Cathepsins K, L, B, X and W are differentially expressed in normal and chronically inflamed gastric mucosa

The expression of cathepsins K, L, B, X and W was studied by quantitative RT-PCR in normal and inflamed gastric mucosa (antrum, corpus, cardia). Cathepsins B, L, K and X were expressed ubiquitously. In contrast, cathepsin W was expressed at very low levels. Infection by Helicobacter pylori caused a significant induction of cathepsin X (p<0.008), whereas the other cathepsins were not or only locally affected by H. pylori infection or reflux disease. Immunohistochemistry revealed specific expression of cathepsin X (macrophages), cathepsin K (parietal cells) and cathepsin W (lymphocytes), whereas cathepsins B and L were predominantly expressed in epithelial cells.     

The heterogeneity of rat kidney lysosomes revealed by immunoelectron microscopic staining for cathepsins B and H

Summary The heterogeneity of lysosomes was studied by analyzing the immunostaining behavior of cathepsins B and H in rat kidney proximal tubule cells. Rat kidneys were fixed by perfusion and embedded in Lowicryl K4M. A protein A-gold technique was applied to serial sections and a double labeling technique to conventional sections. By analyzing the immunostaining behavior of cathepsins B and H in the same lysosomes which were cut into separate sections, four types of lysosomes were found: Type 1 positive for both proteinases; type 2 strongly positive for cathepsin B, but weakly or negative for cathepsin H; type 3 strongly positive for cathepsin H, but weakly or negative for cathepsin B; and type 4 negative for both proteinases. The double labeling by two different sizes of the protein A-gold probes showed these four types of lysosomes. The results indicate that there exists the lysosomal heterogeneity of the proteinase content in the kidney proximal tubule cells.     

The heterogeneity of rat kidney lysosomes revealed by immunoelectron microscopic staining for cathepsins B and H

The heterogeneity of lysosomes was studied by analyzing the immunostaining behavior of cathepsins B and H in rat kidney proximal tubule cells. Rat kidneys were fixed by perfusion and embedded in Lowicryl K4M. A protein A-gold technique was applied to serial sections and a double labeling technique to conventional sections. By analyzing the immunostaining behavior of cathepsins B and H in the same lysosomes which were cut into separate sections, four types of lysosomes were found: Type 1 positive for both proteinases; type 2 strongly positive for cathepsin B, but weakly or negative for cathepsin H; type 3 strongly positive for cathepsin H, but weakly or negative for cathepsin B; and type 4 negative for both proteinases. The double labeling by two different sizes of the protein A-gold probes showed these four types of lysosomes. The results indicate that there exists the lysosomal heterogeneity of the proteinase content in the kidney proximal tubule cells.     

Immunocytochemical localization of prorenin, renin, and cathepsins B, H, and L in juxtaglomerular cells of rat kidney

To examine the correlation of localization of prorenin, renin, and cathepsins B, H, and L, immunocytochemistry was applied to rat renal tissue, using a sequence-specific anti-body (anti-prorenin) that recognizes the COOH terminus of the rat renin prosegment. In serial semi-thin sections, immunodeposits for prorenin, renin, and cathepsins B, H, and L were localized in the same juxtaglomerular (JG) cells. Immunodeposits for renin were detected throughout the cytoplasm of the cells, whereas those for prorenin were detected in the perinuclear region. Immunoreactivity for cathepsin B was stronger than that for cathepsins H and L. By electron microscopy, prorenin was localized in small (immature) granules but not in large mature granules, whereas renin was localized mainly in mature granules. In serial thin sections, prorenin, renin, and cathepsin B were colocalized in the same immature granules containing heterogeneously dense material (intermediate granules). By double immunostaining, co-localization of renin with cathepsins B, H, or L was demonstrated in mature granules. The results suggest the possibility that processing of prorenin to renin occurs in immature granules of rat JG cells, and cathepsin B detected in JG cells may be a major candidate for the maturation of renin.     

Immunocytochemical localization of cathepsins B and H in human pancreatic endocrine cells and insulinoma cells

Cathepsins B and H are representative cysteine proteinases localized to lysosomes of a variety of mammalian cells. Previous studies indicated the presence of these enzymes also in secretory granules of endocrine cells. Therefore, the human endocrine pancreas and human insulinomas were investigated by light microscopical immunohistochemistry on serial semithin plastic sections immunostained sequentially for cathepsins B or H and pancreatic hormones. Out of the four established endocrine cell types, insulin (B-) and glucagon (A-) cells showed immunoreactivities for these cathepsins. Cathepsin B immunoreactivities showed a dot-like appearance in A- and B-cells and in insulinoma cells. Immunoreactivities for cathepsin H additionally were found in cell parts containing secretory granules of B-cells and insulinoma cells. By single and double immunoelectron microscopy the dot-like immunoreactivities for cathepsin B were identified as immunoreactive lysosomes of A- and B-cells and insulinoma cells. In addition, some of the secretory granules of A- and B-cells showed cathepsin B immunoreactivities. Cathepsin H immunoreactivities showed an other pattern: they were found regularly in the secretory granules of A- and B-cells and insulinoma cells, and in lysosomes of A-cells. These findings suggest that cathepsins B and H in lysosomes of A- and/or B-cells are involved in the degradation of lysosomal constituents. In secretory granules of these cells, these cysteine proteinases may participate in the processing of the corresponding hormones from their precursor proteins.     

Immunocytochemical localization of cathepsins B and H in human pancreatic endocrine cells and insulinoma cells

Summary Cathepsins B and H are representative cysteine proteinases localized to lysosomes of a variety of mammalian cells. Previous studies indicated the presence of these enzymes also in secretory granules of endocrine cells. Therefore, the human endocrine pancreas and human insulinomas were investigated by light microscopical immunohistochemistry on serial semithin plastic sections immunostained sequentially for cathepsins B or H and pancreatic hormones. Out of the four established endocrine cell types, insulin (B-) and glucagon (A-) cells showed immunoreactivities for these cathepsins. Cathepsin B immunoreactivities showed a dot-like appearance in A- and B-cells and in insulinoma cells. Immunoreactivities for cathepsin H additionally were found in cell parts containing secretory granules of B-cells and insulinoma cells. By single and double immunoelectron microscopy the dot-like immunoreactivities for cathepsin B were identified as immunoreactive lysosomes of A- and B-cells and insulinoma cells. In addition, some of the secretory granules of A- and B-cells showed cathepsin B immunoreactivities. Cathepsin H immunoreactivities showed an other pattern: they were found regularly in the secretory granules of A- and B-cells and insulinoma cells, and in lysosomes of A-cells. These findings suggest that cathepsins B and H in lysosomes of A- and/or B-cells are involved in the degradation of lysosomal constituents. In secretory granules of these cells, these cystine proteinases may participate in the processing of the corresponding hormones from their precursor proteins.     

Cysteine proteinases in chondrosarcomas

The aim of the present study was to define the role of cathepsins B, H, K, L and S in the pathogenesis of human chondrosarcomas. For this purpose 40 tumour samples obtained from 12 patients with the diagnosis of conventional chondrosarcoma were systematically investigated for the expression of cathepsin mRNAs by Northern hybridisation, and for immunohistochemical localisation of the proteins. Northern analysis demonstrated the highest levels of cathepsins B and L in a recurring grade 1 chondrosarcoma, and in a grade 3 chondrosarcoma and in fibrous histiocytomas. Increased expression of cathepsin K mRNA was seen in seven chondrosarcomas, as well as in control tumours; fibrous histiocytomas, osteosarcomas, enchondromas and a giant cell tumour of bone. Cathepsin L was immunolocalised within the large chondrocytes, while cathepsin K was predominantly localised in large multinucleated osteoclastic cells and in some hypertrophic chondrocytes. These results suggest that chondrosarcoma can be included in the growing list of tumours, where cathepsins may well be involved in tumour progression. The simultaneous upregulation of cathepsins B and L, together with matrix metalloproteinase-13, and the association of cathepsin K with negative prognostic parameters suggests that an aggressive biological behaviour of chondrosarcoma may be related to the synthesis of cysteine proteinases and activation of other proteolytic enzymes. If this turns out to be the case, cathepsin inhibitors could provide the much needed adjuvant therapy for chondrosarcomas.     

Cathepsin expression during skeletal development.

Cysteine proteinases, cathepsins B, H, K, L and S, have been implicated in several proteolytic processes during development, growth, remodeling and aging, as well as in a variety of pathological processes. For systematic analysis of cathepsin gene expression we have produced cDNA clones for mouse and human cysteine cathepsins. Northern analysis of a panel of total RNAs isolated from 16-19 different human and mouse tissues revealed the presence of mRNAs for cathepsin B, H, K, L and S in most tissues, but each with a distinct profile. Of the different cathepsin mRNAs, those for cathepsin K were clearly the highest in bone and cartilage. However, relatively high mRNA levels for the other cathepsins were also present in these tissues. To better understand the roles of different cathepsins during endochondral ossification in mouse long bones, cathepsin mRNAs were localized by in situ hybridization. Cathepsin K mRNAs were predominantly seen in multinucleated chondroclastic and osteoclastic cells at the osteochondral junction and on the surface of bone spicules. The other cathepsin mRNAs were also seen in osteoclasts, and in hypertrophic and proliferating chondrocytes. These observations were confirmed by immunohistochemistry and suggest that all cysteine cathepsins are involved in matrix degradation during endochondral ossification.     

An immunocytochemical study on co-localization of cathepsin B and atrial natriuretic peptides in secretory granules of atrial myoendocrine cells of rat heart

To examine localization of cathepsin B, a representative lysosomal cysteine protease, in atrial myoendocrine cells of the rat heart, immunohistochemistry at the light and electron microscopic level was applied to the atrial tissue, using a monospecific antibody for rat liver cathepsin B. In serial semi-thin sections, immunoreactivity for cathepsin B and atrial natriuretic peptides (ANP) was detected in the para-nuclear region of atrial myoendocrine cells. Several large granules and many fine granules in the region of the cells were positively stained by the cathepsin B antibody. Gold particles indicating cathepsin B antigenicity labeled secretory granules in the cells, which were also labeled by those indicating ANP, using thin sections of the Lowicryl K4M-embedded material. Moreover, some granules labeled densely by immunogold particles for cathepsin B seemed to be lysosomes. By double immunostaining using thin sections of the Epon-embedded material, gold particles indicating cathepsin B and ANP antigenicities were co-localized in secretory granules of the cells. By enzyme assay, activity of cathepsin B was three times higher in atrial tissue than ventricular tissue. The results suggest that co-localization of cathepsin B and ANP in secretory granules is compatible with the possibility that cathepsin B participates in the maturation process of ANP.     

Human Cytomegalovirus Gene Expression in Long-Term Infected Glioma Stem Cells

The most common adult primary brain tumor, glioblastoma (GBM), is characterized by fifteen months median patient survival and has no clear etiology. We and others have identified the presence of human cytomegalovirus (HCMV) gene products endogenously expressed in GBM tissue and primary cells, with a subset of viral genes being consistently expressed in most samples. Among these viral genes, several have important oncomodulatory properties, regulating tumor stemness, proliferation, immune evasion, invasion and angiogenesis. These findings lead us to hypothesize that a specific HCMV gene signature may be associated with GBM pathogenesis. To investigate this hypothesis, we used glioma cell lines and primary glioma stem-like cells (GSC) infected with clinical and laboratory HCMV strains and measured relative viral gene expression levels along several time points up to 15 weeks post-infection. While HCMV gene expression was detected in several infected glioma lines through week 5 post-infection, only HCMV-infected GSC expressed viral gene products 15 weeks post-infection. Efficiency of infection across time was higher in GSC compared to cell lines. Importantly, HCMV-infected GSC outlived their uninfected counterparts, and this extended survival was paralleled by increased tumorsphere frequency and upregulation of stemness regulators, such as SOX2, p-STAT3, and BMX (a novel HCMV target identified in this study). Interleukin 6 (IL-6) treatment significantly upregulated HCMV gene expression in long-term infected glioma cultures, suggesting that pro-inflammatory signaling in the tumor milieu may further augment HCMV gene expression and subsequent tumor progression driven by viral-induced cellular signaling. Together, our data support a critical role for long-term, low-level HCMV infection in promoting survival, stemness, and proliferation of GSC that could significantly contribute to GBM pathogenesis.     

Human kidney cathepsins B and L. Characterization and potential role in degradation of glomerular basement membrane.

Cathepsins B and L were purified from human kidney. SDS/polyacrylamide-gel electrophoresis demonstrated that cathepsins B and L, Mr 27000-30000, consist of disulphide-linked dimers, subunit Mr values 22000-25000 and 5000-7000. The pH optimum for the hydrolysis of methylcoumarylamide (-NHMec) substrates (see below) is approx. 6.0 for each enzyme. Km and kcat. are 252 microM and 364s-1 and 2.2 microM and 25.8 s-1 for the hydrolysis of Z-Phe-Arg-NHMec (where Z- represents benzyloxycarbonyl-) by cathepsins B and L respectively, and 184 microM and 158 s-1 for the hydrolysis of Z-Arg-Arg-NHMec by cathepsin B. A 10 min preincubation of cathepsin B (40 degrees C) or cathepsin L (30 degrees C) with E-64 (2.5 microM) results in complete inhibition. Under identical conditions Z-Phe-Phe-CHN2 (0.56 microM) completely inhibits cathepsin L but has little effect on cathepsin B. Incubation of glomerular basement membrane (GBM) with purified human kidney cathepsin L resulted in dose-dependent (10-40 nM) GBM degradation. In contrast, little degradation of GBM (less than 4.0%) was observed with cathepsin B. The pH optimum for GBM degradation by cathepsin L was 3.5. Cathepsin L was significantly more active in degrading GBM than was pancreatic elastase, trypsin or bacterial collagenase. These data suggest that cathepsin L may participate in the lysosomal degradation of GBM associated with normal GBM turnover in vivo.     

Breast cancer cells express cathepsins B and L but not cathepsins K or H

Lysosomal cysteine proteinases (cathepsins) are considered to play a role in bone degradation mediated by metastatic breast cancers. To evaluate which cathepsin contributes to the osteolysis, we quantitatively determined the expression levels of four cathepsins in two breast cancer cell lines, MCF-7 and MDA-MB-231, by competitive RT-PCR. Cathepsin K, which is the most abundant cathepsin in osteoclasts, was not detected in either cell lines. We also failed to detect cathepsin H mRNA. By contrast, we found significant expression of cathepsins B and L in both cell lines. By Northern blot analysis cathepsin B mRNA was detected in a single form in these cells, whereas osteoclasts contained multiple forms of the mRNA. Cathepsin B protein was also detected by Western blotting as a single immunoreactive band corresponding to its mature enzyme. These findings suggest that osteolysis associated with metastatic breast cancers takes place in a different way from osteoclast-mediated bone resorption.     

Inhibitory properties of cystatin F and its localization in U937 promonocyte cells

Cystatin F is a recently discovered type II cystatin expressed almost exclusively in immune cells. It is present intracellularly in lysosome-like vesicles, which suggests a potential role in regulating papain-like cathepsins involved in antigen presentation. Therefore, interactions of cystatin F with several of its potential targets, cathepsins F, K, V, S, H, X and C, were studied in vitro. Cystatin F tightly inhibited cathepsins F, K and V with Ki values ranging from 0.17 nM to 0.35 nM, whereas cathepsins S and H were inhibited with 100-fold lower affinities (Ki approximately 30 nM). The exopeptidases, cathepsins C and X were not inhibited by cystatin F. In order to investigate the biological significance of the inhibition data, the intracellular localization of cystatin F and its potential targets, cathepsins B, H, L, S, C and K, were studied by confocal microscopy in U937 promonocyte cells. Although vesicular staining was observed for all the enzymes, only cathepsins H and X were found to be colocalized with the inhibitor. This suggests that cystatin F in U937 cells may function as a regulatory inhibitor of proteolytic activity of cathepsin H or, more likely, as a protection against cathepsins misdirected to specific cystatin F containing endosomal/lysosomal vesicles. The finding that cystatin F was not colocalized with cystatin C suggests distinct functions for these two cysteine protease inhibitors in U937 cells.     

The hypoxic peri-arteriolar glioma stem cell niche,an integrated concept of five types of niches in human glioblastoma

Glioblastoma is the most lethal primary brain tumor and poor survival of glioblastoma patients is attributed to the presence of glioma stem cells (GSCs). These therapy-resistant, quiescent and pluripotent cells reside in GSC niches, which are specific microenvironments that protect GSCs against radiotherapy and chemotherapy. We previously showed the existence of hypoxic peri-arteriolar GSC niches in glioblastoma tumor samples. However, other studies have described peri-vascular niches, peri-hypoxic niches, peri-immune niches and extracellular matrix niches of GSCs. The aim of this review was to critically evaluate the literature on these five different types of GSC niches. In the present review, we describe that the five niche types are not distinct from one another, but should be considered to be parts of one integral GSC niche model, the hypoxic peri-arteriolar GSC niche. Moreover, hypoxic peri-arteriolar GSC niches are structural and functional look-alikes of hematopoietic stem cell (HSC) niches in the bone marrow. GSCs are maintained in peri-arteriolar niches by the same receptor-ligand interactions as HSCs in bone marrow. Our concept should be rigidly tested in the near future and applied to develop therapies to expel and keep GSCs out of their protective niches to render them more vulnerable to standard therapies.     

Localization of cathepsins G and L in spontaneous resorption of intervertebral discs in a rat experimental model

To determine the involvement of cathepsins G and L in the mechanism of spontaneous resorption of herniated intervertebral discs, localization of these cathepsins in this process was examined immunohistochemically using a rat model of autologous transplantation of coccygeal discs. Rat coccygeal discs were resected and autotransplanted into the subcutaneous space of the skin of the back. Paraffin-embedded sections of intervertebral disc tissue, harvested at various post-transplantational periods, were immunohistochemically stained with antibodies for cathepsin G, cathepsin L, MMP-1, MMP-3 and ED-2. The number of positive cells was counted in each part of the transplanted discs. Immunolocalization of cathepsins G and L in various types of disc cells was first observed early in the post-transplantation period. From two days after the operation, histology showed invasion by granulation tissue, with many macrophages, in all sections. Subsequently, the number of macrophages in granulation tissue was observed to increase, along with a gradual increase in the percentage of cells positive for MMP-1 and MMP-3. In addition to the ability of cathepsins G and L to degrade major extracellular matrix components of intervertebral discs, cathepsin G is capable of activating latent pro-MMPs. The up-regulation of cathepsins G and L in the intervertebral disc tissue in this spontaneous resorption model suggests that these proteinases may be involved in degradation of extracellular matrix, leading to the natural resorption of herniated discs.     

Immunocytochemical localization of cathepsins B, H, and their endogenous inhibitor, cystatin beta, in islet endocrine cells of rat pancreas

To determine the characteristics of lysosomes in rat islet endocrine cells, we examined the precise localization of cathepsins B, H, and L and their specific inhibitors, cystatins alpha and beta, using immunocytochemical techniques. By use of serial semi-thin sections, we detected immunoreactivity for cathepsin B in insulin-, glucagon-, somatostatin-, and pancreatic polypeptide-positive (PP) cells. Strong immunoreactivity for cathepsin H was seen in A-cells and weak immunoreactivity in PP cells, but none in others. Immunodeposits for cystatin beta were demonstrated in B-cells. Brief dipping of thin sections in 1% sodium methoxide before the following immunocytochemical reaction enhanced specific deposits of immunogold particles on the target organelles. Use of a double-immunostaining technique showed co-localization of insulin with cystatin beta in many secretory granules. This suggests that cystatin beta may regulate converting enzymes participating in the maturation process of insulin. By use of an immunogold technique, heterogeneous localization of cathepsins B and H in lysosomes was also found among islet cells at the light microscopic level. This may be due to the difference in peptides degraded in lysosomes among the cells.     

Expression of cathepsins B, D and L in mouse corneas infected with Pseudomonas aeruginosa.

C57BL/6J na?ve and immunized mice were intracorneally infected with Pseudomonas aeruginosa. Semi-quantitative RT-PCR was performed to detect cathepsin gene expression and the results were further confirmed by immunoblot analysis. The enzymatic activities of cathepsins B, D and L were measured by peptidase assays. Immunohistochemical staining was carried out to localize the expression of the cathepsins. Cathepsins B, D and L were detected in the normal cornea by RT-PCR. A peptidase assay revealed activities of all three cathepsins under normal physiological conditions. In na?ve mice, enzymatic activities of cathepsins B, D and L were all significantly enhanced when the corneas were infected with P. aeruginosa and the peak of the induction appeared around day 6 postinfection. Immunoblot analysis showed increased expression of cathepsins B, D and L. The infected corneal samples from immunized mice exhibited much lower induction of enzymatic activities compared to those from na?ve mice. Immunohistochemistry showed that the expression of cathepsins in the normal cornea was restricted to the epithelial tissue while the induced expression of cathepsins was predominantly in the substantia propria. Our data revealed up-regulated enzymatic activities of cathepsins B, D and L in the na?ve corneas infected with P. aeruginosa, which correlated well with the inflammatory response. Immunization of mice against P. aeruginosa attenuated the inducing effect on cathepsin expression caused by infection. The time sequence for induction of cathepsin proteins and enzymatic activities suggests a mechanism of host proteolytic degradation of the extracellular matrix resulting in corneal destruction after P. aeruginosa infection.     

Biochemical properties and regulation of cathepsin K activity

Cysteine cathepsins (11 in humans) are mostly located in the acidic compartments of cells. They have been known for decades to be involved in intracellular protein degradation as housekeeping proteases. However, the discovery of new cathepsins, including cathepsins K, V and F, has provided strong evidence that they also participate in specific biological events. This review focuses on the current knowledge of cathepsin K, the major bone cysteine protease, which is a drug target of clinical interest. Nevertheless, we will not discuss recent developments in cathepsin K inhibitor design since they have been extensively detailed elsewhere. We will cover features of cathepsin K structure, cellular and tissue distribution, substrate specificity, and regulation (pH, propeptide, glycosaminoglycans, oxidants), and its putative roles in physiological or pathophysiological processes. Finally, we will review the kinetic data of its inhibition by natural endogenous inhibitors (stefin B, cystatin C, H- and L-kininogens).