Cathepsin S Contributes to Lung Inflammation in Acute Respiratory Distress Syndrome.

Rationale: Although the cysteine protease cathepsin S has been implicated in the pathogenesis of several inflammatory lung diseases, its role has not been examined in the context of acute respiratory distress syndrome, a condition that still lacks specific and effective pharmacological treatments. Objectives: To characterize the status of cathepsin S in acute lung inflammation and examine the role of cathepsin S in disease pathogenesis. Methods: Human and mouse model BAL fluid samples were analyzed for the presence and activity of cathepsin S and its endogenous inhibitors. Recombinant cathepsin S was instilled directly into the lungs of mice. The effects of cathepsin S knockout and pharmacological inhibition were examined in two models of acute lung injury. Protease-activated receptor-1 antagonism was used to test a possible mechanism for cathepsin S-mediated inflammation. Measurements and Main Results: Pulmonary cathepsin S concentrations and activity were elevated in acute respiratory distress syndrome, a phenotype possibly exacerbated by the loss of the endogenous antiprotease cystatin SN. Direct cathepsin S instillation into the lungs induced key pathologies of acute respiratory distress syndrome, including neutrophilia and alveolar leakage. Conversely, in murine models of acute lung injury, genetic knockdown and prophylactic or therapeutic inhibition of cathepsin S reduced neutrophil recruitment and protein leakage. Cathepsin S may partly mediate its pathogenic effects via protease-activated receptor-1, because antagonism of this receptor abrogated cathepsin S-induced airway inflammation. Conclusions: Cathepsin S contributes to acute lung injury and may represent a novel therapeutic target for acute respiratory distress syndrome.

Therapeutic Inhibition of Cathepsin S Reduces Inflammation and Mucus Plugging in Adult ßENaC-Tg Mice.

BACKGROUND: Elevated levels of the cysteine protease cathepsin S (CatS) are associated with chronic mucoobstructive lung diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). We have previously demonstrated that prophylactic treatment with a CatS inhibitor from birth reduces inflammation, mucus plugging, and lung tissue damage in juvenile ß-epithelial Na+ channel-overexpressing transgenic (ßENaC-Tg) mice with chronic inflammatory mucoobstructive lung disease. In this study, we build upon this work to examine the effects of therapeutic intervention with a CatS inhibitor in adult ßENaC-Tg mice with established disease. METHODS: ßENaC-Tg mice and wild-type (WT) littermates were treated with a CatS inhibitor from 4 to 6 weeks of age, and CatS-/- ßENaC-Tg mice were analysed at 6 weeks of age. Bronchoalveolar lavage (BAL) fluid inflammatory cell counts were quantified, and lung tissue destruction and mucus obstruction were analysed histologically. RESULTS: At 6 weeks of age, ßENaC-Tg mice developed significant airway inflammation, lung tissue damage, and mucus plugging when compared to WT mice. CatS-/- ßENaC-Tg mice and ßENaC-Tg mice receiving inhibitor had significantly reduced airway mononuclear and polymorphonuclear (PMN) cell counts as well as mucus plugging. However, in contrast to CatS-/- ßENaC-Tg mice, therapeutic inhibition of CatS in ßENaC-Tg mice had no effect on established emphysema-like lung tissue damage. CONCLUSIONS: These results suggest that while early CatS targeting may be required to prevent the onset and progression of lung tissue damage, therapeutic CatS targeting effectively inhibited airway inflammation and mucus obstruction. These results indicate the important role CatS may play in the pathogenesis and progression of mucoobstructive lung disease.

Alzheimer's Disease-Like Neurodegeneration in Porphyromonas gingivalis Infected Neurons with Persistent Expression of Active Gingipains.

BACKGROUND: Porphyromonas gingivalis (P. gingivalis) and its gingipain virulence factors have been identified as pathogenic effectors in Alzheimer's disease (AD). In a recent study we demonstrated the presence of gingipains in over 90% of postmortem AD brains, with gingipains localizing to the cytoplasm of neurons. However, infection of neurons by P. gingivalis has not been previously reported. OBJECTIVE: To demonstrate intraneuronal P. gingivalis and gingipain expression in vitro after infecting neurons derived from human inducible pluripotent stem cells (iPSC) with P. gingivalis for 24, 48, and 72 h. METHODS: Infection was characterized by transmission electron microscopy, confocal microscopy, and bacterial colony forming unit assays. Gingipain expression was monitored by immunofluorescence and RT-qPCR, and protease activity monitored with activity-based probes. Neurodegenerative endpoints were assessed by immunofluorescence, western blot, and ELISA. RESULTS: Neurons survived the initial infection and showed time dependent, infection induced cell death. P. gingivalis was found free in the cytoplasm or in lysosomes. Infected neurons displayed an accumulation of autophagic vacuoles and multivesicular bodies. Tau protein was strongly degraded, and phosphorylation increased at T231. Over time, the density of presynaptic boutons was decreased. CONCLUSION: P. gingivalis can invade and persist in mature neurons. Infected neurons display signs of AD-like neuropathology including the accumulation of autophagic vacuoles and multivesicular bodies, cytoskeleton disruption, an increase in phospho-tau/tau ratio, and synapse loss. Infection of iPSC-derived mature neurons by P. gingivalis provides a novel model system to study the cellular mechanisms leading to AD and to investigate the potential of new therapeutic approaches.

Treatment of Porphyromonas gulae infection and downstream pathology in the aged dog by lysine-gingipain inhibitor COR388.

COR388, a small-molecule lysine-gingipain inhibitor, is currently being investigated in a Phase 2/3 clinical trial for Alzheimer's disease (AD) with exploratory endpoints in periodontal disease. Gingipains are produced by two species of bacteria, Porphyromonas gingivalis and Porphyromonas gulae, typically associated with periodontal disease and systemic infections in humans and dogs, respectively. P. gulae infection in dogs is associated with periodontal disease, which provides a physiologically relevant model to investigate the pharmacology of COR388. In the current study, aged dogs with a natural oral infection of P. gulae and periodontal disease were treated with COR388 by oral administration for up to 90 days to assess lysine-gingipain target engagement and reduction of bacterial load and downstream pathology. In a 28-day dose-response study, COR388 inhibited the lysine-gingipain target and reduced P. gulae load in saliva, buccal cells, and gingival crevicular fluid. The lowest effective dose was continued for 90 days and was efficacious in continuous reduction of bacterial load and downstream periodontal disease pathology. In a separate histology study, dog brain tissue showed evidence of P. gulae DNA and neuronal lysine-gingipain, demonstrating that P. gulae infection is systemic and spreads beyond its oral reservoir, similar to recent observations of P. gingivalis in humans. Together, the pharmacokinetics and pharmacodynamics of COR388 lysine-gingipain inhibition, along with reduction of bacterial load and periodontal disease in naturally occurring P. gulae infection in the dog, support the use of COR388 in targeting lysine-gingipain and eliminating P. gingivalis infection in humans.

Porphyromonas gingivalis in Alzheimer's disease brains: Evidence for disease causation and treatment with small-molecule inhibitors.

Porphyromonas gingivalis, the keystone pathogen in chronic periodontitis, was identified in the brain of Alzheimer's disease patients. Toxic proteases from the bacterium called gingipains were also identified in the brain of Alzheimer's patients, and levels correlated with tau and ubiquitin pathology. Oral P. gingivalis infection in mice resulted in brain colonization and increased production of Aß1-42, a component of amyloid plaques. Further, gingipains were neurotoxic in vivo and in vitro, exerting detrimental effects on tau, a protein needed for normal neuronal function. To block this neurotoxicity, we designed and synthesized small-molecule inhibitors targeting gingipains. Gingipain inhibition reduced the bacterial load of an established P. gingivalis brain infection, blocked Aß1-42 production, reduced neuroinflammation, and rescued neurons in the hippocampus. These data suggest that gingipain inhibitors could be valuable for treating P. gingivalis brain colonization and neurodegeneration in Alzheimer's disease.

Targeting of cathepsin S reduces cystic fibrosis-like lung disease.

Cathepsin S (CatS) is upregulated in the lungs of patients with cystic fibrosis (CF). However, its role in CF lung disease pathogenesis remains unclear.In this study, ß-epithelial Na+ channel-overexpressing transgenic (ßENaC-Tg) mice, a model of CF-like lung disease, were crossed with CatS null (CatS-/-) mice or treated with the CatS inhibitor VBY-999.Levels of active CatS were elevated in the lungs of ßENaC-Tg mice compared with wild-type (WT) littermates. CatS-/-ßENaC-Tg mice exhibited decreased pulmonary inflammation, mucus obstruction and structural lung damage compared with ßENaC-Tg mice. Pharmacological inhibition of CatS resulted in a significant decrease in pulmonary inflammation, lung damage and mucus plugging in the lungs of ßENaC-Tg mice. In addition, instillation of CatS into the lungs of WT mice resulted in inflammation, lung remodelling and upregulation of mucin expression. Inhibition of the CatS target, protease-activated receptor 2 (PAR2), in ßENaC-Tg mice resulted in a reduction in airway inflammation and mucin expression, indicating a role for this receptor in CatS-induced lung pathology.Our data indicate an important role for CatS in the pathogenesis of CF-like lung disease mediated in part by PAR2 and highlight CatS as a therapeutic target.

Analysis of tumour- and stroma-supplied proteolytic networks reveals a brain-metastasis-promoting role for cathepsin S.

Metastasis remains the most common cause of death in most cancers, with limited therapies for combating disseminated disease. While the primary tumour microenvironment is an important regulator of cancer progression, it is less well understood how different tissue environments influence metastasis. We analysed tumour-stroma interactions that modulate organ tropism of brain, bone and lung metastasis in xenograft models. We identified a number of potential modulators of site-specific metastasis, including cathepsin S as a regulator of breast-to-brain metastasis. High cathepsin S expression at the primary site correlated with decreased brain metastasis-free survival in breast cancer patients. Both macrophages and tumour cells produce cathepsin S, and only the combined depletion significantly reduced brain metastasis in vivo. Cathepsin S specifically mediates blood-brain barrier transmigration through proteolytic processing of the junctional adhesion molecule, JAM-B. Pharmacological inhibition of cathepsin S significantly reduced experimental brain metastasis, supporting its consideration as a therapeutic target for this disease.

Extracellular domain dependence of PTPalpha transforming activity.

Two isoforms of the transmembrane protein tyrosine phosphatase PTPalpha, which differ by nine amino acids in their extracellular regions, are expressed in a tissue-specific manner. Over-expression of the shorter isoform transforms rodent cells, and it has previously been reasonable to assume that this was a direct consequence of its dephosphorylation and activation of Src. Transformation by the longer wild-type isoform has not previously been studied. We tested the activities of both isoforms in NIH3T3 cells and found that, while both dephosphorylated and activated Src similarly, only the shorter isoform induced focus formation or anchorage-independent growth. Differences in phosphorylation of PTPalpha at its known regulatory sites, Grb2 binding to PTPalpha, phosphorylation level of focal adhesion kinase by PTPalpha, or overall localization were excluded as possible explanations for the differences in transforming activities. The results suggest that transformation by PTPalpha involves at least one function other than, or in addition to, its activation of Src and that this depends on PTPalpha's extracellular domain. Previous studies have suggested that PTPalpha might be a useful target in breast and colon cancer therapy, and the results presented here suggest that it may be advantageous to develop isoform-specific therapeutic reagents.

Identification and pre-clinical testing of a reversible cathepsin protease inhibitor reveals anti-tumor efficacy in a pancreatic cancer model.

Proteolytic activity is required for several key processes in cancer development and progression, including tumor growth, invasion and metastasis. Accordingly, high levels of protease expression and activity have been found to correlate with malignant progression and poor patient prognosis in a wide variety of human cancers. Members of the papain family of cysteine cathepsins are among the protease classes that have been functionally implicated in cancer. Therefore, the discovery of effective cathepsin inhibitors has considerable potential for anti-cancer therapy. In this study we describe the identification of a novel, reversible cathepsin inhibitor, VBY-825, which has high potency against cathepsins B, L, S and V. VBY-825 was tested in a pre-clinical model of pancreatic islet cancer and found to significantly decrease tumor burden and tumor number. Thus, the identification of VBY-825 as a new and effective anti-tumor drug encourages the therapeutic application of cathepsin inhibitors in cancer.

The oxidative mechanism of action of ortho-quinone inhibitors of protein-tyrosine phosphatase alpha is mediated by hydrogen peroxide.

Here, we report the identification and characterization of five ortho-quinone inhibitors of PTPalpha. We observed that the potency of these compounds in biochemical assays was markedly enhanced by the presence of DTT. A kinetic analysis suggested that they were functioning as irreversible inhibitors and that the inhibition was targeted to the catalytic site of PTPalpha. The inhibition observed by these compounds was sensitive to superoxide dismutase and catalase, suggesting that reactive oxygen species may be mediators of their inhibition. We observed that in the presence of DTT, these compounds would produce up to 2.5mM hydrogen peroxide (H(2)O(2)). The levels of H(2)O(2) produced were sufficient to completely inactivate PTPalpha. In contrast, without a reducing agent the compounds did not generate H(2)O(2) and showed little activity towards PTPalpha. In addition, these compounds inhibited PTPalpha-dependent cell spreading in NIH 3T3 cells at concentrations that were similar to their activity in biochemical assays. The biological implications of these results are discussed as they support growing evidence that H(2)O(2) is a key regulator of PTPs.

The minimal essential core of a cysteine-based protein-tyrosine phosphatase revealed by a novel 16-kDa VH1-like phosphatase, VHZ.

The smallest active protein-tyrosine phosphatase yet (only 16 kDa) is described here and given the name VHZ for VH1-like member Z because it belongs to the group of small Vaccinia virus VH1-related dual specific phosphatases exemplified by VHR, VHX, and VHY. Human VHZ is remarkably well conserved through evolution as it has species orthologs in frogs, fish, fly, and Archaea. The gene for VHZ, which we designate as DUSP25, is located on human chromosome 1q23.1 and consists of only two coding exons. VHZ is broadly expressed in tissues and cells, including resting blood lymphocytes, Jurkat T cells, HL-60, and RAMOS. In transfected cells, VHZ was located in the cytosol and in other cells also in the nucleoli. Endogenous VHZ showed a similar but more granular distribution. We show that VHZ is an active phosphatase and analyze its structure by computer modeling, which shows that in comparison with the 185-amino acid residue VHR, the 150-residue VHZ is a shortened version of VHR and contains the minimal set of secondary structure elements conserved in all known phosphatases from this class. The surface charge distribution of VHZ differs from that of VHR and is therefore unlikely to dephosphorylate mitogen-activated protein kinases. The remarkably high degree of conservation of VHZ through evolution may indicate a role in some ancient and fundamental physiological process.

VHY, a novel myristoylated testis-restricted dual specificity protein phosphatase related to VHX.

The human DUSP15 gene encodes an uncharacterized 235-amino acid member of the subfamily of small dual specificity protein phosphatases related to the Vaccinia virus VH1 phosphatase. Similar to VHR-related MKPX (VHX) (DUSP22), the predicted protein has an N-terminal myristoylation recognition sequence, and we show here that both are indeed modified by the attachment of a myristate to Gly-2. In recognition of this relatedness to VHX, we refer to the DUSP15-encoded protein as VH1-related member Y (VHY). We report that VHY is expressed at high levels in the testis and barely detectable levels in the brain, spinal cord, and thyroid. A VHY-specific antiserum detected a protein with an apparent molecular mass of 26 kDa, and histochemical analysis showed that VHY was readily detectable in pachytene spermatocytes (midstage of meiotic division I) and round spermatids and weakly in Leydig cells (somatic cells outside of the seminiferous tubules). When expressed in 293T or NIH-3T3 cells, VHY was concentrated at the plasma membrane with some staining of vesicular structures in the Golgi region. Mutation of the myristoylation site Gly-2 abrogated membrane location. Finally, we demonstrate that VHY is an active phosphatase in vitro. We conclude that VHY is a new member of a subgroup of myristoylated VH1-like small dual specificity phosphatases.

The transmembrane receptor protein tyrosine phosphatase DEP1 interacts with p120(ctn).

The receptor-like protein tyrosine phosphatase DEP1, also known as CD148, is expressed predominantly in epithelial cells, in a variety of tumor cell lines, and in lymphocytes. Expression of DEP1 is enhanced at high cell density, and this observation suggests that DEP1 may function in the regulation of cell adhesion and possibly contact inhibition of cell growth. In order to investigate the function of DEP1, substrate-trapping mutants of the phosphatase were used to identify potential substrates. GST-fusion proteins containing the DEP1 catalytic domain with a substrate-trapping D/A mutation were found to interact with p120(ctn), a component of adherens junctions. DEP1 also interacted with other members of the catenin gene family including beta-catenin and gamma-catenin. The interaction with p120(ctn) is likely to be direct, as the interaction occurs in K562 cells lacking functional adherens junctions and E-cadherin expression. Catalytic domains of the tyrosine phosphatases PTP-PEST, CD45, and PTPbeta did not interact with proteins of the catenin family to detectable levels, suggesting that the interaction of DEP1 with these proteins is specific. DEP1 expression was concentrated at sites of cell-cell contact in A549 cells. p120(ctn) was found to colocalize with these structures. Together these data suggest an important role for DEP-1 in the function of cell-cell contacts and adherens junctions.