TNF-α–induced apoptosis is thought to involve mediators from acidic vesicles. Cathepsin B (cat B), a lysosomal cysteine protease, has recently been implicated in apoptosis. To determine whether cat B contributes to TNF-α–induced apoptosis, we exposed mouse hepatocytes to the cytokine in vitro and in vivo. Isolated hepatocytes treated with TNF-α in the presence of the transcription inhibitor actinomycin D (AcD) accumulated cat B in their cytosol. Further experiments using cell-free systems indicated that caspase-8 caused release of active cat B from purified lysosomes and that cat B, in turn, increased cytosol-induced release of cytochrome c from mitochondria. Consistent with these observations, the ability of TNF-α/AcD to induce mitochondrial release of cytochrome c, caspase activation, and apoptosis of isolated hepatocytes was markedly diminished in cells from CatB^–/– mice. Deletion of the CatB gene resulted in diminished liver injury and enhanced survival after treatment in vivo with TNF-α and an adenovirus construct expressing the IκB superrepressor. Collectively, these observations suggest that caspase-mediated release of cat B from lysosomes enhances mitochondrial release of cytochrome c and subsequent caspase activation in TNF-α–treated hepatocytes.

Degradation of invariant chain (Ii) is a critical step in major histocompatibility complex class II–restricted antigen presentation. Cathepsin L was found to be necessary for Ii degradation in cortical thymic epithelial cells (cTECs), but not in bone marrow (BM)–derived antigen-presenting cells (APCs). Consequently, positive selection of CD4 ⁺ T cells was reduced. Because different cysteine proteinases are responsible for specific Ii degradation steps in cTECs and BM-derived APCs, the proteolytic environment in cells mediating positive and negative selection may be distinct. The identification of a protease involved in class II presentation in a tissue-specific manner suggests a potential means of manipulating CD4 ⁺ T cell responsiveness in vivo.

Autodigestion of the pancreas by its own prematurely activated digestive proteases is thought to be an important event in the onset of acute pancreatitis. The mechanism responsible for the intrapancreatic activation of digestive zymogens is unknown, but a recent hypothesis predicts that a redistribution of lysosomal cathepsin B (CTSB) into a zymogen-containing subcellular compartment triggers this event. To test this hypothesis, we used CTSB-deficient mice in which the ctsb gene had been deleted by targeted disruption. After induction of experimental secretagogue–induced pancreatitis, the trypsin activity in the pancreas of ctsb^–/– animals was more than 80% lower than in ctsb^+/+ animals. Pancreatic damage as indicated by serum activities of amylase and lipase, or by the extent of acinar tissue necrosis, was 50% lower in ctsb^–/– animals. These experiments provide the first conclusive evidence to our knowledge that cathepsin B plays a role in intrapancreatic trypsinogen activation and the onset of acute pancreatitis.

Proteolysis in close vicinity of tumor cells is a hallmark of cancer invasion and metastasis. We show here that mouse mammary tumor virus–polyoma middle T antigen (PyMT) transgenic mice deficient for the cysteine protease cathepsin B (CTSB) exhibited a significantly delayed onset and reduced growth rate of mammary cancers compared with wild-type PyMT mice. Lung metastasis volumes were significantly reduced in PyMT;ctsb^+/−, an effect that was not further enhanced in PyMT;ctsb^−/− mice. Furthermore, lung colonization studies of PyMT cells with different CTSB genotypes injected into congenic wild-type mice and in vitro Matrigel invasion assays confirmed a specific role for tumor-derived CTSB in invasion and metastasis. Interestingly, cell surface labeling of cysteine cathepsins by the active site probe DCG-04 detected up-regulation of cathepsin X on PyMT;ctsb^−/− cells. Treatment of cells with a neutralizing anti-cathepsin X antibody significantly reduced Matrigel invasion of PyMT;ctsb^−/− cells but did not affect invasion of PyMT;ctsb^+/+ or PyMT;ctsb^+/− cells, indicating a compensatory function of cathepsin X in CTSB-deficient tumor cells. Finally, an adoptive transfer model, in which ctsb^+/+, ctsb^+/−, and ctsb^−/− recipient mice were challenged with PyMT;ctsb^+/+ cells, was used to address the role of stroma-derived CTSB in lung metastasis formation. Notably, ctsb^−/− mice showed reduced number and volume of lung colonies, and infiltrating macrophages showed a strongly up-regulated expression of CTSB within metastatic cell populations. These results indicate that both cancer cell–derived and stroma cell–derived (i.e., macrophages) CTSB plays an important role in tumor progression and metastasis. (Cancer Res 2006; 66(10): 5242-50)