Regular ArticleSynthesis and Secretion of Procathepsin B and Cystatin C by Human Bronchial Epithelial Cells in Vitro: Modulation of Cathepsin B Activity by Neutrophil Elastase
Abstract
Procathepsin B and cystatin C are found in human lung secretions. We investigated the capacity of human bronchial epithelial cells to synthesize and secrete these proteins. Immunoprecipitation of [35S]methionine-labeled proteins from cultured bronchial epithelial cell lysates, followed by denaturing gel electrophoresis and autoradiography, showed the presence of newly synthesized procathepsin B of Mr 42,000; no mature form was detected. Cathepsin B in conditioned medium from epithelial cells was tagged with benzyloxycarbonyl-125I-tyrosyl-alanine-diazomethane before and after treatment of the medium with neutrophil elastase. Control medium again showed a predominant form of cathepsin B with a Mr of 42,000, but upon treatment with neutrophil elastase this protein was converted to a Mr of 38,000, similar to the active form previously found in lung secretions, and cathepsin B activity was generated. The medium also contained the cathepsin B inhibitor, cystatin C, but cystatins A, B, S, SN, SA, and kininogen were not detected. After removal of cystatin C from the medium, elastase was still required to activate procathepsin B. These results suggest that bronchial epithelial cells are a source of procathepsin B and cystatin C in lung secretions. Cleavage both of cystatin C and procathepsin B by neutrophil elastase is essential for the generation of cathepsin B activity in the medium.
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Cathepsin B
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2007, xPharm: The Comprehensive Pharmacology ReferenceCathepsin B is a member of the …
PROTEINASE INHIBITORS | Cystatins
2006, Encyclopedia of Respiratory Medicine: Volume 1-4The cystatins comprise a superfamily of proteins related primarily by virtue of DNA and amino acid sequence homology. The superfamily consists so far of four distinct types of molecules ranging from the simpler low-molecular-weight type I and II cystatins, which function primarily to inhibit lysosomal cysteine proteinases (CPs), to the higher-molecular-weight type III and IV cystatins, which possess additional latent functions expressed only during episodes of injury and inflammation, or have evolved entirely novel inhibitory functions.
The role cystatins play in respiratory diseases such as asthma and COPD is poorly understood. However, they do modulate the immune response by acting directly on neutrophils, macrophages, and antigen presenting cells. It is also clear that they do not function independently of other proteolytic pathways involved in remodeling of the lung. Limited proteolysis inactivates cystatins allowing lysosomal CP activity to directly contribute to lung tissue degradation and also liberates kinins which signal through G-protein-coupled receptors to cause both constriction and dilation of the bronchioles, pain via stimulation of sensory nerves, mucus secretion, cough, and edema.
Proteinase Inhibitors: Cystatins
2006, Encyclopedia of Respiratory Medicine, Four-Volume SetThe cystatins comprise a superfamily of proteins related primarily by virtue of DNA and amino acid sequence homology. The superfamily consists so far of four distinct types of molecules ranging from the simpler low-molecular-weight type I and II cystatins, which function primarily to inhibit lysosomal cysteine proteinases (CPs), to the higher-molecular-weight type III and IV cystatins, which possess additional latent functions expressed only during episodes of injury and inflammation, or have evolved entirely novel inhibitory functions.
The role cystatins play in respiratory diseases such as asthma and COPD is poorly understood. However, they do modulate the immune response by acting directly on neutrophils, macrophages, and antigen presenting cells. It is also clear that they do not function independently of other proteolytic pathways involved in remodeling of the lung. Limited proteolysis inactivates cystatins allowing lysosomal CP activity to directly contribute to lung tissue degradation and also liberates kinins which signal through G-protein-coupled receptors to cause both constriction and dilation of the bronchioles, pain via stimulation of sensory nerves, mucus secretion, cough, and edema.
Bone microenvironment modulates expression and activity of cathepsin B in prostate cancer
2005, NeoplasiaProstate cancers metastasize to bone leading to osteolysis. Here we assessed proteolysis of DOcollagen I (a bone matrix protein) and, for comparison, DO-collagen IV, by living human prostate carcinoma cells in vitro. Both collagens were degraded, this degradation was reduced by inhibitors of matrix metallo, serine, cysteine proteases. Because secretion of the cysteine protease cathepsin B is increased in human breast fibroblasts grown on collagen I gels, we analyzed cathepsin B levels and secretion in prostate cells grown on collagen I gels. Levels and secretion were increased only in DU145 cells-cells that expressed the highest baseline levels of cathepsin B. Secretion of cathepsin B was also elevated in DU145 cells grown in vitro on human bone fragments. We further investigated the effect of the bone microenvironment on cathepsin B expression and activity in vivo in a SCID-human model of prostate bone metastasis. High levels of cathepsin B protein and activity were found in DU145, PC3, LNCaP bone tumors, although the PC3 and LNCaP cells had exhibited low cathepsin B expression in vitro. Our results suggest that tumor-stromal interactions in the context of the bone microenvironment can modulate the expression of the cysteine protease cathepsin B.
Is the cystatin-like domain of TSL functionally active in external ocular infections and during the normal diurnal cycle?
2004, Experimental Eye ResearchPurpose. To test whether the cystatin-like functional domain in tear specific lipocalin (TSL) is functionally active in tears during the normal diurnal cycle and during external ocular infections.
Methods. Capillary tube collected reflex (RTF), open (OTF) and closed (CTF) eye tear samples were recovered from six normals and semi-quantitatively western blot assayed for cystatin C and TSL. CTF samples were immunoprecipitated with antibodies raised against TSL, cystatin C and other antiproteases and screened for the co-precipitation of proteases by casein and gelatin zymography. OTF samples recovered from individuals with viral, fungal and bacterial keratitis were similarly screened for TSL-bound proteases. Human tissue was subjected to immunohistochemical study.
Results. Western blot analysis reveals a progressive increase in cystatin C in going from RTF to OTF to CTF samples (∼3, 7 and 30 ng μl−1, respectively). In contrast, the concentration of TSL remains constant (∼1500 ng μl−1). Immunocytochemistry data show staining of the apical surface of the human conjunctiva and some intra-cellular staining for cystatin C, but not for cystatin A. Zymography confirms earlier data that CTF contains exceptionally high levels of proteases bound to a wide range of specific inhibitors. However, only trace amounts of proteases are complexed with cystatin C and no protease can be detected bound to TSL in either the pathological or CTF samples.
Conclusion. Although TSL contains a functional cystatin-like domain, it is not physiologically active during the normal diurnal cycle or during external ocular infections. Reactive proteases in CTF are most likely controlled by the presence of excess levels of more reactive cystatins, especially cystatin C, which accumulates during prolonged eye closure. Immunohistochemical data suggest that the apical conjunctiva may be a contributing source for the accumulating cystatin C.