Yoshiko Okamoto

Cathepsin E Prevents Tumor Growth and Metastasis by Catalyzing the Proteolytic Release of Soluble TRAIL from Tumor Cell Surface

The aspartic proteinase cathepsin E is expressed predominantly in cells of the immune system and highly secreted by activated phagocytes, and deficiency of cathepsin E in mice results in a phenotype affecting immune responses. However, because physiologic substrates for cathepsin E have not yet been identified, the relevance of these observations to the physiologic functions of this protein remains speculative. Here, we show that cathepsin E specifically induces growth arrest and apoptosis in human prostate carcinoma tumor cell lines without affecting normal cells by catalyzing the proteolytic release of soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) from the cell surface. The antitumor activity of cathepsin E was corroborated by in vivo studies with mice bearing human and mouse tumor transplants. Administration of purified cathepsin E into human tumor xenografts in nude mice dose-dependently induced apoptosis in the tumor cells to inhibit tumor growth. The growth, viability, and metastasis of mouse B16 melanoma cells were also more profound in cathepsin E-deficient mice compared with those in the syngeneic wild-type and transgenic mice overexpressing cathepsin E. Taken together, the number of apoptotic tumor cells, as well as tumor-infiltrating activated macrophages, was apparently reduced in cathepsin E-deficient mice compared with those in the other two groups, implying the positive correlation of endogenous cathepsin E levels with the extent of tumor suppression in vivo. These results thus indicate that cathepsin E plays a substantial role in host defense against tumor cells through TRAIL-dependent apoptosis and/or tumor-associated macrophage-mediated cytotoxicity.

Cathepsin E Deficiency Induces a Novel Form of Lysosomal Storage Disorder Showing the Accumulation of Lysosomal Membrane Sialoglycoproteins and the Elevation of Lysosomal pH in Macrophages

Cathepsin E, an endolysosomal aspartic proteinase predominantly expressed in cells of the immune system, has an important role in immune responses. However, little is known about the precise roles of cathepsin E in this system. Here we report that cathepsin E deficiency (CatE-/-) leads to a novel form of lysosome storage disorder in macrophages, exhibiting the accumulation of the two major lysosomal membrane sialoglycoproteins LAMP-1 and LAMP-2 and the elevation of lysosomal pH. These striking features were also found in wild-type macrophages treated with pepstatin A and Ascaris inhibitor. Whereas there were no obvious differences in their expression, biosynthesis, and trafficking between wild-type and CatE-/- macrophages, the degradation rates of these two membrane proteins were apparently decreased as a result of cathepsin E deficiency. Because there was no difference in the vacuolar-type H+-ATPase activity in both cell types, the elevated lysosomal pH in CatE-/- macrophages is most likely due to the accumulation of these lysosomal membrane glycoproteins highly modified with acidic monosaccharides, thereby leading to the disruption of non-proton factors controlling lysosomal pH. Furthermore, the selective degradation of LAMP-1 and LAMP-2, as well as LIMP-2, was also observed by treatment of the lysosomal membrane fraction isolated from wild-type macrophages with purified cathepsin E at pH 5. Our results thus suggest that cathepsin E is important for preventing the accumulation of these lysosomal membrane sialoglycoproteins that can induce a new form of lysosomal storage disorder.

Impaired chemotaxis and cell adhesion due to decrease in several cell-surface receptors in cathepsin E-deficient macrophages

Cathepsin E is an endo-lysosomal aspartic proteinase exclusively present in immune system cells. Previous studies have shown that cathepsin E-deficient (CatE(-/-)) mice display aberrant immune responses such as atopic dermatitis and higher susceptibility to bacterial infection. However, the mechanisms underlying abnormal immune responses induced by cathepsin E deficiency are still unclear. In this study, we found that the cell-surface levels of chemotactic receptors, including chemokine receptor (CCR)-2 and N-formyl peptide receptors (FPRs), were clearly diminished in CatE(-/-)macrophages compared with those in wild-type cells. Consistently, chemotaxis of CatE(-/-)macrophages to MCP-1 and N-formyl-methionyl-leucyl-phenylalanine was also decreased. Similar to the chemotactic receptors, the surface expressions of the adhesion receptors CD18 (integrin beta(2)) and CD 29 (integrin beta(1)) in CatE(-/-) macrophages were significantly decreased, thereby reducing cell attachment of CatE(-/-) macrophages. These results indicate that the defects in chemotaxis and cell adhesion are likely to be involved in the imperfect function of CatE(-/-)macrophages.

Cathepsin E Deficiency Impairs Autophagic Proteolysis in Macrophages

Cathepsin E is an endosomal aspartic proteinase that is predominantly expressed in immune-related cells. Recently, we showed that macrophages derived from cathepsin E-deficient (CatE −/−) mice display accumulation of lysosomal membrane proteins and abnormal membrane trafficking. In this study, we demonstrated that CatE −/− macrophages exhibit abnormalities in autophagy, a bulk degradation system for aggregated proteins and damaged organelles. CatE −/− macrophages showed increased accumulation of autophagy marker proteins such as LC3 and p62, and polyubiquitinated proteins. Cathepsin E deficiency also altered autophagy-related signaling pathways such as those mediated by the mammalian target of rapamycin (mTOR), Akt, and extracellular signal-related kinase (ERK). Furthermore, immunofluorescence microscopy analyses showed that LC3-positive vesicles were merged with acidic compartments in wild-type macrophages, but not in CatE −/− macrophages, indicating inhibition of fusion of autophagosome with lysosomes in CatE −/− cells. Delayed degradation of LC3 protein was also observed under starvation-induced conditions. Since the autophagy system is involved in the degradation of damaged mitochondria, we examined the accumulation of damaged mitochondria in CatE −/− macrophages. Several mitochondrial abnormalities such as decreased intracellular ATP levels, depolarized mitochondrial membrane potential, and decreased mitochondrial oxygen consumption were observed. Such mitochondrial dysfunction likely led to the accompanying oxidative stress. In fact, CatE −/− macrophages showed increased reactive oxygen species (ROS) production and up-regulation of oxidized peroxiredoxin-6, but decreased antioxidant glutathione. These results indicate that cathepsin E deficiency causes autophagy impairment concomitantly with increased aberrant mitochondria as well as increased oxidative stress.

Role of the transcription factor Sp1 in regulating the expression of the murine cathepsin E gene

Cathepsin E (CE) is an intracellular aspartic proteinase that is exclusively expressed in cells of the gastrointestinal tracts, lymphoid tissues, urinary organs and red blood cells. However, the molecular mechanism by which CE is predominantly expressed in these cells remains unknown. Here, we report the identification of several transcription start sites of the CE gene and their regulatory factors in gastric adenosarcoma cells. We first identified several unique transcription start sites in mouse CE genes by an oligo cap method. Their analysis also revealed the existence of a non-coding region ∼24-kb upstream of exon 1 in the CE gene and also the existence of two transcripts for CE. Luciferase analyses in upstream of exon 1 revealed that this site contained putative binding regions for the transcription factors Sp1, AP-1 and cEts-1 essential for the expression of CE gene. Moreover, electrophoretic mobility shift assays revealed that the protein-oligonucleotides complex of the Sp1 site were supershifted by an anti-Sp1 antibody. The chromatin immunoprecipitation assay showed that Sp1 bound to the CE promoter region. In addition, overexpression of the Sp1 protein increased the expression of the CE protein. Altogether, these results suggest that Sp1 binding plays a particularly important role in the regulation of CE gene expression.

Studies on the function mechanism of a Formosan grey mullet protamine—mugiline β M6: interaction of the M6 and M6 fragments with DNA

The interaction of three peptide segments of one component of Formosan grey mullet protamine (mugiline beta M6), obtained by chemical and enzymatic cleavage, with DNA was studied by spectroscopic measurement, thermal denaturation and circular dichroism. The data obtained were then compared with those of whole M6 and other fish protamines such as salmine of salmon and clupeine of herring. M6-B-I, which lacks C-terminal 11 amino acids in M6, showed significantly different properties. It showed remarkably high DNA aggregating ability which was due to a conformational change of DNA from B to A form. The conformational change of DNA induced by the binding of M6-B-I was reproduced by the carboxypeptidase B digestion of DNA-M6 complex. From these results, the arginine-rich, C-terminal domain of the M6 molecule was estimated to be essential for natural DNA binding.