A comparison of cell death mechanisms of antioxidants, butylated hydroxyanisole and butylated hydroxytoluene.

Butylated hydroxyanisole (BHA) and the chemically similar butylated hydroxytoluene (BHT) are widely used as antioxidants. Toxicity of BHA and BHT has been reported under in vitro and in vivo experimental conditions. However, the mechanism of BHA-induced toxic effects in cells is unclear. In this study, the cytotoxic effects of BHA and differences in cell death mechanism for BHA and BHT were investigated in rat thymocytes by flow cytometric analysis using a fluorescent probe. We observed a significant increase in propidium iodide fluorescence in the population of cells treated with 100 µM and 300 µM BHA (dead cells). Thymocytes treated with 100 µM BHA showed increased intracellular Ca2+ and Zn2+ levels and depolarized cell membranes. BHA (30-100 µM) decreased non-protein thiol content of cells, indicating decreased glutathione content. Co-stimulation with 100 µM BHA and 300 µM H2O2 acted synergistically to increase cell lethality. Moreover, BHA significantly increased caspase-3 activity and the number of annexin-V-positive cells in a concentration-dependent manner, indicating apoptosis. However, BHT reduced caspase-3 activity and increased the number of annexin-V-negative dead cells, indicating non-apoptotic cell death. Our results reveal the toxicity of BHA could be attributed to increased levels of intracellular Ca2+ and Zn2+, resulting in an increased vulnerability of rat thymocytes to oxidative stress. In addition, we demonstrate that whereas BHA induced apoptosis, BHT induced non-apoptotic cell death in rat thymocytes. Therefore, these results may support the safety of BHA, but also demonstrate the importance of performing toxicity evaluation at the cellular level besides the tissue level.

Ubiquitin ligase Cbl-b and inhibitory Cblin peptides.

This review focuses on the Cbl-b muscle atrophy-associated ubiquitin ligase and its inhibitors. Herein, the role of E3 ubiquitin ligase-associated muscle atrophy genes (atrogenes), including MAFbx-1/agrogin-1 and MuRF-1, as well as another ubiquitin ligase, Cbl-b and its inhibitors, is discussed. Cbl-b plays an important role in unloading muscle atrophy caused by spaceflight and in bedridden patients: Cbl-b ubiquitinated and induced the degradation of IRS-1, a key intermediate in the IGF-1 signaling. Furthermore, a pentapetpide (DGpYMP), inhibited Cbl-b-mediated IRS-1 ubiquitination. This peptide-based Cbl-b inhibitor Cblin and its homologous peptides in foods presumably affect muscle atrophy under such conditions.

Expression of Cathepsin L and Its Intrinsic Inhibitors in Glomeruli of Rats With Puromycin Aminonucleoside Nephrosis.

Cathepsin L, a lysosomal cysteine proteinase, may have a key role in various biological and disease processes by intracellular and extracellular degradation of proteins. We examined the levels of cathepsin L and its intrinsic inhibitors in glomeruli of rats with puromycin aminonucleoside (PAN) nephrosis. In contrast to the weak levels of cathepsin L in normal glomeruli, on days 4 and 8, strong immunostaining was detected in almost all podocytes when proteinuria and pathological changes of the podocytes developed. Cathepsin L was reduced after day 28, but remained in a focal and segmental manner. Cystatin ß, an intracellular inhibitor, was not detected in podocytes. However, cystatin C, an extracellular inhibitor, was detected in podocytes after day 4, coincident with cathepsin L. Cystatin C levels were gradually reduced but sustained in many podocytes on day 28, while cystatin C was not detected in podocytes sustained cathepsin L. These results demonstrated that cathepsin L levels are not always accompanied by the levels of its inhibitors in podocytes of PAN nephrosis, suggesting a potential role of cathepsin L in podocyte injury, which is a critical process for the development and progression of tuft adhesion and sclerosis.

Acid Sphingomyelinase Promotes Cellular Internalization of Clostridium perfringens Iota-Toxin.

Clostridium perfringens iota-toxin is a binary actin-ADP-ribosylating toxin composed of the enzymatic component Ia and receptor binding component Ib. Ib binds to a cell surface receptor, forms Ib oligomer in lipid rafts, and associates with Ia. The Ia-Ib complex then internalizes by endocytosis. Here, we showed that acid sphingomyelinase (ASMase) facilitates the cellular uptake of iota-toxin. Inhibitions of ASMase and lysosomal exocytosis by respective blockers depressed cell rounding induced by iota-toxin. The cytotoxicity of the toxin increased in the presence of Ca2+ in extracellular fluids. Ib entered target cells in the presence but not the absence of Ca2+. Ib induced the extracellular release of ASMase in the presence of Ca2+. ASMase siRNA prevented the cell rounding induced by iota-toxin. Furthermore, treatment of the cells with Ib resulted in the production of ceramide in cytoplasmic vesicles. These observations showed that ASMase promotes the internalization of iota-toxin into target cells.

Inhibition of autophagy blocks cathepsins-tBid-mitochondrial apoptotic signaling pathway via stabilization of lysosomal membrane in ischemic astrocytes.

Our previous study and others have demonstrated that autophagy is activated in ischemic astrocytes and contributes to astrocytic cell death. However, the mechanisms of ischemia-induced autophagy remain largely unknown. In this study, we established a rat's model of permanent middle cerebral artery occlusion (pMCAO) and an in vitro oxygen and glucose deprivation (OGD) model. Autophagy was inhibited by either pharmacological treatment with 3-methyladenine (3-MA) and wortmannin (Wort) or genetic treatment with knockdown of Atg5 in primary cultured astrocytes and knockout of Atg5 in mouse embryonic fibroblast (MEF) cells, respectively. We found that pharmacological or genetic inhibition of autophagy reversed pMCAO or OGD-induced increase in LC3-II, active cathepsin B and L, tBid, active caspase-3 and cytoplastic cytochrome c (Cyt-c), and suppressed the injury-induced reduction in mitochondrial Cyt-c in ischemic cortex, in injured astrocytes and MEF cells. Immunofluorescence analysis showed that 3-MA or Wort treatment reversed OGD-induced release of cathepsin B and L from the lysosome to the cytoplasm and activation of caspase-3 in the astrocytes. Furthermore, treatment of 3-MA or Wort decreased OGD-induced increase in lysosomal membrane permeability and enhanced OGD-induced upregulation of lysosomal heat shock protein 70.1B (Hsp70.1B) in astrocytes. Inhibition of autophagy by 3-MA or Wort reduced infarction volume in rats and protected OGD-induced astrocytic cell injury. A non-selective caspase inhibitor z-VAD-fmk or a specific caspase-3 inhibitor Q-DEVD-OPh also rescued OGD-induced astrocytic cell injury. In conclusion, our presenting data suggest that inhibition of autophagy blocks cathepsins-tBid-mitochondrial apoptotic signaling pathway via stabilization of lysosomal membranes, possibly due to upregulation of the lysosomal Hsp70.1B in ischemic astrocytes.

Type II cGMP-dependent protein kinase negatively regulates fibroblast growth factor signaling by phosphorylating Raf-1 at serine 43 in rat chondrosarcoma cells.

Although type II cGMP-dependent protein kinase (PKGII) is a major downstream effector of cGMP in chondrocytes and attenuates the FGF receptor 3/ERK signaling pathway, its direct target proteins have not been fully explored. In the present study, we attempted to identify PKGII-targeted proteins, which are associated with the inhibition of FGF-induced MAPK activation. Although FGF2 stimulation induced the phosphorylation of ERK1/2, MEK1/2, and Raf-1 at Ser-338 in rat chondrosarcoma cells, pretreatment with a cell-permeable cGMP analog strongly inhibited their phosphorylation. On the other hand, Ser-43 of Raf-1 was phosphorylated by cGMP in a dose-dependent manner. Therefore, we examined the direct phosphorylation of Raf-1 by PKGII. Wild-type PKGII phosphorylated Raf-1 at Ser-43 in a cGMP-dependent manner, but a PKGII D412A/R415A mutant, which has a low affinity for cGMP, did not. Finally, we found that a phospho-mimic mutant, Raf-1 S43D, suppressed FGF2-induced MAPK pathway. These results suggest that PKGII counters FGF-induced MEK/ERK activation through the phosphorylation of Raf-1 at Ser-43 in chondrocytes.

Inhibition of cysteine cathepsin B and L activation in astrocytes contributes to neuroprotection against cerebral ischemia via blocking the tBid-mitochondrial apoptotic signaling pathway.

The roles of cathepsins in the ischemic astrocytic injury remain unclear. Here, we test the hypothesis that activation of cathepsin B and L contributes to the ischemic astrocyte injury via the tBid-mitochondrial apoptotic signaling pathways. In the rat models of pMCAO, CA-074Me or Clik148, a selective inhibitor of cathepsin B or cathepsin L, reduced the infarct volume, improved the neurological deficits and increased the MAP2 and GFAP levels. In OGD-induced astrocyte injury, CA-074Me or Clik148 decreased the LDH leakage and increased the GFAP levels. In the ischemic cortex or OGD-induced astrocytes injury, Clik148 or CA-074Me reversed pMCAO or OGD-induced increase in active cathepsin L or cathepsin B at 3 h or 6 h, increase in tBid, reduction in mitochondrial cytochrome-c (Cyt-c) and increase in cytoplastic Cyt-c and active caspase-3 at 12-24 h of the late stage of pMCAO or OGD. CA-074Me or Clik148 also reduced cytosolic and mitochondrial tBid, increased mitochondrial Cyt-c and decreased cytoplastic Cyt-c and active caspase-3 at 6 h of the early stage of Bid activation. CA-074Me or Clik148 blocked the pMCAO-induced release of cathepsin B or L from the lysosomes into the cytoplasm and activation of caspase-3 in ischemic astrocytes at 12 h after ischemia. Concurrent inhibition of cathepsin B and cathepsin L provided better protection on the OGD-induced astrocytic apoptosis than obtained with separate use of each inhibitor. These results suggest that inhibition of the cysteine cathepsin B and cathepsin L activation in ischemic astrocytes contributes to neuroprotection via blocking the tBid-mitochondrial apoptotic signaling pathway.

Intracellular trafficking of Clostridium botulinum C2 toxin.

Clostridium botulinum C2 toxin is a binary toxin composed of an enzymatic component (C2I) and binding component (C2II). The activated binding component (C2IIa) forms heptamers and the oligomer with C2I is taken up by receptor-mediated endocytosis. We investigated the intracellular trafficking of C2 toxin. When MDCK cells were incubated with C2I and C2IIa at 37 °C, C2I colocalized with C2IIa in cytoplasmic vesicles at 5 min, and C2I then disappeared (15 min incubation and later), and C2IIa was observed in the vesicles. Internalized C2I and C2IIa were transported to early endosomes. Some of both components were returned to the plasma membrane through recycling endosomes, whereas the rest of C2IIa was transported to late endosomes and lysosomes for degradation. Bafilomycin A1, an endosomal acidification inhibitor, caused the accumulation of C2IIa in endosomes, and both nocodazole and colchicine, microtubule-disrupting agents, restricted C2IIa's movement in the cytosol. These results indicated that an internalized C2I and C2IIa complex was delivered to early endosomes, and that subsequent delivery of C2I to the cytoplasm occurred in early endosomes. C2IIa was either sent back to the plasma membranes through recycling endosomes or transported to late endosomes and lysosomes for degradation.

Autophagy and cathepsin L are involved in the antinociceptive effect of DMBC in a mouse acetic acid-writhing model.

AIM: 2-(3',5'-Dimethoxybenzylidene) cyclopentanone (DMBC) is a novel synthetic compound with antinociceptive activities. The aim of this study was to investigate the roles of the autophagic-lysosomal pathway in the antinociceptive effect of DMBC in a mouse acetic acid-writhing model. METHODS: Mouse acetic acid-writhing test and hotplate test were used to assess the antinociceptive effects of DMBC, 3-MA (autophagy inhibitor) and Clik148 (cathepsin L inhibitor). The drugs were administered peripherally (ip) or centrally (icv). RESULTS: Peripheral administration of 3-MA (7.5-30 mg/kg) or Clik148 (10-80 mg/kg) produced potent antinociceptive effect in acetic acid-writhing test. Central administration of 3-MA or Clik148 (12.5-50 nmol/L) produced comparable antinociceptive effect in acetic acid-writhing test. Peripheral administration of DMBC (25-50 mg/kg) produced potent antinociceptive effects in both acetic acid-writhing and hotplate tests. Furthermore, the antinociceptive effect produced by peripheral administration of DMBC (50 mg/kg) in acetic acid-writhing test was antagonized by low doses of 3-MA (3.75 mg/kg) or Clik148 (20 mg/kg) peripherally administered, but was not affected by 3-MA or Clik148 (25 nmol/L) centrally administered. CONCLUSION: Activation of central autophagy and cathepsin L is involved in nociception in mice, whereas peripheral autophagy and cathepsin L contributes, at least in part, to the antinociceptive effect of DMBC in mice.

Intracellular trafficking of Clostridium perfringens iota-toxin b.

Clostridium perfringens iota-toxin is composed of an enzymatic component (Ia) and a binding component (Ib). Ib binds to a cell surface receptor, undergoes oligomerization in lipid rafts, and binds Ia. The resulting complex is then endocytosed. Here, we show the intracellular trafficking of iota-toxin. After the binding of the Ib monomer with cells at 4°C, oligomers of Ib formed at 37°C and later disappeared. Immunofluorescence staining of Ib revealed that the internalized Ib was transported to early endosomes. Some Ib was returned to the plasma membrane through recycling endosomes, whereas the rest was transported to late endosomes and lysosomes for degradation. Degraded Ib was delivered to the plasma membrane by an increase in the intracellular Ca(2+) concentration caused by Ib. Bafilomycin A1, an endosomal acidification inhibitor, caused the accumulation of Ib in endosomes, and both nocodazole and colchicine, microtubule-disrupting agents, restricted Ib's movement in the cytosol. These results indicated that an internalized Ia and Ib complex was delivered to early endosomes and that subsequent delivery of Ia to the cytoplasm occurs mainly in early endosomes. Ib was either sent back to the plasma membranes through recycling endosomes or transported to late endosomes and lysosomes for degradation. Degraded Ib was transported to plasma membranes.

L-tryptophan suppresses rise in blood glucose and preserves insulin secretion in type-2 diabetes mellitus rats.

Ample evidence indicates that a high-protein/low-carbohydrate diet increases glucose energy expenditure and is beneficial in patients with type-2 diabetes mellitus (T2DM). The present study was designed to investigate the effects of L-tryptophan in T2DM. Blood glucose was measured by the glucose dehydrogenase assay and serum insulin was measured with ELISA in both normal and hereditary T2DM rats after oral glucose administration with or without L-D-tryptophan and tryptamine. The effect of tryptophan on glucose absorption was examined in the small intestine of rats using the everted-sac method. Glucose incorporation in adipocytes was assayed with [(3)H]-2-deoxy-D-glucose using a liquid scintillation counter. Indirect computer-regulated respiratory gas-assay calorimetry was applied to assay energy expenditure in rats. L-Tryptophan suppressed both serum glucose and insulin levels after oral glucose administration and inhibited glucose absorption from the intestine. Tryptamine, but not L-tryptophan, enhanced insulin-stimulated [(3)H]-glucose incorporation into differentiated adipocytes. L-Tryptophan increased glucose-associated energy expenditure in rats in vivo. L-Tryptophan-rich chow consumed from a young age preserved the secretion of insulin and delayed the progression of T2DM in hereditary diabetic rats. The results suggested that L-tryptophan suppresses the elevation of blood glucose and lessens the burden associated with insulin secretion from ß-cells.

Structural basis of free reduced flavin generation by flavin reductase from Thermus thermophilus HB8.

Free reduced flavins are involved in a variety of biological functions. They are generated from NAD(P)H by flavin reductase via co-factor flavin bound to the enzyme. Although recent findings on the structure and function of flavin reductase provide new information about co-factor FAD and substrate NAD, there have been no reports on the substrate flavin binding site. Here we report the structure of TTHA0420 from Thermus thermophilus HB8, which belongs to flavin reductase, and describe the dual binding mode of the substrate and co-factor flavins. We also report that TTHA0420 has not only the flavin reductase motif GDH but also a specific motif YGG in C terminus as well as Phe-41 and Arg-11, which are conserved in its subclass. From the structure, these motifs are important for the substrate flavin binding. On the contrary, the C terminus is stacked on the NADH binding site, apparently to block NADH binding to the active site. To identify the function of the C-terminal region, we designed and expressed a mutant TTHA0420 enzyme in which the C-terminal five residues were deleted (TTHA0420-ΔC5). Notably, the activity of TTHA0420-ΔC5 was about 10 times higher than that of the wild-type enzyme at 20-40 °C. Our findings suggest that the C-terminal region of TTHA0420 may regulate the alternative binding of NADH and substrate flavin to the enzyme.

Clostridium perfringens iota-toxin b induces rapid cell necrosis.

Clostridium perfringens iota-toxin is a binary toxin composed of an enzyme component (Ia) and a binding component (Ib). Each component alone lacks toxic activity, but together they produce cytotoxic effects. We examined the cytotoxicity of iota-toxin Ib in eight cell lines. A431 and A549 cells were susceptible to Ib, but MDCK, Vero, CHO, Caco-2, HT-29, and DLD-1 cells were not. Ib bound and formed oligomers in the membranes of A431 and MDCK cells. However, Ib entered MDCK cells but not A431 cells, suggesting that uptake is essential for cellular survival. Ib also induced cell swelling and the rapid depletion of cellular ATP in A431 and A549 cells but not the insensitive cell lines. In A431 cells, Ib binds and oligomerizes mainly in nonlipid rafts in the membranes. Disruption of lipid rafts by methyl-ß-cyclodextrin did not impair ATP depletion or cell death caused by Ib. Ib induced permeabilization by propidium iodide without DNA fragmentation in A431 cells. Ultrastructural studies revealed that A431 cells undergo necrosis after treatment with Ib. Ib caused a disruption of mitochondrial permeability and the release of cytochrome c. Staining with active-form-specific antibodies showed that the proapoptotic Bcl-2-family proteins Bax and Bak were activated and colocalized with mitochondria in Ib-treated A431 cells. We demonstrate that Ib by itself produces cytotoxic activity through necrosis.

"Hybrid exercise" prevents muscle atrophy in association with a distinct gene expression pattern.

"Hybrid exercise" utilizing combined electrical stimulation and voluntary muscle contraction has been developed as a muscle exercise method. Although our previous studies have confirmed the effectiveness of the procedure, the mechanisms of its efficacy still remain unclear. In the present study, we identified genes that are specifically expressed in disused muscles, using the semitendinosus muscle from patients who underwent anterior cruciate ligament (ACL) reconstruction. Preoperative exercise was performed by four ACL-injured patients, who were subjected either to hybrid exercise (n=2), electrical stimulation (n=1), or no electrical stimulation (n=1), in addition to standard weight training for 4 weeks. Cross-sectional area (CSA) of the semitendinosus muscle was measured before and after the exercise by magnetic resonance imaging (MRI). A piece of the semitendinosus muscle was isolated during the surgery, and comprehensive analysis of the gene expression in this sample was performed using DNA microarray analysis. CSA increased in size by 4.2 and 14.7%, respectively, after hybrid exercise, and by 1.4% after electrical stimulation. However it shrunk by 7.7% without electrical stimulation. DNA microarray analysis revealed that hybrid exercise was more effective at stimulating the expression of signal transduction-, transcription- and cytoskeleton-related genes in semitendinosus muscles than electrical stimulation alone. In particular, gene ontology analysis revealed that hybrid exercise induced significantly higher expression of eukaryotic translation initiation factor 5A (EIFSA), peroxisomal biogenesis factor 6 (PEX6) and histone cluster 1 H4 (HIST1H4), compared with electrical stimulation alone. The expression of signal transduction-, transcription- and cytoskeleton-related genes may play an important role in muscle bulk increasing mechanisms in hybrid exercise.

Induction of T regulatory cells by cytotoxic T-lymphocyte antigen-2α on corneal endothelial cells.

PURPOSE: To determine whether murine corneal endothelial (CE) cells can promote the generation of T regulatory (Treg) cells in vitro. METHODS: To induce Treg cells in vitro by CE cell lines, T cells exposed to CE cells were used as Treg cells. T cells exposed to CE cells in the presence of anti-mouse CD3 antibody were harvested and added to target bystander T cells in vitro. T-cell activation was assessed for proliferation by [(3)H]-thymidine incorporation. Expression of CD25 or Foxp3 on Treg cells was evaluated by flow cytometry. Expression of cytotoxic T-lymphocyte antigen-2 alpha (CTLA-2α) on CE cells was evaluated by flow cytometry, RT-PCR, immunohistochemistry, or in situ hybridization. Anti-CTLA-2α neutralizing antibodies, CTLA-2α siRNA, or pro-cathepsin L blocking proteins were used to abolish the CE-inhibitory function. RESULTS: Cultured CE cells produced CTLA-2α on their surfaces, thereby enabling bystander CD4(+) T cells to be converted to Treg cells by TGFß promotion. CE-induced Treg cells had immunosuppressive capacities by highly expressing CD25(high) and Foxp3. When mRNA downregulation (siRNA transfection), neutralizing antibodies, or blocking proteins were used to block CTLA-2α expression on CE cells, CE-induced Treg cells failed to acquire Treg function. CONCLUSIONS: These findings indicate that cell surface CTLA-2α contributes to the CE-dependent suppression of bystander T cells. Thus, ocular resident tissue-exposed T cells can be induced to become regulators within the peripheral microenvironment.

Nobuhiko Katunuma: an outstanding scientist in the field of proteolysis and warm-hearted 'Kendo Fighter' biochemist.

Professor Nobuhiko Katunuma is well known for his outstanding contribution to the understanding of proteolysis in general and cysteine proteinases and their inhibitors in mammals. In fact, he is a world pioneer in the field. In 1963, he started his highly successful scientific career as a Professor at the Institute for Enzyme Research, the University of Tokushima. During the initial 30 years of his career, he was interested in vitamin B6 metabolism and discovered the acceleration of turnover rates of pyridoxal enzyme in apoprotein formation. After this period, his interest expanded to lysosomal cystein proteinases and their endogenous inhibitors. After determining the crystal structure of human cathepsin B, he generated a series of chemically synthesized specific inhibitors of cathepsins. These inhibitors are currently used throughout the world and some of them have been applied therapeutically in various diseases. During his career and even at present, Professor Katunuma has been studying Biochemistry in Medicine and also practicing to become a 'Kendo sword fencing Fighter'.

Quinolinate phosphoribosyl transferase, a key enzyme in de novo NAD(+) synthesis, suppresses spontaneous cell death by inhibiting overproduction of active-caspase-3.

Quinolinate phosphoribosyl transferase (QPRT) is a key enzyme in de novo NAD(+) synthesis. QPRT enzyme activity has a restricted tissue distribution, although QPRT mRNA is expressed ubiquitously. This study was designed to elucidate the functions of QPRT protein in addition to NAD(+) synthesis. QPRT was identified as a caspase-3 binding protein using double layer fluorescent zymography, but was not a substrate for caspase-3. Surface plasmon resonance analysis using recombinant proteins showed interaction of QPRT with active-caspase-3 in a dose dependent manner at 55 nM of the dissociation constant. The interaction was also confirmed by immunoprecipitation analysis of actinomycin D-treated QPRT-FLAG expressing cells using anti-FLAG-agarose. QPRT-depleted cells showed increased sensitivity to spontaneous cell death, upregulated caspase-3 activity and strong active-caspase-3 signals. Considered together, the results suggested that QPRT protein acts as an inhibitor of spontaneous cell death by suppressing overproduction of active-caspase-3.

Acquisition of T regulatory function in cathepsin L-inhibited T cells by eye-derived CTLA-2alpha during inflammatory conditions.

Pigment epithelium isolated from the eye possesses immunosuppressive properties such as regulatory T (Treg) cell induction; e.g., cultured retinal pigment epithelium (RPE) converts CD4(+) T cells into Treg cells in vitro. RPE constitutively expresses a novel immunosuppressive factor, CTLA-2alpha, which is a cathepsin L (CathL) inhibitor, and this molecule acts via RPE to induce Treg cells. To clarify CTLA-2alpha's role in the T cell response to RPE in ocular inflammation, we used the experimental autoimmune uveitis (EAU) animal model to examine this new immunosuppressive property of RPE. In EAU models, TGF-beta, but not IFN-gamma inflammatory cytokines, promotes the up-regulation of the expression of CTLA-2alpha in RPE. Similarly, CTLA-2alpha via RPE was able to promote TGF-beta production by the CD4(+) T cells. The RPE-exposed T cells (RPE-induced Treg cells) greatly produced TGF-beta and suppressed bystander effector T cells. There was less expression of CathL by the RPE-exposed T cells, and CathL-inhibited T cells were able to acquire the Treg phenotype. Moreover, CathL-deficient mice spontaneously produced Treg cells, with the increase in T cells potentially providing protection against ocular inflammation. More importantly, CD4(+) T cells from EAU in CathL knockout mice or rCTLA-2alpha from EAU animals were found to contain a high population of forkhead box p3(+) T cells. In both EAU models, there was significant suppression of the ocular inflammation. These results indicate that RPE secretes CTLA-2alpha, thereby enabling the bystander T cells to be converted into Treg cells via TGF-beta promotion.

Inhibition of collagenolytic cathepsins by beta-lactoglobulin in milk and its suppressive effect on bone resorption.

It is well known that the collagenolytic cathepsins play an important role in the degradation of bone matrix. Therefore, the purpose of this study was to clarify the prevention effect of bone resorption by milk components. Using double-layer reverse zymography, we found a 20 kDa protein in milk which inhibited cysteine proteases. This inhibitory protein was identified as beta-lactoglobulin B. The inhibitory activity of beta-lactoglobulin B against cathepsin K was stronger than that of beta-lactoglobulin A. Beta-lactoglobulin B specifically inhibited papain type cysteine proteases such as cathepsins K and L, but not serine proteases, aspartic proteases or metallo proteases. Beta-lactoglobulin B inhibited cathepsin K non-competitively and the Ki value was 10(-5) M. The formation of osteoclastic pits in the culture system was effectively inhibited by 10(-4)-10(-5) M beta-lactoglobulin B in vitro. Furthermore, we demonstrated that beta-lactoglobulin B inhibited degradation of type I-collagen by collagenolytic cathepsins. Using the everted-sac method in rat small intestines, it was found that beta-lactoglobulin was more easily absorbed from the intestines of young rats (5 wk-old) than from those of older rats (more than 20 wk-old). The digested products of beta-lactoglobulin B with lysyl-endopeptidase showed a similar inhibitory activity against cathepsin K to the intact beta-lactoglobulin B did. Therefore, peroral intake of beta-lactoglobulin in milk and its digested peptides are expected to help protect osteoclastic bone resorption via inhibition of collagenolytic cathepsins K and L.

Ubiquitin ligase Cbl-b is a negative regulator for insulin-like growth factor 1 signaling during muscle atrophy caused by unloading.

Skeletal muscle atrophy caused by unloading is characterized by both decreased responsiveness to myogenic growth factors (e.g., insulin-like growth factor 1 [IGF-1] and insulin) and increased proteolysis. Here, we show that unloading stress resulted in skeletal muscle atrophy through the induction and activation of the ubiquitin ligase Cbl-b. Upon induction, Cbl-b interacted with and degraded the IGF-1 signaling intermediate IRS-1. In turn, the loss of IRS-1 activated the FOXO3-dependent induction of atrogin-1/MAFbx, a dominant mediator of proteolysis in atrophic muscle. Cbl-b-deficient mice were resistant to unloading-induced atrophy and the loss of muscle function. Furthermore, a pentapeptide mimetic of tyrosine(608)-phosphorylated IRS-1 inhibited Cbl-b-mediated IRS-1 ubiquitination and strongly decreased the Cbl-b-mediated induction of atrogin-1/MAFbx. Our results indicate that the Cbl-b-dependent destruction of IRS-1 is a critical dual mediator of both increased protein degradation and reduced protein synthesis observed in unloading-induced muscle atrophy. The inhibition of Cbl-b-mediated ubiquitination may be a new therapeutic strategy for unloading-mediated muscle atrophy.