Distinct roles of cathepsin K and cathepsin L in osteoclastic bone resorption

The role of cathepsin K (CAK), cloned as a novel collagenolytic cysteine protease (CCP), cathepsin L (CAL) and cathepsin B (CAB) in bone resorption was investigated. In mouse calvarial organ culture medium, CAL, detected in trace amounts in control conditions, and CCP activity were increased by stimulants of bone resorption: 1alpha,25-(OH)2D3 parathyroid hormone (PTH), IL-1alpha, IL-6 or TNF-alpha. CAK was the most abundantly detected CCP and was not increased by these stimulants. In the absence of the stimulants, only antisense oligodeoxynucleotide (AS-ODN) for CAK suppressed collagenolysis and CCP activity. On the other hand, in the presence of the stimulants, AS-ODN for both CAK and CAL suppressed collagenolysis and CCP activity, and these activities were additive. AS-ODN for CAB did not suppress collagenolysis. These results suggested CAK was constitutively synthesized as the main CCP, and CAL was synthesized as an inducible CCP in osteoclasts to degrade type-I collagen in combination with CAK.     

组织蛋白酶B、L在非小细胞肺癌中的表达及意义

目的探讨组织蛋白酶B、L在非小细胞肺癌中的表达及意义。方法收集44例非小细胞肺癌标本，石蜡包埋切片，通过免疫组化的方法检测组织蛋白酶B、L的表达。结果组织蛋白酶B、L随着肿瘤分期的进展其表达阳性率明显升高，Ⅲ期～Ⅳ期的病人组织蛋白酶B、L的表达与I期-Ⅱ期的病人有显著差异（P〈0．05）。组织蛋白酶B和L的表达高度相似。结论组织蛋白酶B、L的表达与非小细胞肺癌的进展和预后有关。     

Normal neutrophil function in cathepsin G-deficient mice

Cathepsin G is a neutral serine protease that is highly expressed at the promyelocyte stage of myeloid development. We have developed a homologous recombination strategy to create a loss-of-function mutation for murine cathepsin G. Bone marrow derived from mice homozygous for this mutation had no detectable cathepsin G protein or activity, indicating that no other protease in bone marrow cells has the same specificity. Hematopoiesis in cathepsin G-/- mice is normal, and the mice have no overt abnormalities in blood clotting. Neutrophils derived from cathepsin G-/- mice have normal morphology and azurophil granule composition; these neutrophils also display normal phagocytosis and superoxide production and have normal chemotactic responses to C5a, fMLP, and interleukin-8. Although cathepsin G has previously shown to have broad spectrum antibiotic properties, challenges of mice with Staphylococcus aureus, Klebsiella pneumoniae, or Escherichia coli yielded survivals that were not different from those of wild-type animals. In sum, cathepsin G-/- neutrophils have no obvious defects in function; either cathepsin G is not required for any of these normal neutrophil functions or related azurophil granule proteases with different specificities (ie, neutrophil elastase, proteinase 3, azurocidin, and/or others) can substitute for it in vivo.     

Renal cathepsin G and angiotensin II generation

BACKGROUND: Alternative pathways of angiotensin II biosynthesis play a significant role in the renin-angiotensin system. In this study porcine renal tissue was investigated for angiotensin II-generating enzymes. METHODS AND RESULTS: Protein extracts from porcine renal tissue were fractionated by liquid chromatography and tested for their angiotensin II-generating activity by the mass-spectrometry-assisted enzyme screening system (MES) and the active fractions were purified to near homogeneity. In one of these active fractions, inhibitable by an angiotensin-converting enzyme specific inhibitor, purified by anion-exchange chromatography, followed by hydroxyapatite chromatography, lectin affinity chromatography, size-exclusion chromatography and two-dimensional electrophoresis, angiotensin-converting enzyme was identified by a tryptic peptide matrix-assisted-laser-desorption/ionization (MALDI) mass fingerprint analysis. In a second active fraction, which was inhibited by chymostatin and antipain, yielded by anion-exchange chromatography, followed by hydroxyapatite chromatography, lectin affinity chromatography, chymostatin-antipain chromatography and one-dimensional electrophoresis, cathepsin G was identified by electro-spray ionization (ESI)-ion-trap mass spectrometry. The angiotensin-generating activities of the fraction containing angiotensin-converting enzyme and the fraction containing cathepsin G were in the same order of magnitude, thus showing that the contribution of cathepsin G towards the production of angiotensin II is significant. CONCLUSION: This is the first time that cathepsin G has been identified in mammalian renal tissue.     

Serum cathepsin activity in chronic liver diseases

Serum cathepsin activity and serum level of glycoproteins were determined in patients with chronic liver disorders (cirrhosis, chronic persistent hepatitis, chronic active hepatitis, hepatoma. An increase of cathepsin activity was found in chronic persistent hepatitis and cirrhosis of the liver. Increased level of glycoproteins was observed, the most evident in patients with chronic active hepatitis. An increase of the ratio cathepsin activity/glycoprotein concentration was found in chronic persistent hepatitis, and a decrease of this ratio was observed in chronic active hepatitis.     

Synergistic antitumor effects of combined cathepsin B and cathepsin Z deficiencies on breast cancer progression and metastasis in mice

The lysosomal cysteine proteases cathepsin B (Ctsb) and cathepsin Z (Ctsz, also called cathepsin X/P) have been implicated in cancer pathogenesis. Compensation of Ctsb by Ctsz in Ctsb ^−/− mice has been suggested. To further define the functional interplay of these proteases in the context of cancer, we generated Ctsz null mice, crossed them with Ctsb-deficient mice harboring a transgene for the mammary duct–specific expression of polyoma middle T oncogene (PymT), and analyzed the effects of single and combined Ctsb and Ctsz deficiencies on breast cancer progression. Single Ctsb deficiency resulted in delayed detection of first tumors and reduced tumor burden, whereas Ctsz-deficient mice had only a prolonged tumor-free period. However, only a trend toward reduced metastatic burden without statistical significance was detected in both single mutants. Strikingly, combined loss of Ctsb and Ctsz led to additive effects, resulting in significant and prominent delay of early and advanced tumor development, improved histopathologic tumor grading, as well as a 70% reduction in the number of lung metastases and an 80% reduction in the size of these metastases. We conclude that the double deficiency of Ctsb and Ctsz exerts significant synergistic anticancer effects, whereas the single deficiencies demonstrate at least partial reciprocal compensation.     

Enzymatic activity of cathepsin B,cathepsin B and L,plasmin, trypsin and collagenase in hepatocellular carcinoma

Hepatocellular carcinoma is one of the most common malignancies worldwide. Invasiveness of this tumour seems to be related to degradation of extracellular matrix. Such proteolytic enzymes as: cathepsin B and L, plasmin, collagenase and trypsin are thought to play a pivotal role in this process. Enzymatic activity depends on balance between enzymes and their inhibitors and--moreover--on interactions among these enzymes. The purpose of our study was to evaluate enzymatic activity of cathepsin B, cathepsin B and L, plasmin, collagenase and trypsin in patients with hepatocellular carcinoma in liver tissue and in peripheral blood. Then correlations between activity of enzymes (mentioned above) and clinical status, pathological findings and laboratory tests were assessed. Our study was conducted on 14 patients who underwent surgery because of hepatocellular carcinoma. Tissue samples were obtained during surgery from neoplastic area and from non-neoplastic area. Peripheral blood was withdrawn before surgery and within early post-operative period. Proteolytic activity of these enzymes was determined with use of fluorometric assay. Enzymatic activity in tissue samples was referred to protein concentration (BCA assay) and to DNA concentration (fluorometric assay). RESULTS: Proteolytic activity of plasmin and trypsin in neoplastic tissue were significantly lower as compared to non-neoplastic area of these patients (p = 0.0356; p = 0.0412, respectively). Activity of the remaining enzymes: cathepsin B, cathepsin B and L and collagenase did not differ significantly. No difference was demonstrated between activity of enzymes in peripheral blood withdrawn before surgery and in postoperative period. There was a statistically significant inverse correlation between serum AFP level and enzymatic activity of cathepsin B, cathepsin B and L and collagenase in tumor tissue. Lower activity of all investigated enzymes was observed in tumor tissue of HBV related hepatocellular carcinoma in comparison with the remaining tissue samples. Correlation between patients age and activity of enzymes was not statistically significant. CONCLUSION: Although the evaluation of presented enzymatic profile did not allow for the assessment of associations between investigated enzymes, our results demonstrated correlations between proteolytic activity of enzymes and serum AFP level, viral status, but it requires further investigations.     

Increased cathepsin B activity in human lung tumors

The occurrence and levels of cathepsin B activity were investigated in primary human lung tumors and lung metastases of renal, colorectal and urinary bladder carcinomas as well as in the associated apparently normal lung parenchyma using a continuous rate enzyme assay with Ac-Leu-Arg-Arg-NHMec (7-(N-acetyl-L-leucyl-L-arginyl-L-arginylamido)-4-methylcoumarin) as the fluorogenic substrate. The inhibition studies of the enzymic hydrolysis of the substrate provided evidence for the catalytic action of the cysteine proteinase cathepsin B (CB) in the lung tumor tissues and the lung parenchyma under the assay conditions used. In the studied group of twenty-four patients with primary lung tumors of all major histological types, the level of CB activity in the tumor tissue was increased twofold and more over that in the associated lung parenchyma in 83% and 75% of cases, when expressed on the basis of wet tissue weight and tissue DNA, respectively. In patients with primary lung adenocarcinoma, the activity of the enzyme in the tumor tissue was elevated over that in the lung parenchyma in all cases studied. In both subgroups of patients with squamous cell lung carcinoma and adenocarcinoma, the mean cathepsin B activity was significantly higher in the tumor tissue than in the lung parenchyma. No obvious correlation was found between the tissue level of cathepsin B activity and the stage of primary lung tumor disease. In a limited number of patients with lung metastases, the level of cathepsin B activity was also higher in the tumor tissue than in the lung parenchyma.     

Histologic assessment of cathepsin D in osteoarthritic cartilage

The lysosomal endopeptidase cathepsin D is the most abundant proteinase in chondrocytes. Its significance in the pathogenesis of cartilage matrix proteoglycan (PG) degradation in osteoarthritis (OA) is unclear. The extracellular localization of cathepsin D and its potential spatial relationship to areas of PG depletion has been studied in human femoral head OA cartilage. Enzyme was identified by indirect immunofluorescence using rabbit antisera developed against a highly purified cathepsin D preparation. PG distribution was assessed in parallel sections by safranin O staining. Specimens were selected to include regions of cartilage having minimal structural and cellular alterations, severe reduction in thickness, hypocellularity, multicellular chondrocyte clusters and varying degrees of PG loss. Cathepsin D was identified in chondrocytes. When overlying fibrous connective tissue pannus was present, extracellular enzyme was predominantly localized to the cartilage-pannus interface. Cathepsin D could not be demonstrated extracellularly in areas of cartilage that were partially or totally devoid of PG. Chondrocytes in damaged regions, particularly in the superficial and upper transitional zones showing diffuse hypercellularity and/or "brood" clusters, contained increased enzyme staining. Results fail to support a role for cathepsin D in extracellular matrix PG degradation. The potential significance of this enzyme in the pathogenesis of OA would appear relegated to intracellular catabolism. Its intracellular increase at pathologic sites is consistent with enhanced catabolic activity in such regions.     

Collagenase, cathepsin B and cathepsin L gene expression in the synovial membrane of patients with early inflammatory arthritis.

OBJECTIVE: To examine the expression of the matrix metalloproteinase, MMP-1, and the cysteine proteases, cathepsin B (CB) and cathepsin L (CL), in the synovial membrane (SM) of patients with early inflammatory arthritis. METHODS: Samples of SM were obtained by blind needle biopsy or needle arthroscopy from inflamed knees of 28 patients with early inflammatory arthritis (mean disease duration 10.2 months, range 2 weeks-18 months). Sixteen patients had rheumatoid arthritis (RA), nine psoriatic arthritis and there was one each with ankylosing spondylitis, gout and an undifferentiated arthritis. Comparison was made with tissue from two patients with established erosive RA and three normal synovial tissue samples. In situ hybridization was performed using digoxigenin-labelled RNA probes. RESULTS: MMP-1, CB and CL were expressed in all patients with early arthritis and in established erosive RA, whereas normal synovium showed only scanty expression. The three proteases were prominent in perivascular infiltrates and endothelial cells of early arthritis tissue. MMP-1 was observed primarily in the lining layer, but was also evident in the sublining area. CB and CL were expressed to a lesser extent in the lining layer, and were present mainly in the subintima. The three proteases were not found in lymphoid aggregrates. No differences were observed between the disease categories. CONCLUSIONS: The detection of MMP-1, CB and CL in the synovium shortly after symptom onset implies that the potential for joint destruction exists at a very early stage in the disease. In addition, the perivascular and endothelial cell expression suggests a role for these proteases in mononuclear cell influx to the inflamed synovium and in angiogenesis.     

In vitro conversion of proinsulin to insulin by cathepsin B in isolated islets and its inhibition by cathepsin B antibodies

Summary Purified bovine cathepsin B, when incubated with isolated rat islets of Langerhans, completely converts proinsulin to insulin as demonstrated by the incorporation of¹⁴C-leucine into islet proteins, releasing lysine as the only basic amino acid. Cathepsin B antibodies raised in rabbit inhibited the above conversion. CDRI Communication No. 2703     

Defensins- and cathepsin G-ANCA in systemic lupus erythematosus

In this study, we examined the content of antineutrophil cytoplasmic antibodies (ANCA) against defensins and cathepsin G in sera from systemic lupus erythematosus (SLE) patients and their significance in estimating the activity of SLE. Defensins- and cathepsin G-ANCA in sera from 28 patients with SLE, eight patients with rheumatoid arthritis (RA) and eight patients with microscopic polyangitis (mPA) were measured by ELISA. Significantly increased defensins- and cathepsin G-ANCA were found in sera of patients with SLE and mPA when compared with the value of normal controls. Though significantly higher defensins- and cathepsin G-ANCA were detected in both active and inactive SLE patients, the value in active SLE patients was significantly higher than inactive SLE patients. After the therapy with high dose of prednisolone, the serum level of defensins- and cathepsin G-ANCA was decreased, and this decrease was sustained for at least 16 weeks. This study suggests that defensins- and cathepsin G-ANCA may serve as useful markers of the disease activity of SLE.     

Cathepsin K的研究进展

Cathepsin K基因定位于1q21.2,翻译产物为前组织蛋白酶原K,属于溶酶体半胱氨酸蛋白酶中的番木瓜蛋白酶超家族。敲除cathepsin K基因的小鼠表现为骨样硬化症。人类缺失此基因会导致致密性成骨不全症(Pyknodysostosis);而过量表达cathepsin K会引起戈谢病(Gaucher病)。它在多种病理现象中发挥作用,尤其与骨质疏松症关系最密切。作用机制可能为降解多种骨基质蛋白,包括Ⅰ型胶原、骨桥蛋白、骨连接素。Cathepsin K的抑制剂可用于治疗骨质疏松症,具有广阔的市场前景。     

Immunohistochemical localization of cathepsin D in colorectal tumors

PURPOSE: Although it has been suggested that cathepsin D, a lysosomal protease, is involved in tumor invasion and metastasis in human colorectal cancers, conflicting studies have also been reported recently. In addition, this issue has been only rarely studied in human colorectal tumors by use of immunohistochemical methods. The aim of the study presented here was to clarify not only the correlation between cathepsin D expression and tumor invasion or metastasis but also the correlation between the intracellular immunostaining pattern of cathepsin D and tumor invasion and metastasis in human colorectal tumors. METHODS: Thirty-four primary colorectal adenocarcinomas and 24 adenomas were immunostained by use of an anticathepsin D antibody. Both the incidence and the immunostaining patterns of cathepsin D were investigated in all tissue samples. RESULTS: Three different immunostaining patterns,i.e., supranuclear, basal, and diffuse, were observed in samples containing cathepsin D. Although the incidence of cathepsin D-positive carcinomas was not correlated with tumor progression, invasion, or metastasis, the immunostaining pattern was significantly correlated with lymphatic invasion. CONCLUSIONS: The results of this study suggest that abnormal cathepsin D immunostaining patterns (basal or diffuse) can be used to predict a potential for lymphatic invasion in colorectal carcinoma.Supported in part by a Grant-in-Aid for Cancer Research (9–24) from the Ministry of Health and Welfare, Japan.     

Immunohistochemical study on expression of cathepsin in gastric carcinoma

AIM To study the expression of cathepsin B in gastric carcinoma and its relationship with pathologic type.METHODS The cathepsin B expression in 54 specimens of human gastric adenocarcinoma was studied byimmunohistochemistry.RESULTS The cathepsin B expression was detected in 33/54 (61.1%) specimens of human gastriccarcinoma and in 3/54 (5.6%) of normal tissue (P<0.01). There was no obvious correlation between theexpression of cathepsin B and pathologic type of gastric adenocarcinoma.CONCLUSION There is a high expression of cathepsin B in human gastric adenocarcinoma.     

Renin-like and cathepsin D activities in bovine pineal glands

Pineal extract is shown to contain both renin-like and cathepsin D activities. Evidence of renin-like activity in the bovine pineal gland was brought by incubation with natural and synthetic renin substrates and by inhibition by pepstatin. Cathepsin D activity was demonstrated by incubation with hemoglobin and synthetic fluorogenic peptide. The separation of both activities was performed by affinity chromatography on a caseinyl-Sepharose gel. The elution of the extract on affinity chromatography allowed to separate the renin-like activity, which is not retained by the gel at acid pH, from cathepsin D activity, which binds to the column at acid pH and is eluted at alkaline pH. The isolated pineal renin-like activity was found higher on tetradecapeptide renin substrate than on angiotensinogen at pH 5.5. The pH dependence of pineal renin-like activity showed two peaks of activity. One broad peak between pH 6 and 8 and one sharp peak at pH 3.5-4. These results demonstrate the existence of renin-like and cathepsin D activities in bovine pineal gland. They suggest moreover that the renin-like activity measured might represent a mixture of at least two different enzymatic activities.