs (DMARDs) on the activities of rheumatoid arthritis-associated cathepsins K and S.

Rheumatoid arthritis is an inflammatory and disabling joint disease affecting 0.5-1.5% of the population. Although various anti-inflammatory (NSAIDs) and disease-modifying (DMARDs) drugs are in clinical use, their precise mechanisms of action are not always defined. In this report, we discuss the effects of widely used DMARDs such as gold derivatives and chloroquine on cathepsins K and S, which have been implicated as critical mediators of inflammation and joint erosion in rheumatoid arthritis. We demonstrate that clinically potent gold derivatives inhibit cathepsins K and S in in vitro and cell-based assays. An X-ray analysis of the gold thiomalate/cathepsin K complex reveals that the inhibitor is bound to the active-site cysteine residue of the protease. Chloroquine, a lysosomotropic agent of lower clinical potency than gold derivatives, inhibits neutral pH-labile cathepsins intracellularly, but does not affect the neutral pH-stable cathepsin S. The potent inhibition of cathepsins implicated in the pathogenesis of rheumatoid arthritis by gold derivatives may explain the therapeutic efficacy of these drugs.

Characterization of rat cathepsin E and mutants with changed active-site residues and lacking propeptides and N-glycosylation, expressed in human embryonic kidney 293T cells.

To study the roles of the catalytic activity, propeptide, and N-glycosylation of the intracellular aspartic proteinase cathepsin E in biosynthesis, processing, and intracellular trafficking, we constructed various rat cathepsin E mutants in which active-site Asp residues were changed to Ala or which lacked propeptides and N-glycosylation. Wild-type cathepsin E expressed in human embryonic kidney 293T cells was mainly found in the LAMP-1-positive endosomal organelles, as determined by immunofluorescence microscopy. Consistently, pulse-chase analysis revealed that the initially synthesized pro-cathepsin E was processed to the mature enzyme within a 24 h chase. This process was completely inhibited by brefeldin A and bafilomycin A, indicating its transport from the endoplasmic reticulum (ER) to the endosomal acidic compartment. Mutants with Asp residues in the two active-site consensus motifs changed to Ala and lacking the propeptide (Leu23-Phe58) and the putative ER-retention sequence (Ser59-Asp98) were neither processed nor transported to the endosomal compartment. The mutant lacking the ER-retention sequence was rapidly degraded in the ER, indicating the importance of this sequence in correct folding. The single (N92Q or N324D) and double (N92Q/N324D) N-glycosylation-deficient mutants were neither processed into a mature form nor transported to the endosomal compartment, but were stably retained in the ER without degradation. These data indicate that the catalytic activity, propeptides, and N-glycosylation of this protein are all essential for its processing, maturation, and trafficking.

The role of the cathepsin E propeptide in correct folding, maturation and sorting to the endosome.

Cathepsin E (CE) is an endosomal aspartic proteinase of the A1 family that is highly homologous to the lysosomal aspartic proteinase cathepsin D (CD). Newly synthesized CE undergoes several proteolytic processing events to yield mature CE, from which the N-terminal propeptide usually comprising 39 amino acids is removed. To define the role of the propeptide of CE in its biosynthesis and processing, we constructed two fusion proteins using chimeric DNAs encoding the CE propeptide fused to the mature CD tagged with HA at the COOH terminus (termed ED-HA) and encoding the CD propeptide fused to the mature CE (termed DE). Pulse-chase analysis revealed that wild-type CE expressed in human embryonic kidney cells is autoproteolytically processed into mature CE within a 12-h chase, whereas the chimeric DE failed to be converted into mature CE even after a 24-h chase. The DE chimera was nevertheless capable of acid-dependent autoactivation in vitro to yield a catalytically active form, although its specificity constants (kcat/Km) were considerably high but less (35%) than those of the wild-type CE. By contrast, the chimeric ED-HA expressed in HeLa cells underwent neither processing into a catalytically active enzyme nor acid-dependent autoactivation in vitro. The ED-HA protein was less stable than wt-CD-HA, as determined on pulse-chase analysis and on trypsin digestion. These data indicate that the propeptide of CE is essential for the correct folding, maturation, and targeting of this protein to its final destination.

The role of cathepsins in osteoporosis and arthritis: rationale for the design of new therapeutics.

Human cysteine proteases of the papain family have been recognized as potential drug targets for musculoskeletal diseases. Most of the interest is focused on cathepsins S and K, which display selective expression in cells of the immune system and cells capable to efficiently degrade extracellular matrix proteins, in particular collagens. The predominant expression of cathepsin K in osteoclasts has rendered the enzyme into a major target for the development of novel anti-resorptive drugs in osteoporosis whereas cathepsin S appears to be an attractive drug target candidate for various inflammatory diseases including rheumatoid arthritis. Since rheumatoid arthritis is at the same time an inflammatory and joint destructive disorder, the combined inhibition of both cathepsins S and K should be beneficial. This review will outline the rationale and recent progress for targeting cathepsins in arthritis and osteoporosis.

A new selective substrate for cathepsin E based on the cleavage site sequence of alpha2-macroglobulin.

Cathepsin E is an intracellular aspartic proteinase of the pepsin family predominantly expressed in cells of the immune system and believed to contribute to homeostasis by participating in host defense mechanisms. Studies on its enzymatic properties, however, have been limited by a lack of sensitive and selective substrates. For a better understanding of the importance of this enzyme in vivo, we designed and synthesized a highly sensitive peptide substrate for cathepsin E based on the sequence of the specific cleavage site of alpha2-macroglobulin. The substrate constructed, MOCAc-Gly-Ser-Pro-Ala-Phe-Leu-Ala-Lys(Dnp)-D-Arg-NH2 [where MOCAc is (7-methoxycoumarin-4-yl)acetyl and Dnp is dinitrophenyl], derived from the cleavage site sequence of human alpha2-macroglobulin, was the most sensitive and selective for cathepsin E, with k(cat)/K(m) values of 8-11 microM(-1) s(-1), whereas it was resistant to hydrolysis by the analogous aspartic proteinases cathepsin D and pepsin, as well as the lysosomal cysteine proteinases cathepsins B, L, and H. The assay allows the detection of a few fmol of cathepsin E, even in the presence of plasma and cell lysate, and gives accurate results over a wide enzyme concentration range. This substrate might represent a useful tool for monitoring and accurately quantifying cathepsin E, even in crude enzyme preparations.