Cathepsin B deficiency ameliorates liver lipid deposition, inflammatory cell infiltration, and fibrosis after diet-induced nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is a severe form of nonalcoholic fatty liver disease characterized by fat accumulation and inflammation in liver. Yet, the mechanistic insight and diagnostic and therapeutic options of NASH remain incompletely understood. This study tested the roles of cysteine protease cathepsin B (CatB) in mouse NASH development. Immunoblot revealed increased liver CatB expression in NASH mice. Fructose-palmitate-cholesterol diet increased body weight gain, liver to body weight ratio, blood fasting glucose, plasma total cholesterol and alanine transaminase levels, and liver triglyceride, but decreased plasma high-density lipoprotein in wild-type mice. All these changes were blunted in CatB-deficient (Ctsb-/-) mice. In parallel to reduced expression of genes involved in liver lipid transport and lipogenesis, liver CD36, FABP4, and PPARγ protein levels were also significantly decreased in Ctsb-/- mice, although CatB deficiency did not affect liver gluconeogenesis and fatty acid beta-oxidation-associated gene expression. Mechanistic studies showed that CatB deficiency decreased liver expression of adhesion molecules, inflammatory cytokine, and chemokine, along with reduced liver inflammatory cell infiltration. CatB deficiency also promoted M2 macrophage polarization and reduced liver TGF-ß1 signaling and fibrosis. Together, CatB deficiency improves liver function in NASH mice by suppressing de novo lipogenesis and liver inflammation and fibrosis.

Cathepsin B inhibition blocks neurite outgrowth in cultured neurons by regulating lysosomal trafficking and remodeling.

Lysosomes are known to mediate neurite outgrowth in neurons. However, the principal lysosomal molecule controlling that outgrowth is unclear. We studied primary mouse neurons in vitro and found that they naturally develop neurite outgrowths over time and as they did so the lysosomal cysteine protease cathepsin B (CTSB) mRNA levels dramatically increased. Surprisingly, we found that treating those neurons with CA-074Me, which inhibits CTSB, prevented neurites. As that compound also inhibits another protease, we evaluated a N2a neuronal cell line in which the CTSB gene was deleted (CTSB knockout, KO) using CRISPR technology and induced their neurite outgrowth by treatment with retinoic acid. We found that CTSB KO N2a cells failed to produce neurite outgrowths but the wild-type (WT) did. CA-074Me is a cell permeable prodrug of CA-074, which is cell impermeable and a specific CTSB inhibitor. Neurite outgrowth was and was not suppressed in WT N2a cells treated with CA-074Me and CA-074, respectively. Lysosome-associated membrane glycoprotein 2-positive lysosomes traffic to the plasma cell membrane in WT but not in CTSB KO N2 a cells. Interestingly, no obvious differences between WT and CTSB KO N2a cells were found in neurite outgrowth regulatory proteins, PI3K/AKT, ERK/MAPK, cJUN, and CREB. These findings show that intracellular CTSB controls neurite outgrowth and that it does so through regulation of lysosomal trafficking and remodeling in neurons. This adds valuable information regarding the physiological function of CTSB in neural development.

Cathepsin B-Mediated Activation of Trypsinogen in Endocytosing Macrophages Increases Severity of Pancreatitis in Mice.

BACKGROUND & AIMS: Acute pancreatitis is characterized by premature intracellular activation of digestive proteases within pancreatic acini and a consecutive systemic inflammatory response. We investigated how these processes interact during severe pancreatitis in mice. METHODS: Pancreatitis was induced in C57Bl/6 wild-type (control), cathepsin B (CTSB)-knockout, and cathepsin L-knockout mice by partial pancreatic duct ligation with supramaximal caerulein injection, or by repetitive supramaximal caerulein injections alone. Immune cells that infiltrated the pancreas were characterized by immunofluorescence detection of Ly6g, CD206, and CD68. Macrophages were isolated from bone marrow and incubated with bovine trypsinogen or isolated acinar cells; the macrophages were then transferred into pancreatitis control or cathepsin-knockout mice. Activities of proteases and nuclear factor (NF)-κB were determined using fluorogenic substrates and trypsin activity was blocked by nafamostat. Cytokine levels were measured using a cytometric bead array. We performed immunohistochemical analyses to detect trypsinogen, CD206, and CD68 in human chronic pancreatitis (n = 13) and acute necrotizing pancreatitis (n = 15) specimens. RESULTS: Macrophages were the predominant immune cell population that migrated into the pancreas during induction of pancreatitis in control mice. CD68-positive macrophages were found to phagocytose acinar cell components, including zymogen-containing vesicles, in pancreata from mice with pancreatitis, as well as human necrotic pancreatic tissues. Trypsinogen became activated in macrophages cultured with purified trypsinogen or co-cultured with pancreatic acini and in pancreata of mice with pancreatitis; trypsinogen activation required macrophage endocytosis and expression and activity of CTSB, and was sensitive to pH. Activation of trypsinogen in macrophages resulted in translocation of NF-kB and production of inflammatory cytokines; mice without trypsinogen activation (CTSB-knockout mice) in macrophages developed less severe pancreatitis compared with control mice. Transfer of macrophage from control mice to CTSB-knockout mice increased the severity of pancreatitis. Inhibition of trypsin activity in macrophages prevented translocation of NF-κB and production of inflammatory cytokines. CONCLUSIONS: Studying pancreatitis in mice, we found activation of digestive proteases to occur not only in acinar cells but also in macrophages that infiltrate pancreatic tissue. Activation of the proteases in macrophage occurs during endocytosis of zymogen-containing vesicles, and depends on pH and CTSB. This process involves macrophage activation via NF-κB-translocation, and contributes to systemic inflammation and severity of pancreatitis.

Cathepsin B-mediated CD18 shedding regulates leukocyte recruitment from angiogenic vessels.

Cathepsin B (CtsB) contributes to atherosclerosis and cancer progression by processing the extracellular matrix and promoting angiogenesis. Although CtsB was reported to promote and reduce angiogenesis, there is no mechanistic explanation that reconciles this apparent discrepancy. CtsB cleaves CD18 from the surface of immune cells, but its contribution to angiogenesis has not been studied. We developed an in vivo technique for visualization of immune cell transmigration from corneal vessels toward implanted cytokines. Wild-type (WT) leukocytes extravasated from limbal vessels, angiogenic stalks, and growing tip vessels and migrated toward the cytokines, indicating immune competence of angiogenic vessels. Compared to WT leukocytes, CtsB-/- leukocytes accumulated in a higher number in angiogenic vessels, but extravasated less toward the implanted cytokine. The accumulated CtsB-/- leukocytes in angiogenic vessels expressed more CD18. CD18-/- leukocytes extravasated later than WT leukocytes. However, once extravasated, CD18-/- leukocytes transmigrated more rapidly than their WT counterparts. These results suggest that, although CD18 facilitates efficient extravasation, outside of the vessel CD18 interaction with the extracellular matrix, it reduced transmigration velocity. Our results reveal an unexpected role for CtsB in leukocyte extravasation and transmigration, which advances our understanding of the complex contribution of CtsB to angiogenesis.-Nakao, S., Zandi, S., Sun, D., Hafezi-Moghadam, A. Cathepsin B-mediated CD18 shedding regulates leukocyte recruitment from angiogenic vessels.

Cathepsin B-Deficient Mice Resolve Leishmania major Inflammation Faster in a T Cell-Dependent Manner.

A critical role for intracellular TLR9 has been described in recognition and host resistance to Leishmania parasites. As TLR9 requires endolysosomal proteolytic cleavage to achieve signaling functionality, we investigated the contribution of different proteases like asparagine endopeptidase (AEP) or cysteine protease cathepsins B (CatB), L (CatL) and S (CatS) to host resistance during Leishmania major (L. major) infection in C57BL/6 (WT) mice and whether they would impact on TLR9 signaling. Unlike TLR9-/-, which are more susceptible to infection, AEP-/-, CatL-/- and CatS-/- mice are as resistant to L. major infection as WT mice, suggesting that these proteases are not individually involved in TLR9 processing. Interestingly, we observed that CatB-/- mice resolve L. major lesions significantly faster than WT mice, however we did not find evidence for an involvement of CatB on either TLR9-dependent or independent cytokine responses of dendritic cells and macrophages or in the innate immune response to L. major infection. We also found no difference in antigen presenting capacity. We observed a more precocious development of T helper 1 responses accompanied by a faster decline of inflammation, resulting in resolution of footpad inflammation, reduced IFNγ levels and decreased parasite burden. Adoptive transfer experiments into alymphoid RAG2-/-γc-/- mice allowed us to identify CD3+ T cells as responsible for the immune advantage of CatB-/- mice towards L. major. In vitro data confirmed the T cell intrinsic differences between CatB-/- mice and WT. Our study brings forth a yet unappreciated role for CatB in regulating T cell responses during L. major infection.

Cathepsin B deficiency attenuates cardiac remodeling in response to pressure overload via TNF-α/ASK1/JNK pathway.

Cathepsin B (CTSB), a member of the lysosomal cathepsin family that is expressed in both murine and human hearts, was previously shown to participate in apoptosis, autophagy, and the progression of certain types of cancers. Recently, CTSB has been linked to myocardial infarction. Given that cathepsin L, another member of the lysosomal cathepsin family, ameliorates pathological cardiac hypertrophy, we hypothesized that CTSB plays a role in pressure overload-induced cardiac remodeling. Here we report that CTSB was upregulated in cardiomyocytes in response to hypertrophic stimuli both in vivo and in vitro. Moreover, knockout of CTSB attenuated pressure overload-induced cardiac hypertrophy, fibrosis, dysfunction, and apoptosis. Furthermore, the aortic banding-induced activation of TNF-α, apoptosis signal-regulating kinase 1 (ASK1), c-Jun NH2-terminal kinases (JNK), c-Jun, and release of cytochrome c was blunted by CTSB deficiency, which was further confirmed in in vitro studies induced by angiotensin II. In cardiomyocytes pretreatment with SP600125, a JNK inhibitor, suppressed the cardiomyocytes hypertrophy by inhibiting the ASK1/JNK pathway. Altogether, these data indicate that the CTSB protein functions as a necessary modulator of hypertrophic response by regulating TNF-α/ASK1/JNK signaling pathway involved in cardiac remodeling.

Cathepsin B-deficient mice as source of monoclonal anti-cathepsin B antibodies.

Cathepsin B has been demonstrated to be involved in several proteolytic processes that support tumor progression and metastasis and neurodegeneration. To further clarify its role, defined monoclonal antibodies are needed. As the primary structure of human cathepsin B is almost identical to that of the mouse, cathepsin B-deficient mice were used in a novel approach for generating such antibodies, providing the chance of an increased immune response to the antigen, human cathepsin B. Thirty clones were found to produce cathepsin B-specific antibodies. Seven of these antibodies were used to detect cathepsin B in MCF10-DCIS human breast cancer cells by immunocytochemistry and immunoblotting. Five different binding sites were identified by epitope mapping giving the opportunity to combine these antibodies in oligoclonal antibody mixtures for an improved detection of cathepsin B.

CA-074Me compound inhibits osteoclastogenesis via suppression of the NFATc1 and c-FOS signaling pathways.

The osteoclast is an integral cell of bone resorption. Since osteolytic disorders hinge on the function and dysfunction of the osteoclast, understanding osteoclast biology is fundamental to designing new therapies that curb osteolytic disorders. The identification and study of lysosomal proteases, such as cathepsins, have shed light on mechanisms of bone resorption. For example, Cathepsin K has already been identified as a collagen degradation protease produced by mature osteoclasts with high activity in the acidic osteoclast resorption pits. Delving into the mechanisms of cathepsins and other osteoclast related compounds provides new targets to explore in osteoclast biology. Through our anti-osteoclastogenic compound screening experiments we encountered a modified version of the Cathepsin B inhibitor CA-074: the cell membrane-permeable CA-074Me (L-3-trans-(Propylcarbamoyl) oxirane-2-carbonyl]-L-isoleucyl-L-proline Methyl Ester). Here we confirm that CA-074Me inhibits osteoclastogenesis in vivo and in vitro in a dose-dependent manner. However, Cathepsin B knockout mice exhibited unaltered osteoclastogenesis, suggesting a more complicated mechanism of action than Cathepsin B inhibition. We found that CA-074Me exerts its osteoclastogenic effect within 24 h of osteoclastogenesis stimulation by suppression of c-FOS and NFATc1 pathways.

Cathepsin B in antigen-presenting cells controls mediators of the Th1 immune response during Leishmania major infection.

Resistance and susceptibility to Leishmania major infection in the murine model is determined by the capacity of the host to mount either a protective Th1 response or a Th2 response associated with disease progression. Previous reports involving the use of cysteine cathepsin inhibitors indicated that cathepsins B (Ctsb) and L (Ctsl) play important roles in Th1/Th2 polarization during L. major infection in both susceptible and resistant mouse strains. Although it was hypothesized that these effects are a consequence of differential patterns of antigen processing, the mechanisms underlying these differences were not further investigated. Given the pivotal roles that dendritic cells and macrophages play during Leishmania infection, we generated bone-marrow derived dendritic cells (BMDC) and macrophages (BMM) from Ctsb-/- and Ctsl-/- mice, and studied the effects of Ctsb and Ctsl deficiency on the survival of L. major in infected cells. Furthermore, the signals used by dendritic cells to instruct Th cell polarization were addressed: the expression of MHC class II and co-stimulatory molecules, and cytokine production. We found that Ctsb-/- BMDC express higher levels of MHC class II molecules than wild-type (WT) and Ctsl-/- BMDC, while there were no significant differences in the expression of co-stimulatory molecules between cathepsin-deficient and WT cells. Moreover, both BMDC and BMM from Ctsb-/- mice significantly up-regulated the levels of interleukin 12 (IL-12) expression, a key Th1-inducing cytokine. These findings indicate that Ctsb-/- BMDC display more pro-Th1 properties than their WT and Ctsl-/- counterparts, and therefore suggest that Ctsb down-regulates the Th1 response to L. major. Moreover, they propose a novel role for Ctsb as a regulator of cytokine expression.

Double deficiency of cathepsins B and L results in massive secretome alterations and suggests a degradative cathepsin-MMP axis.

Endolysosomal cysteine cathepsins functionally cooperate. Cathepsin B (Ctsb) and L (Ctsl) double-knockout mice die 4 weeks after birth accompanied by (autophago-) lysosomal accumulations within neurons. Such accumulations are also observed in mouse embryonic fibroblasts (MEFs) deficient for Ctsb and Ctsl. Previous studies showed a strong impact of Ctsl on the MEF secretome. Here we show that Ctsb alone has only a mild influence on extracellular proteome composition. Protease cleavage sites dependent on Ctsb were identified by terminal amine isotopic labeling of substrates (TAILS), revealing a prominent yet mostly indirect impact on the extracellular proteolytic cleavages. To investigate the cooperation of Ctsb and Ctsl, we performed a quantitative secretome comparison of wild-type MEFs and Ctsb (-/-) Ctsl (-/-) MEFs. Deletion of both cathepsins led to drastic alterations in secretome composition, highlighting cooperative functionality. While many protein levels were decreased, immunodetection corroborated increased levels of matrix metalloproteinase (MMP)-2. Re-expression of Ctsl rescues MMP-2 abundance. Ctsl and to a much lesser extent Ctsb are able to degrade MMP-2 at acidic and neutral pH. Addition of active MMP-2 to the MEF secretome degrades proteins whose levels were also decreased by Ctsb and Ctsl double deficiency. These results suggest a degradative Ctsl-MMP-2 axis, resulting in increased MMP-2 levels upon cathepsin deficiency with subsequent degradation of secreted proteins such as collagen α-1 (I).

TNFα-induced lysosomal membrane permeability is downstream of MOMP and triggered by caspase-mediated NDUFS1 cleavage and ROS formation.

When NF-κB activation or protein synthesis is inhibited, tumor necrosis factor alpha (TNFα) can induce apoptosis through Bax- and Bak-mediated mitochondrial outer membrane permeabilization (MOMP) leading to caspase-3 activation. Additionally, previous studies have implicated lysosomal membrane permeability (LMP) and formation of reactive oxygen species (ROS) as early steps of TNFα-induced apoptosis. However, how these two events connect to MOMP and caspase-3 activation has been largely debated. Here, we present the novel finding that LMP induced by the addition of TNFα plus cycloheximide (CHX), the release of lysosomal cathepsins and ROS formation do not occur upstream but downstream of MOMP and require the caspase-3-mediated cleavage of the p75 NDUFS1 subunit of respiratory complex I. Both a caspase non-cleavable p75 mutant and the mitochondrially localized antioxidant MitoQ prevent LMP mediated by TNFα plus CHX and partially interfere with apoptosis induction. Moreover, LMP is completely blocked in cells deficient in both Bax and Bak, Apaf-1, caspase-9 or both caspase-3 and -7. Thus, after MOMP, active caspase-3 exerts a feedback action on complex I to produce ROS. ROS then provoke LMP, cathepsin release and further caspase activation to amplify TNFα apoptosis signaling.

Silencing of Cathepsin B suppresses the proliferation and invasion of endometrial cancer.

The molecular mechanism involved in the metastasis of endometrial cancer (EC) remains unclear. The lysosomal cysteine protease Cathepsin B has been implicated in the progression of various human tumors. In the present study, we assessed the expression of Cathepsin B and its functions in EC. Immunohistochemistry was used to examine Cathepsin B expression in 76 paraffin-embedded endometrial tumor tissues. Lentiviral packing short hairpin RNA (shRNA) was transfected into HEC-1A cells to build a stable Cathepsin B knockdown cell line. The cellular levels of Cathepsin B mRNA and protein were detected by real-time PCR and western immunoblotting. The functions of Cathepsin B in EC cells were measured by MTT, migration and invasion assays. In additon, tumorigenicity assays were established in nude mice to study tumor growth in vivo. The results of our study showed that Cathepsin B was overexpressed in EC tissues compared with normal endometrium and endometrial atypical hyperplasia. Depletion of Cathepsin B in vitro inhibited cell proliferation, migration and invasion. Tumor formation assays confirmed that suppression of Cathepsin B inhibited the proliferation potential of HEC-1A cells in vivo, demonstrated by lower proliferation rates. These results suggest that Cathepsin B may act as an oncogene in EC, with the potential to provide a new therapeutic target for treating endometrial malignancy.

Erythropoietin-directed erythropoiesis depends on serpin inhibition of erythroblast lysosomal cathepsins.

Erythropoietin (EPO) and its cell surface receptor (EPOR) are essential for red blood cell production and exert important cytoprotective effects on select vascular, immune, and cancer cells. To discover novel EPO action modes, we profiled the transcriptome of primary erythroid progenitors. We report Serpina3g/Spi2A as a major new EPO/EPOR target for the survival of erythroid progenitors. In knockout mice, loss of Spi2A worsened anemia caused by hemolysis, radiation, or transplantation. EPO-induced erythropoiesis also was compromised. In particular, maturing erythroblasts required Spi2A for cytoprotection, with iron and reactive oxygen species as cytotoxic agents. Spi2A defects were ameliorated by cathepsin-B/L inhibition, and by genetic co-deletion of lysosomal cathepsin B. Pharmacological inhibition of cathepsin B/L enhanced EPO-induced red cell formation in normal mice. Overall, we define an unexpected EPO action mode via an EPOR-Spi2A serpin-cathepsin axis in maturing erythroblasts, with lysosomal cathepsins as novel therapeutic targets.

Deletion of cysteine cathepsins B or L yields differential impacts on murine skin proteome and degradome.

Numerous studies highlight the fact that concerted proteolysis is essential for skin morphology and function. The cysteine protease cathepsin L (Ctsl) has been implicated in epidermal proliferation and desquamation, as well as in hair cycle regulation. In stark contrast, mice deficient in cathepsin B (Ctsb) do not display an overt skin phenotype. To understand the systematic consequences of deleting Ctsb or Ctsl, we determined the protein abundances of >1300 proteins and proteolytic cleavage events in skin samples of wild-type, Ctsb(-/-), and Ctsl(-/-) mice via mass-spectrometry-based proteomics. Both protease deficiencies revealed distinct quantitative changes in proteome composition. Ctsl(-/-) skin revealed increased levels of the cysteine protease inhibitors cystatin B and cystatin M/E, increased cathepsin D, and an accumulation of the extracellular glycoprotein periostin. Immunohistochemistry located periostin predominantly in the hypodermal connective tissue of Ctsl(-/-) skin. The proteomic identification of proteolytic cleavage sites within skin proteins revealed numerous processing sites that are underrepresented in Ctsl(-/-) or Ctsb(-/-) samples. Notably, few of the affected cleavage sites shared the canonical Ctsl or Ctsb specificity, providing further evidence of a complex proteolytic network in the skin. Novel processing sites in proteins such as dermokine and Notch-1 were detected. Simultaneous analysis of acetylated protein N termini showed prototypical mammalian N-alpha acetylation. These results illustrate an influence of both Ctsb and Ctsl on the murine skin proteome and degradome, with the phenotypic consequences of the absence of either protease differing considerably.

Cathepsin B & L are not required for ebola virus replication.

Ebola virus (EBOV), family Filoviridae, emerged in 1976 on the African continent. Since then it caused several outbreaks of viral hemorrhagic fever in humans with case fatality rates up to 90% and remains a serious Public Health concern and biothreat pathogen. The most pathogenic and best-studied species is Zaire ebolavirus (ZEBOV). EBOV encodes one viral surface glycoprotein (GP), which is essential for replication, a determinant of pathogenicity and an important immunogen. GP mediates viral entry through interaction with cellular surface molecules, which results in the uptake of virus particles via macropinocytosis. Later in this pathway endosomal acidification activates the cysteine proteases Cathepsin B and L (CatB, CatL), which have been shown to cleave ZEBOV-GP leading to subsequent exposure of the putative receptor-binding and fusion domain and productive infection. We studied the effect of CatB and CatL on in vitro and in vivo replication of EBOV. Similar to previous findings, our results show an effect of CatB, but not CatL, on ZEBOV entry into cultured cells. Interestingly, cell entry by other EBOV species (Bundibugyo, Côte d'Ivoire, Reston and Sudan ebolavirus) was independent of CatB or CatL as was EBOV replication in general. To investigate whether CatB and CatL have a role in vivo during infection, we utilized the mouse model for ZEBOV. Wild-type (control), catB(-/-) and catL(-/-) mice were equally susceptible to lethal challenge with mouse-adapted ZEBOV with no difference in virus replication and time to death. In conclusion, our results show that CatB and CatL activity is not required for EBOV replication. Furthermore, EBOV glycoprotein cleavage seems to be mediated by an array of proteases making targeted therapeutic approaches difficult.

Microglial cathepsin B contributes to the initiation of peripheral inflammation-induced chronic pain.

Interleukin (IL)-1ß and IL-18 play critical roles in the induction of chronic pain hypersensitivity. Their inactive forms are activated by caspase-1. However, little is known about the mechanism underlying the activation of pro-caspase-1. There is increasing evidence that cathepsin B (CatB), a typical lysosomal cysteine protease, is involved in the pro-caspase-1 activation and the subsequent maturation of IL-1ß and IL-18. In this context, CatB is considered to be an important molecular target to control chronic pain. However, no information is currently available about the role of CatB in chronic pain hypersensitivity. We herein show that CatB deficiency or the intrathecal administration of CA-074Me, a specific CatB inhibitor, significantly inhibited the induction of complete Freund's adjuvant-induced tactile allodynia in mice without affecting peripheral inflammation. In contrast, CatB deficiency did not affect the nerve injury-induced tactile allodynia. Furthermore, CatB deficiency or CA-074Me treatment significantly inhibited the maturation and secretion of IL-1ß and IL-18 by cultured microglia following treatment with the neuroactive glycoprotein chromogranin A (CGA), but not with ATP. Moreover, the IL-1ß expression in spinal microglia and the induction of tactile allodynia following the intrathecal administration of CGA depended on CatB, whereas those induced by the intrathecal administration of ATP or lysophosphatidic acid were CatB independent. These results strongly suggest that CatB is an essential enzyme for the induction of chronic inflammatory pain through its activation of pro-caspase-1, which subsequently induces the maturation and secretion of IL-1ß and IL-18 by spinal microglia. Therefore, CatB-specific inhibitors may represent a useful new strategy for treating inflammation-associated pain.

Inhibition or deficiency of cathepsin B leads defects in HIV-1 Gag pseudoparticle release in macrophages and HEK293T cells.

Human immunodeficiency virus type 1 (HIV-1) egresses from infected cells through utilizing the host membrane budding mechanisms. Assembly of HIV-1 Gag particles occurs on membranes where the Gag multimers subsequently bud off and form enveloped viral particles. In certain cell types such as macrophages, HIV-1 Gag particles have shown to be released into intracellular virus containing compartments (VCC) such as late endosomes, multivesicular bodies (MVBs) or invaginated plasma membrane pockets. Here, we showed that macrophages or HEK293T cells treated with the cathepsin B (CTSB)-specific inhibitor CA-074Me or cells deficient in CTSB failed to release HIV-1 Gag pseudoparticles into the extracellular environment. Based on immunofluorescence and electron microscopy, these cells retained the pseudoparticles in heterogeneous intracellular VCC. CA-074Me was also able to inhibit propagation of two enveloped viruses, herpes simplex virus and influenza A virus, but not non-enveloped enterovirus. These results suggest that CTSB is required for the efficient release of HIV-1 Gag pseudoparticles and targeting CTSB can be a new therapeutic strategy for inhibiting egress of HIV-1 and other enveloped viruses.

uPAR/cathepsin B overexpression reverse angiogenesis by rescuing FAK phosphorylation in uPAR/cathepsin B down regulated meningioma.

BACKGROUND: Meningiomas are the most commonly occurring intracranial tumors and account for approximately 15-20% of central nervous system tumors. Surgery and radiation therapy is a common treatment for brain tumors, however, patients whose tumors recur after such treatments have limited therapeutic options. Earlier studies have reported important roles of uPA, uPAR and cathepsin B in tumor progression. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we examined the therapeutic significance of RNAi-mediated simultaneous down regulation of these proteolytic networks using two bicistronic siRNA constructs, pUC (uPAR/cathepsin B) and pU2 (uPA/uPAR) either alone or in combination with radiation in two different meningioma cell lines. Transfection of meningioma cells with pUC and pU2 significantly reduced angiogenesis as compared to control treatment both in vitro and in vivo nude mice model. This effect is mediated by inhibiting angiogenic molecules (Ang-1, Ang-2 and VEGF). Expression of focal adhesion kinase (FAK) is elevated in malignant meningioma, yet the role of intrinsic FAK activity in promoting tumor progression remains undefined. We found that pUC treatment reduced FAK phosphorylation at Y925 more efficiently compared to pU2 treatment. In immunoprecipitation assay, we found pronounced reduction of FAK (Y925) interaction with Grb2 in meningioma cells transfected with pUC with and without irradiation. Transient over-expression of uPAR and cathepsin B by full length uPAR/cathepsin B (FLpU/C) in pUC transfected meningioma cells promoted vascular phenotype, rescued expression of Ang-1, Ang-2, VEGF, FAK (Y925) and Grb2 both in vitro and in vivo mice model. CONCLUSION/SIGNIFICANCE: These studies provide the first direct proof that bicistronic siRNA construct for uPAR and cathepsin B (pUC) reduces Y925-FAK activity and this inhibition is rescued by overexpression of both uPAR and cathepsin B which clearly demonstrates that pUC could thus be a potential therapeutic approach as an anti-angiogenic agent in meningioma.

CpG inhibits pro-B cell expansion through a cathepsin B-dependent mechanism.

TLR9 is expressed in cells of the innate immune system, as well as in B lymphocytes and their progenitors. We investigated the effect of the TLR9 ligand CpG DNA on the proliferation of pro-B cells. CpG DNA inhibits the proliferation of pro-B, but not pre-B, cells by inducing caspase-independent cell death through a pathway that requires the expression of cathepsin B. This pathway is operative in Rag-deficient mice carrying an SP6 transgene, in which B lymphopoiesis is compromised, to reduce the size of the B lymphocyte precursor compartments in the bone marrow. Thus, TLR9 signals can regulate B lymphopoiesis in vivo.