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This study investigated the effects of blockade of IL-6 on bone loss induced by modeled microgravity (MG). Adult male mice were exposed to hind-limb suspension (HLS) and treated with IL-6-neutralizing antibody (IL-6 nAb) for 4 weeks. HLS in mice led to upregulation of IL-6 expression in both sera and femurs. IL-6 nAb treatment in HLS mice significantly alleviated bone loss, evidenced by increased bone mineral density of whole tibia, trabecular thickness and number, bone volume fraction of proximal tibiae, and ultimate load and stiffness of femoral diaphysis. IL-6 nAb treatment in HLS mice significantly enhanced levels of osteocalcin in sera and reduced levels of deoxypyridinoline. In MC3T3-E1 cells exposed to MG in vitro, IL-6 nAb treatment increased mRNA expression and activity of alkaline phosphatase, mRNA expression of osteopontin and runt-related transcription factor 2, and protein levels of osteoprotegerin and decreased protein levels of receptor activator of the NF-[kappa]B ligand. In RAW254.7 cells exposed to MG, IL-6 nAb treatment downregulated mRNA expression of cathepsin K and tartrate-resistant acid phosphatase (TRAP) and reduced numbers ofTRAP-positive multinucleated osteoclasts. In conclusion, blockade of IL-6 alleviated the bone loss induced by MG. Key words: microgravity, bone loss, IL-6, osteoblastogenesis, osteoclastogenesis. Cette etude portait sur les effets de l'inhibition de l'IL-6 sur la perte osseuse dans un modele de microgravite (MG). Nous avons mis la patte posterieure de souris adultes males en suspension (SPP) et administre un anticorps neutralisant l'IL-6 (IL-6 nAb) pendant 4 semaines. La SPP chez les souris a mene a une regulation a la hausse de l'expression de l'IL-6 dans le serum comme dans le femur. L'administration d'IL-6 nAb chez les souris SPP a permis d'attenuer la perte osseuse de facon marquee, phenomene mis en evidence par une augmentation de la densite minerale osseuse du tibia entier, de l'epaisseur des trabecules et de leur nombre, de la fraction de volume osseux dans le tibia proximal, ainsi que de la charge ultime et de la rigidite de la diaphyse femorale. L'administration d'IL-6 nAb chez les souris SPP a entraine une hausse notable des taux d'osteocalcine dans le serum, avec une diminution des taux de desoxypyridinoline. Dans les cellules MC3T3-E1 exposees a la MG in vitro, l'administration d'IL-6 nAb entrainait une augmentation de l'expression en ARNm et de l'activite de la phosphatase alcaline, de l'expression en ARNm de l'osteopontine et du facteur de transcription lie a Runt 2, et des taux en proteines d'osteoproteregine, avec une diminution des taux en proteines de l'activateur du recepteur du ligand du NF-[kappa]B. Dans les cellules RAW254.7 exposees a la MG, l'administration d'IL-6 nAb entrainait une regulation a la baisse de l'expression en ARNm de la cathepsine K et de la phosphatase acide resistante au tartrate (ou TRAP pour << tartrate-resistant acid phosphatase >>) avec une diminution du nombre d'osteoclastes multinuclees reagissant a la TRAP. En conclusion, l'inhibition de l'IL-6 permettait d'attenuer la perte osseuse engendree par la MG. [Traduit par la Redaction] Mots-cles: microgravite, perte osseuse, IL-6, osteoblastogenese, osteoclastogenese., Introduction Bone loss caused by microgravity in astronauts poses a significant risk to the health and is known as a significant barrier for long-duration spaceflight (Grimm et al. 2016). The [...]

Chorda tympani denervation (CTD) of rats was earlier shown to result in loss of submandibular gland (SMG) weight (at only 1 wk) and in continued reduction in aquaporin 5 (AQP5) protein expression (until 4 wk), without affecting its mRNA synthesis (Li X, Azlina A, Karabasil MR, Purwanti N, Hasegawa T, Yao C, Akamatsu T, Hosoi K. Am J Physiol Gastrointest Liver Physiol 295:G112-G123, 2008). The present study indicated that despite elevation of bax, a proapoptosis protein, by CTD, the operation also increased the level of bcl-2, an antiapoptosis protein, in the SMG. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL assay) showed no increase in the number of apoptotic cells in the SMG. CTD, however, induced strongly and transiently (at 1-3 days) the protein expression of LC3B-II, a marker protein of autophagosomes, suggesting that the reduction in the gland weight was due to onset of autophagy by CTD. Upon CTD, Lamp2, a lysosomal marker, gradually increased in amount, reaching a peak at the 14th day. Immunohistochemical analysis revealed an increase in the number of lysosome-like structures positive for both AQP5 and Lamp2 in the acinar cells of the SMG after CTD; similar changes were observed also for AQP5 and LC3Bs. These data suggest that AQP5 in the SMG entered autophagosomes and/or lysosomes for degradation upon CTD. In vitro AQP5-degrading activity was found in the SMG extracts, and such activity was shown to be increased by CTD. Inhibitor experiments implied cathepsins B and L to be candidate enzymes for this degradation under normal and CTD conditions, respectively. protein degradation; autophagy; lysosomal proteolysis; LC3B; apoptosis doi: 10.1152/ajpgi.00194.2010.

Renin, an aspartyl protease that catalyzes the rate-limiting step in the renin-angiotensin system (RAS), is proteolytically activated by a second protease [referred to as the prorenin processing enzyme (PPE)] before its secretion from the juxtaglomerular cells of the kidney. Although several enzymes are capable of activating renin in vitro, the leading candidate for the PPE in the kidney is cathepsin B (CTSB) due to is colocalization with the renin precursor (prorenin) in juxtaglomerular cell granules and because of its site-selective activation of human prorenin both in vitro and in transfected tissue culture cell models. To verify the role of CTSB in prorenin processing in vivo, we tested the ability of CTSB-deficient (CTSB-/-) mice to generate active renin. CTSB-/- mice do not exhibit any overt symptoms (renal malformation, preweaning mortality) typical of an RAS deficiency and have normal levels of circulating active renin, which, like those in control animals, rise more than 15-fold in response to pharmacologic inhibition of the RAS. The mature renin enzyme detected in kidney lysates of CTSB-/- mice migrates at the same apparent molecular weight as that in control mice, and the processing to active renin is not affected by chloroquine treatment of the animals. Finally, the distribution and morphology of renin-producing cells in the kidney is normal in CTSB-/- mice. In conclusion, CTSB-deficient mice exhibit no differences compared with controls in their ability to generate active renin, and our results do not support CTSB as the PPE in mice. blood pressure; renin-angiotensin system; renin; enzyme precursor; mouse knock out doi: 10.1152/ajpregu.00830.2009.

Previous studies have shown that the protozoan parasite Blastocystis exhibits apoptotic features with caspase-like activity upon exposure to a cytotoxic monoclonal antibody or the anti-parasitic drug metronidazole. The present study reports that staurosporine (STS), a common apoptosis inducer in mammalian cells, also induces cytoplasmic and nuclear features of apoptosis in Blastocystis, including cell shrinkage, phosphatidylserine (PS) externalization, maintenance of plasma membrane integrity, extensive cytoplasmic vacuolation, nuclear condensation and DNA fragmentation. STS-induced PS exposure and DNA fragmentation were abolished by the mitochondrial transition pore blocker cyclosporine A and significantly inhibited by the broad-range cysteine protease inhibitor iodoacetamide. Interestingly, the apoptosis phenotype was insensitive to inhibitors of caspases and cathepsins B and L, while calpain-specific inhibitors augmented the STS-induced apoptosis response. While the identities of the proteases responsible for STS-induced apoptosis warrant further investigation, these findings demonstrate that programmed cell death in Blastocystis is complex and regulated by multiple mediators. DOI 10.1099/mic.0.034025-0

The X-linked disorder Lowe syndrome arises from mutations in OCRLl, a lipid phosphatase that hydrolyzes phosphatidylinositol 4,5-bisphosphate ([PIP.sub.2]). Most patients with Lowe syndrome develop proteinuria very early in life. PIP2 dynamics are known to modulate numerous steps in membrane trafficking, and it has been proposed that OCRL1 activity regulates the biogenesis or trafficking of the multiligand receptor megalin. To examine this possibility, we investigated the effects of siRNA-mediated OCRL1 knockdown on biosynthetic and postendocytic membrane traffic in canine and human renal epithelial cells. Cells depleted of OCRL1 did not have significantly elevated levels of cellular PIP2 but displayed an increase in actin comets, as previously observed in cultured cells derived from Lowe patients. Using assays to independently quantitate the endocytic trafficking of megalin and of megalin ligands, we could observe no defect in the trafficking or function of megalin upon OCRL1 knockdown. Moreover, apical delivery of a newly synthesized marker protein was unaffected. OCRL1 knockdown did result in a significant increase in secretion of the lysosomal hydrolase cathepsin D, consistent with a role for OCRL1 in membrane trafficking between the trans-Golgi network and endosomes. Together, our studies suggest that OCRL1 does not directly modulate endocytosis or postendocytic membrane traffic and that the renal manifestations observed in Lowe syndrome patients are downstream consequences of the loss of OCRL1 function. proximal tubule; Lowe syndrome; phosphatidylinositol; megalin; CLC-5; endocytosis; cathepsin D doi: 10.1152/ajprenal.00453.2009

Cathepsin L is a lysosomal enzyme thought to play a key role in malignant transformation. Recent work from our laboratory has demonstrated that this enzyme may also regulate cancer cell resistance to chemotherapy. The present study was undertaken to define the relevance of targeting cathepsin L in the suppression of drug resistance in vitro and in vivo and also to understand the mechanism(s) of its action. In vitro experiments indicated that cancer cell adaptation to increased amounts of doxorubicin over time was prevented in the presence of a cathepsin L inhibitor, suggesting that inhibition of this enzyme not only reverses but also prevents the development of drug resistance. The combination of the cathepsin L inhibitor with doxorubicin also strongly suppressed the proliferation of drug-resistant tumors in nude mice. An investigation of the underlying mechanism(s) led to the finding that the active form of this enzyme shuttles between the cytoplasm and nucleus. As a result, its inhibition stabilizes and enhances the availability of cytoplasmic and nuclear protein drug targets including estrogen receptor-[alpha], Bcr-Abl, topoisomerase-II[alpha], histone deacetylase 1, and the androgen receptor. In support of this, the cellular response to doxorubicin, tamoxifen, imatinib, trichostatin A, and flutamide increased in the presence of the cathepsin L inhibitor. Together, these findings provided evidence for the potential role of cathepsin L as a target to suppress cancer resistance to chemotherapy and uncovered a novel mechanism by which protease inhibition-mediated drug target stabilization may enhance cellular visibility and, thus, susceptibility to anticancer agents. drug resistance; topoisomerase; histone deacetylase 1; estrogen receptor

Cell-death programs executed in the pancreas under pathological conditions remain largely undetermined, although the severity of experimental pancreatitis has been found to depend on the ratio of apoptosis to necrosis. We have defined mechanisms by which apoptosis is induced in pancreatic acinar cells by the oxidant stressor menadione. Real-time monitoring of initiator caspase activity showed that caspase-9 (66% of cells) and caspase-8 (15% of cells) were activated within 30 min of menadione administration, but no activation of caspase-2, -10, or -12 was detected. Interestingly, when caspase-9 activation was inhibited, activation of caspase-8 was increased. Half-maximum activation ([t.sub.0.5]) of caspase-9 occurred within ~2 min and was identified at or in close proximity to mitochondria, whereas [t.sub.0.5] for caspase-8 occurred within ~26 min of menadione application and was distributed homogeneously throughout cells. Caspase-9 but not caspase-8 activation was blocked completely by the calcium chelator BAPTA or bongkrekic acid, an inhibitor of the mitochondrial permeability transition pore. In contrast, caspase-8 but not caspase-9 activation was blocked by the destruction of lysosomes (preincubation with Gly-Phe [beta]-naphthylamide, a cathepsin C substrate), loss of lysosomal acidity (bafilomycin A1), or inhibition of cathepsin L or D. Using pepstatin A-BODIPY FL conjugate, we confirmed translocation of cathepsin D out of lysosomes in response to menadione. We conclude that the oxidative stressor menadione induces two independent apoptotic pathways within pancreatic acinar cells: the classical mitochondrial calcium-dependent pathway that is initiated rapidly in the majority of cells, and a slower, caspase-8-mediated pathway that depends on the lysosomal activities of cathepsins and is used when the caspase-9 pathway is disabled. pancreas; acinar cells; menadione doi:10.1152/ajpgi.00103.2007

We have hypothesized that the colocalization of digestive zymogens with lysosomal hydrolases, which occurs during the early stages of every experimental pancreatitis model, facilitates activation of those zymogens by lysosomal hydrolases such as cathepsin B and that this activation triggers acute pancreatitis by leading to acinar cell injury. Some, however, have argued that the colocalization phenomenon may be the result, rather than the cause, of zymogen activation during pancreatitis. To resolve this controversy and explore the causal relationships between zymogen activation and other early pancreatitis events, we induced pancreatitis in mice by repeated supramaximal secretagogue stimulation with caerulein. Some animals were pretreated with the cathepsin B inhibitor CA-074me to inhibit cathepsin B, prevent intrapancreatic activation of digestive zymogens, and reduce the severity of pancreatitis. We show that inhibition of cathepsin B by pretreatment with CA-074me prevents intrapancreatic zymogen activation and reduces organellar fragility, but it does not alter the caerulein-induced colocalization phenomenon or subcellular F-actin redistribution or prevent caerulein-induced activation of NF-[kappa]B, ERK1/2, and JNK or upregulated expression of cytochemokines. We conclude 1) that the colocalization phenomenon, F-actin redistribution, activation of proinflammatory transcription factors, and upregulated expression of cytochemokines are not the results of zymogen activation, and 2) that these early events in pancreatitis are not dependent on cathepsin B activity. In contrast, zymogen activation and increased subcellular organellar fragility during caerulein-induced pancreatitis are dependent on cathepsin B activity. trypsinogen activation; cathepsin B; colocalization; lysosomes doi:10.1152/ajpgi.00543.2006

Sensitivity to TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis and the lysosomal pathway of cell death are features of cancer cells. However, it is unknown if TRAIL cytotoxic signaling engages the lysosomal pathway of cell death. Our aim, therefore, was to ascertain if TRAIL killing involves lysosomal permeabilization. TRAIL-induced apoptosis of hepatocellular carcinoma cells (HuH-7, Hep3B) was associated with lysosomal permeabilization, as demonstrated by redistribution of the lysosomal protease cathepsin B into the cytosol. Pharmacological and short hairpin RNA-targeted inhibition of cathepsin B reduced apoptosis. Because cellular FLICE-inhibitory protein (cFLIP) inhibits TRAIL-induced cell death and is frequently overexpressed by human cancers, the ability of cFLIP to prevent lysosomal permeabilization during TRAIL treatment was examined. Enforced long-form cFLIP (cFLI[P.sub.L]) expression reduced release of cathepsin B from lysosomes and attenuated apoptosis, cFLI[P.sub.L] overexpression was also associated with robust p42/44 MAPK activation following exposure to TRAIL. In contrast, cFLI[P.sub.L] overexpression attenuated p38 MAPK activation and had no significant effect on JNK and NF-[kappa]B activation. Inhibition of p42/44 MAPK by PD98059 restored TRAIL-mediated lysosomal permeabilization and apoptosis in cFLIP-overexpressing cells. In conclusion, these results demonstrate that lysosomal permeabilization contributes to TRAIL-induced apoptosis of hepatocellular carcinoma cells and suggest that cFLI[P.sub.L] cytoprotection is, in part, due to p42/44 MAPK-dependent inhibition of lysosomal breakdown. caspase-8; cathepsin B; PD98059; p42/44 mitogen-activated protein kinase doi:10.1152/ajpgi.00497.2006.

Lysosomal permeabilization is a key feature of hepatocyte lipotoxicity, yet the mechanisms mediating this critical cellular event are unclear. This study examined the mechanisms involved in free fatty acid (FFA)-induced lysosomal permeabilization and the role of Bax, a Bc1-2 family member, in this event. Exposure of liver cells to palmitate induced Bax activation and translocation to lysosomes. Studies to suppress Bax activation either by pharmacological approaches or small interfering-RNA-mediated inhibition of Bax expression showed that lysosomal permeabilization is Bax dependent. In addition, palmitate treatment resulted in a significant decrease in Bc1-[X.sub.L], a Bax antagonist. Moreover, forced Bc1-[X.sub.L] expression blocked lysosomal permeabilization. Lysosomal permeabilization by FFA was ceramide and caspase independent. Finally, paradigms that inhibit lysosomal permeabilization also reduced apoptosis. In conclusion, these data strongly support a regulatory role for Bax in FFA-mediated lysosomal permeabilization and subsequent cell death. nonalcoholic fatty liver disease; lysosomes; Bax; Bc1-2 family: cathepsin B

Role of nuclear factor of activated T-cells in heart valve leaflet morphogenesis and endocardial expression of receptor activator of NF?B ligand is presented.

A comparative genomic study was conducted using Caenorhabditis elegans as a model organism to properly understand the biology of the member of the serpin family. Analysis of SRP-3 inhibitory activity indicated that SRP-3 was a potent inhibitor of the serine peptidases, chymotrypsin and cathepsin G.

Latency and reactivation are a significant problem that contributes to the incidence, transmission and pathogenesis of tuberculosis. The mechanisms involved in these processes, at the level of both the bacillus and the host, are poorly understood. In Mycobacterium tuberculosis the [alpha]-crystallin (acr) gene has been linked to latency, because it is highly expressed during hypoxic growth conditions. Deletion of the acr gene in M. tuberculosis H37Rv ([Delta]acr strain) was previously shown to reduce the intracellular growth of bacilli in macrophages; however, its impact on pathogenesis in vivo was unknown. This study demonstrated that infection of C57BL6 mice with [Delta]acr results in lung bacillary loads 1-2 log units higher in comparison to parental H37Rv. Haematoxylin/eosin staining of lungs revealed exacerbated pathology characterized by extensive obliteration of alveolar air spaces by granulomatous inflammation. RT-PCR analysis and immunostaining of lungs showed that infection with either H37Rv or [Delta]acr results in the differential expression of lysosomal cathepsin proteases. A slight increase in the expression of the matrix-degrading acidic-type cathepsins B, D and H was noted in [Delta]acr-infected mice and was associated with clusters of macrophages within lung granulomas. [Delta]acr-infected mice also showed high serum levels of TNF-[alpha], IFN-[gamma] and G-CSF, suggesting that Acr may play a role in modulating the host response to infection.

Research has been conducted on the cathepsin V, a lysosomal cysteine protease expressed in the thymus. The crystal structure of this cathepsin which is associated with the irreversible vinyl sulfone inhibitor has been investigated.

Research has been conducted on the myelin basic protein (MBP) deimination. The degradation of MBP by cathepsin D has been investigated.

Research has been conducted on the arginyl residues in myelin basic protein (MBP). The deimination effect of these residues on the MBP's digestion susceptibility by cathepsin D has been investigated.

Research was conducted to examine the inhibition of cathepsin G by native and oxidized mucus proteinase inhibitor (MPI) in the absence and presence of heparin. The stopped-flow kinetic investigation reveals that, within the inhibitor concentration range used, the mechanism of inhibition of cathepsin G an chymotrypsin by oxidized MPI is consistent with a one-step reaction while the inhibition of elastase takes place in two steps. Results demonstrate that oxidation of MPI during chronic bronchitis may result in cathepsin G-mediated lung tissue degradation.

Research was conducted to compare the susceptibilities to degradation by cathepsin D of myelin basic protein (MBP) Cit6 and MBP-C1, which are both from normal and multiple sclerosis brain tissue, and Marburg MBP Cit18. The rapidity of digestion of MBP Cit6 correlates with the amount of citrulline present in the molecule, whereby the greater the amount of citrulline, the more rapid the digestion. Results suggest that the generation of MBP peptides is directly related to deimination of arginine to citrulline in MBP.

Research was conducted to identify UDP-N-acetylglucosamine-phosphotransferase-binding sites on the lysosomal proteases, cathepsins A, B, and D. Four structurally conserved surface patches were found in the nonrelated lysosomal proteases, cathepsins A, B and D. Results suggest that cathepsin A and cathepsin D possess one closely related phosphotransferase recognition site represented by a topologically and structurally conserved beta-hairpin loop.

Stable BHK cell lines expressing wild-type or dominant negative mutant forms of the GTPase, rab7, have been isolated, to examined the role of a rab7-regulated pathway in lyosome biogenesis. Cation-independent mannose 6-phosphate receptor (CI-MPR) was dramatically redistributed on expression of mutant rab7N1251 protein. Newly synthesized CI-MPR and cathepsin D travelled through an early endocytic compartment, and functional rab7 was found to be essential for delivery to later compartments.

The major late promoter element (MLPE) was characterized as a second cathepsin D gene E2-reponsive region. Results identify an imperfect palindromic ERE (CGCCCACGTACC) DNA sequence that binds the ER to make a retarded band in an electrophoretic mobility gel shift assay. Different biological and physiological processes was regulated by steroid hormone receptors by modulating tissue-specific expression of target genes.

Cathepsin D has fibrinolytic and fibrinogenolytic properties with a perfect pH profile for lysosomal action. The properties of cathepsin D include pepstatin A inactivation, urea denaturation resistance and acid pH dependence of enzyme activity. The existence of plasmin-independent fibrinolytic pathway in activated human monocytoid cells reveals the importance of cathepsin D in pathophysiology and thrombic disorder treatment.

Analyses of RNA extracted from MCF-7 cells is used to study the effect of structural changes to the estradiol-17 beta (E2) molecule on the indirection of pS2 and Cathepsin D (Cath D) mRNAs. The level of expression of these estrogen-sensitive genes is unaffected by the exposure of cultures to estratriene. The ligands (2- or 4-hydroxyestratrien-17 beta-ol) which have affinities for the estrogen receptor that are unrelated to their ability to control some responsive genes (pS2 or Cath D) are generated by the discrete alterations in the structure of estradiol. The changes in the receptor-analogue complex are responsible for the discrimination between these two responsive genes.

The kinetics of inactivation of human cathepsin L was studied. The inactivation rate was determined as a function of pH and temperature and compared with the rate constant of cystatin-induced enzyme inactivation. Cathepsin L is an unstable lysosomal cysteine proteinase at neutral or alkaline pH. The results indicate that the inactivation is a first-order process and is inversely related to substrate concentration.

The purification of tachykinin and enkephalin precursor cleaving enzymes from bovine chromaffin granules was reported. Proteolytic activity was assayed by cleavage of tachykinin and enkephalin precursors in the form of 35S-(Met)-beta-PPT and 35S-(Met)-PPE, respectively. Purification resulted in the isolation of a chromaffin granule aspartyl protease (CGAP) that resembles cathepsin D and cleaves the tachykinin but not the enkephalin precursor. The CGAP was then characterized with respect to pH optimum, cleavage sites, NH2-terminal microsequencing and amino acid composition.

A study was conducted on procathepsin Dusing a nonglycosylated human fibroblast procathepsin D form expressed in Escherichia coli. Sequence analysis performed to determine its autoproteolytic cleavage site revealed a similarity between the sequence enveloping the cleavage site between the LeuP26 and IleP27 residues and the cleavage site observed when other aspartyl proteases are activated. Pseudocathepsin D and mature cathepsin D both had the same Km toward peptide substrates and cleaved them at the same sites.