Differential expression of Cathepsin E in transthyretin amyloidosis: from neuropathology to the immune system.

BACKGROUND: Increasing evidence supports a key role for inflammation in the neurodegenerative process of familial amyloidotic polyneuropathy (FAP). While there seems to be an overactivation of the neuronal interleukin-1 signaling pathway, the immune response is apparently compromised in FAP. Accordingly, little immune cell infiltration is observed around pre-fibrillar or fibrillar amyloid deposits, with the underlying mechanism for this phenomenon remaining poorly understood. Cathepsin E (CtsE) is an important intermediate for antigen presentation and chemotaxis, but its role in the pathogenesis of FAP disease remains unknown. METHODS: In this study, we used both mouse primary macrophages and in vivo studies based on transgenic models of FAP and human samples to characterize CtsE expression in different physiological systems. RESULTS: We show that CtsE is critically decreased in bone marrow-derived macrophages from a FAP mouse model, possibly contributing for cell function impairment. Compromised levels of CtsE were also found in injured nerves of transgenic mice and, most importantly, in naïve peripheral nerves, sensory ganglia, murine stomach, and sural nerve biopsies derived from FAP patients. Expression of CtsE in tissues was associated with transthyretin (TTR) deposition and differentially regulated accordingly with the physiological system under study. Preventing deposition with a TTR small interfering RNA rescued CtsE in the peripheral nervous system (PNS). In contrast, the expression of CtsE increased in splenic cells (mainly monocytes) or peritoneal macrophages, indicating a differential macrophage phenotype. CONCLUSION: Altogether, our data highlights the potential of CtsE as a novel FAP biomarker and a possible modulator for innate immune cell chemotaxis to the disease most affected tissues-the peripheral nerve and the gastrointestinal tract.

High Expression of Cathepsin E is Associated with the Severity of Airflow Limitation in Patients with COPD.

BACKGROUND: It was reported that Cathepsin E (Cat E) plays a critical role in antigen processing and in the development of pulmonary emphysema. The aim of this study was to investigate the role of Cat E and airflow limitation in the pathogenesis of COPD. METHODS: Sixty-five patients with COPD, 20 smoking control subjects without COPD and 15 non-smoking healthy control subjects were enrolled. Cat E and EIC (Elastase inhibitory capacity) expressions were measured by ELISA in sputum and serum samples and compared according to different subgroups. RESULTS: Cat E concentrations were significantly higher in patients with COPD than smoking control and non-smoking control subjects (P < 0.01). The levels of CatE were inversely correlated with FEV1% predicted in COPD patients (r = -0.95, P < 0.01). The levels of EIC were inversely positively correlated with FEV1% predicted in COPD patients (r = 0.926, P < 0.01). Levels of Cat E were also inversely correlated with the levels of EIC (r = -0.922, P < 0.01). CONCLUSIONS: Cat E contributes to the severity of airflow limitation during progression of COPD.

Monitoring pancreatic carcinogenesis by the molecular imaging of cathepsin E in vivo using confocal laser endomicroscopy.

The monitoring of pancreatic ductal adenocarcinoma (PDAC) in high-risk populations is essential. Cathepsin E (CTSE) is specifically and highly expressed in PDAC and pancreatic intraepithelial neoplasias (PanINs), and its expression gradually increases along with disease progression. In this study, we first established an in situ 7,12-dimethyl-1,2-benzanthracene (DMBA)-induced rat model for PanINs and PDAC and then confirmed that tumorigenesis properties in this model were consistent with those of human PDAC in that CTSE expression gradually increased with tumor development using histology and immunohistochemistry. Then, using in vivo imaging of heterotopically implanted tumors generated from CTSE- overexpressing cells (PANC-1-CTSE) in nude mice and in vitro imaging of PanINs and PDAC in DMBA-induced rats, the specificity of the synthesized CTSE-activatable probe was verified. Quantitative determination identified that the fluorescence signal ratio of pancreatic tumor to normal pancreas gradually increased in association with progressive pathological grades, with the exception of no significant difference between PanIN-II and PanIN-III grades. Finally, we monitored pancreatic carcinogenesis in vivo using confocal laser endomicroscopy (CLE) in combination with the CTSE-activatable probe. A prospective double-blind control study was performed to evaluate the accuracy of this method in diagnosing PDAC and PanINs of all grades (>82.7%). This allowed us to establish effective diagnostic criteria for CLE in PDAC and PanINs to facilitate the monitoring of PDAC in high-risk populations.

Over-expression of cathepsin E and trefoil factor 1 in sessile serrated adenomas of the colorectum identified by gene expression analysis.

Sessile serrated adenomas are now recognised as precursor lesions of a substantial subset of colorectal cancers arising via a so-called "serrated pathway". However, their biological markers remain to be defined. The aim of our study was to identify differentially expressed genes in sessile serrated adenomas and conventional adenomas. Gene expression analysis demonstrated molecular differences between polyp types. Further studies using quantitative real-time polymerase chain reaction on cathepsin E (CTSE) demonstrated a significantly (p < 0.05) higher expression in sessile serrated adenomas as compared to hyperplastic polyp and tubular adenomas. Trefoil Factor 1 showed the same trend of expression for sessile serrated adenomas as compared to hyperplastic polyps and was significantly higher in both polyps compared to tubular adenomas. Immunohistochemistry for both proteins demonstrated strong cytoplasmic staining of abnormal crypts in all sessile serrated adenomas, while staining in tubular adenomas and hyperplastic polyps was absent or weak and focal. BRAF and KRAS mutation analysis were employed to further validate polyp discrimination. The findings demonstrated the positive association of the BRAF mutation, V600E, with sessile serrated adenomas and KRAS mutations with tubular adenomas (p < 0.05). This study demonstrates the over-expression in CTSE, in particular, and TFF1 in sessile serrated adenomas compared to both hyperplastic polyps and tubular adenomas.

An essential role for the antiviral endoribonuclease, RNase-L, in antibacterial immunity.

Type I IFNs were discovered as the primary antiviral cytokines and are now known to serve critical functions in host defense against bacterial pathogens. Accordingly, established mediators of IFN antiviral activity may mediate previously unrecognized antibacterial functions. RNase-L is the terminal component of an RNA decay pathway that is an important mediator of IFN-induced antiviral activity. Here, we identify a role for RNase-L in the host antibacterial response. RNase-L(-/-) mice exhibited a dramatic increase in mortality after challenge with Bacillus anthracis and Escherichia coli; this increased susceptibility was due to a compromised immune response resulting in increased bacterial load. Investigation of the mechanisms of RNase-L antibacterial activity indicated that RNase-L is required for the optimal induction of proinflammatory cytokines that play essential roles in host defense from bacterial pathogens. RNase-L also regulated the expression of the endolysosomal protease, cathepsin-E, and endosome-associated activities, that function to eliminate internalized bacteria and may contribute to RNase-L antimicrobial action. Our results reveal a unique role for RNase-L in the antibacterial response that is mediated through multiple mechanisms. As a regulator of fundamental components of the innate immune response, RNase-L represents a viable therapeutic target to augment host defense against diverse microbial pathogens.

Cathepsin E regulates the presentation of tetanus toxin C-fragment in PMA activated primary human B cells.

Processing of antigens by proteases in the endocytic compartments of antigen presenting cells (APC) is essential to make them suitable for presentation as antigenic peptides to T lymphocytes. Several proteases of the cysteine, aspartyl and serine classes are involved in this process. It has been speculated, that the aspartyl protease cathepsin E (CatE) is involved in antigen processing in B cell line, monocyte-derived dendritic cells (DC) and murine DC. Here we show the expression of CatE in primary human B cells and DC, which was only elevated in B cells after induction with phorbol 12-myristate 13-acetate (PMA), resulted in enhanced presentation of tetanus toxin C-fragment (TTC) to the respective T cells. Inhibition of aspartyl proteases using pepstatin-A-penetratin (PepA-P), a highly efficient, cell-permeable aspartyl protease inhibitor, reduced significantly T cell activation in PMA activated B cells but not in PMA activated myeloid DC (mDC). Thus we suggest that CatE is important in the processing of TTC in primary human B cells.

Regulation of the human cathepsin E gene by the constitutive androstane receptor.

Cathepsin E (CTSE) is an aspartic protease that has been linked to antigen processing and innate immunity. Elevated levels of CTSE expression have also been associated with several forms of cancer, including carcinomas exhibiting highly invasive character. In this study, we performed DNA microarray experiments, together with quantitative reverse transcriptase PCR analyses and enzymatic activity determinations to identify human CTSE as a novel target gene for regulation by the constitutive androstane receptor (CAR), a nuclear receptor activated by the liver tumor promoting agent, phenobarbital. In particular, two motifs within the 5'-flanking region of the human CTSE gene were identified as direct sites of interaction with CAR/RXRalpha heterodimers, a direct repeat-3 site at position -766 and a direct repeat-4 site at position -1407. Thus, these studies demonstrate CAR-mediated regulation of CTSE within primary hepatocyte cultures from several individual donors and suggest that elevated CTSE activity may play a functional role in the etiology of hepatocarcinogenesis.

Ligand activation of peroxisome proliferator-activated receptor beta inhibits colon carcinogenesis.

There is considerable debate whether peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) ligands potentiate or suppress colon carcinogenesis. Whereas administration of a PPARbeta ligand causes increased small intestinal tumorigenesis in Apc(min/+) mice, PPARbeta-null (Pparb-/-) mice exhibit increased colon polyp multiplicity in colon cancer bioassays, suggesting that ligand activation of this receptor will inhibit colon carcinogenesis. This hypothesis was examined by treating wild-type (Pparb+/+) and Pparb-/- with azoxymethane, coupled with a highly specific PPARbeta ligand, GW0742. Ligand activation of PPARbeta in Pparb+/+ mice caused an increase in the expression of mRNA encoding adipocyte differentiation-related protein, fatty acid-binding protein, and cathepsin E. These findings are indicative of colonocyte differentiation, which was confirmed by immunohistochemical analysis. No PPARbeta-dependent differences in replicative DNA synthesis or expression of phosphatase and tensin homologue, phosphoinositide-dependent kinase, integrin-linked kinase, or phospho-Akt were detected in ligand-treated mouse colonic epithelial cells although increased apoptosis was found in GW0742-treated Pparb+/+ mice. Consistent with increased colonocyte differentiation and apoptosis, inhibition of colon polyp multiplicity was also found in ligand-treated Pparb+/+ mice, and all of these effects were not found in Pparb-/- mice. In contrast to previous reports suggesting that activation of PPARbeta potentiates intestinal tumorigenesis, here we show that ligand activation of PPARbeta attenuates chemically induced colon carcinogenesis and that PPARbeta-dependent induction of cathepsin E could explain the reported disparity in the literature about the effect of ligand activation of PPARbeta in the intestine.

Cathepsin E is a specific marker of dysplasia in APC mouse intestine.

Transcriptional profiling of APC(Min/+) mouse intestinal epithelial tissue has revealed that cathepsin E (catE) manifests high relative expression in adenomas and carcinomas relative to normal epithelium. Real-time RT-PCR data presented previously confirm the presence of catE transcript in APC(Min/+) adenomatous cells compared with samples derived from normal APC(Min/+) and wild-type tissue. At the protein level, strong, highly specific immunohistochemical staining for catE is displayed in dysplastic lesions of APC(Min/+) mice. Using Western immunoblot analyses, it was additionally established that the urine of tumor-bearing mice contains higher levels of the monomeric form of catE than their wild-type counterparts. These results authenticate the relationship between transcript abundance and protein levels in transformed tissue and suggest potential utility for catE as a marker for the inception and progression of intestinal cancers.

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

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

The expression and function of cathepsin E in dendritic cells.

Cathepsin E is an aspartic proteinase that has been implicated in Ag processing within the class II MHC pathway. In this study, we document the presence of cathepsin E message and protein in human myeloid dendritic cells, the preeminent APCs of the immune system. Cathepsin E is found in a perinuclear compartment, which is likely to form part of the endoplasmic reticulum, and also a peripheral compartment just beneath the cell membrane, with a similar distribution to that of Texas Red-dextran within 2 min of endocytosis. To investigate the function of cathepsin E in processing, a new soluble targeted inhibitor was synthesized by linking the microbial aspartic proteinase inhibitor pepstatin to mannosylated BSA via a cleavable disulfide linker. This inhibitor was shown to block cathepsin D/E activity in cell-free assays and within dendritic cells. The inhibitor blocked the ability of dendritic cells from wild-type as well as cathepsin D-deficient mice to present intact OVA, but not an OVA-derived peptide, to cognate T cells. The data therefore support the hypothesis that cathepsin E has an important nonredundant role in the class II MHC Ag processing pathway within dendritic cells.

Cathepsin E: a novel target for regulation by class II transactivator.

The aspartic proteinase cathepsin E (CatE) has been implicated in Ag processing. In this study we report that CatE expression is negatively regulated by the MHC class II transactivator (CIITA). CIITA-deficient murine and human B cells expressed greater CatE than wild-type B cells, whereas overexpression of CIITA in a human gastric carcinoma cell line, AGS, resulted in decreased CatE mRNA and protein. AGS cells expressing CIITA also exhibited decreased processing of OVA Ag. Inhibition of CatE expression is specific to the type III CIITA isoform and maps to the acidic and proline/serine/threonine-rich (PST) protein domains of CIITA. We found that CatE expression is inducible by PU.1 and p300, and that this induction can be reversed by CIITA. These findings demonstrate a novel phenomenon: regulation of CatE Ag processing by CIITA in an isoform-dependent manner.

Association of cathepsin E deficiency with development of atopic dermatitis.

Atopic dermatitis (AD) is a pruritic inflammatory skin diseases associated with a family history of atropy. Here we show that mice lacking the endolysosomal aspartic proteinase cathepsin E spontaneously develop skin lesions similar to those of humans with AD when reared under conventional conditions but not under specific pathogen-free conditions. These mice showed the increase in the ratio of CD4+/CD8+ T cells, the strong polarization of naïve T cells to T helper 2 cells, and the systemic accumulation of IL-18 and IL-1beta accompanied by a marked increase in IL-4, IL-5, and IgE. The relative rates of degradation of IL-18 and IL-1beta were significantly lower in cathepsin E-deficient mice than wild-type mice. These results strongly suggest that the development of AD in cathepsin E-deficient mice is initiated by systemic accumulation of IL-18 and IL-1beta, mainly due to their reduced turnover rates. In addition, the reduced expression of cathepsin E was also observed in erythrocytes of both humans with AD and the AD mouse model NC/Nga. Cathepsin E deficiency might thus be responsible for the induction of AD in humans and mice.

Pronapsin A and B gene expression in normal and malignant human lung and mononuclear blood cells.

The two human pronapsin genes which are located immediately adjacent to one another on chromosome 19 were shown, using quantitative RT-PCR, to have distinctly different expression patterns. Transcription of the pronapsin A gene in normal human lung tissue was not appreciably altered in lung carcinomas and comparable pronapsin A mRNA levels were also quantified in lung epithelial cell lines and in tumour cell lines originating from human lung epithelium. Pronapsin A may thus have considerable diagnostic value as a marker for primary lung cancer. In contrast, the pronapsin B gene, which lacks an in-frame stop codon and so may be a transcribed pseudogene, is expressed at comparable levels in normal human spleen, thymus, cytotoxic and helper T-lymphocytes, natural killer (NK) cells and B-lymphocytes. The mRNA levels quantified in B-cells from adults with chronic lymphoblastic leukaemia were not significantly different from those measured in B-cells from healthy individuals. Similarly, comparable levels of expression were quantified in monocytes from healthy individuals and from a patient with acute myeloid leukaemia, whereas in alveolar macrophages, which are terminally differentiated myeloid cells, transcription of the pronapsin B gene was down-regulated by approximately one order of magnitude. Reciprocally, an approximately 20-fold up-regulation in expression of the procathepsin D gene in the macrophages relative to the circulating monocytes was revealed by quantitative measurements made in parallel throughout all of this study for the respective mRNAs encoding the intracellular aspartic proteinases, procathepsin D and procathepsin E. Thus, while there appears to be little difference in expression of the pronapsin A and B genes between healthy and malignant human cells and tissues, the reciprocal alterations in the levels of transcription of the pronapsin B and procathepsin D genes may have significance in the developmental processes associated with myeloid cell differentiation into macrophages.

Production of angiotensin II by homogeneous cultures of vascular smooth muscle cells from spontaneously hypertensive rats.

Production of angiotensin II (Ang II) in spontaneously hypertensive rats (SHR)-derived vascular smooth muscle cells (VSMC) has now been investigated. A nonpeptide antagonist (CV-11974) of Ang II type 1 receptors inhibited basal DNA synthesis in VSMC from SHR, but it had no effect on cells from Wistar-Kyoto (WKY) rats. Ang II-like immunoreactivity, determined by radioimmunoassay after HPLC, was readily detected in conditioned medium and extracts of SHR-derived VSMC, whereas it was virtually undetectable in VSMC from WKY rats. Isoproterenol increased the amount of Ang II-like immunoreactivity in conditioned medium and extracts of SHR-derived VSMC, whereas the angiotensin-converting enzyme inhibitor delapril significantly reduced the amount of Ang II-like immunoreactivity in conditioned medium and extracts of these cells. Reverse transcription-polymerase chain reaction analysis revealed that the abundance of mRNAs encoding angiotensinogen, cathepsin D, and angiotensin-converting enzyme was greater in VSMC from SHR than in cells from WKY rats. The abundance of cathepsin D protein by Western blotting was greater in VSMC from SHR than in cells from WKY rats. Ang I-generating and acid protease activities were detected in VSMC from SHR, but not in cells from WKY rats. These results suggest that SHR-derived VSMC generate Ang II with increases in angiotensinogen, cathepsin D, and angiotensin-converting enzyme, which contribute to the basal growth. Production of Ang II by homogeneous cultures of VSMC is considered as a new mechanism of hypertensive vascular disease.