Guo-Ping Shi

Pycnodysostosis, a lysosomal disease caused by cathepsin K deficiency

Pycnodysostosis, an autosomal recessive osteochondrodysplasia characterized by osteosclerosis and short stature, maps to chromosome 1q21. Cathepsin K, a cysteine protease gene that is highly expressed in osteoclasts, localized to the pycnodysostosis region. Nonsense, missense, and stop codon mutations in the gene encoding cathepsin K were identified in patients. Transient expression of complementary DNA containing the stop codon mutation resulted in messenger RNA but no immunologically detectable protein. Thus, pycnodysostosis results from gene defects in a lysosomal protease with highest expression in osteoclasts. These findings suggest that cathepsin K is a major protease in bone resorption, providing a possible rationale for the treatment of disorders such as osteoporosis and certain forms of arthritis.

Expression of the elastolytic cathepsins S and K in human atheroma and regulation of their production in smooth muscle cells.

Formation of the atherosclerotic intima must involve altered metabolism of the elastin-rich arterial extracellular matrix. Proteases potentially involved in these processes remain unclear. This study examined the expression of the potent elastases cathepsins S and K in human atheroma. Normal arteries contained little or no cathepsin K or S. In contrast, macrophages in atheroma contained abundant immunoreactive cathepsins K and S. Intimal smooth muscle cells (SMC), especially cells appearing to traverse the internal elastic laminae, also contained these enzymes. Extracts of atheromatous tissues had approximately twofold greater elastase-specific activity than extracts of uninvolved arteries, mostly due to cysteine proteases. Cultured human SMC displayed no immunoreactive cathepsins K and S and exhibited little or no elastolytic activity when incubated with insoluble elastin. SMC stimulated with the atheroma-associated cytokines IL-1beta or IFN-gamma secreted active cathepsin S and degraded substantial insoluble elastin (15-20 microg/10(6) cells/24 h). A selective inhibitor of cathepsin S blocked > 80% of this elastolytic activity. The presence of cathepsins K and S at sites of vascular matrix remodeling and the ability of SMC and macrophages to use these enzymes to degrade elastin supports a role for elastolytic cathepsins in vessel wall remodeling and identifies novel therapeutic targets in regulating plaque stability.

Genetic deficiency and pharmacological stabilization of mast cells reduce diet-induced obesity and diabetes in mice

Although mast cell functions have classically been related to allergic responses, recent studies indicate that these cells contribute to other common diseases such as multiple sclerosis, rheumatoid arthritis, atherosclerosis, aortic aneurysm and cancer. This study presents evidence that mast cells also contribute to diet-induced obesity and diabetes. For example, white adipose tissue (WAT) from obese humans and mice contain more mast cells than WAT from their lean counterparts. Furthermore, in the context of mice on a Western diet, genetically induced deficiency of mast cells, or their pharmacological stabilization, reduces body weight gain and levels of inflammatory cytokines, chemokines and proteases in serum and WAT, in concert with improved glucose homeostasis and energy expenditure. Mechanistic studies reveal that mast cells contribute to WAT and muscle angiogenesis and associated cell apoptosis and cathepsin activity. Adoptive transfer experiments of cytokine-deficient mast cells show that these cells, by producing interleukin-6 (IL-6) and interferon-γ (IFN-γ), contribute to mouse adipose tissue cysteine protease cathepsin expression, apoptosis and angiogenesis, thereby promoting diet-induced obesity and glucose intolerance. Our results showing reduced obesity and diabetes in mice treated with clinically available mast cell-stabilizing agents suggest the potential of developing new therapies for these common human metabolic disorders.

Cathepsin S Required for Normal MHC Class II Peptide Loading and Germinal Center Development

Major histocompatibility complex (MHC) class II molecules acquire antigenic peptides after degradation of the invariant chain (Ii), an MHC class II-associated protein that otherwise blocks peptide binding. Antigen-presenting cells of mice that lack the protease cathepsin S fail to process Ii beyond a 10 kDa fragment, resulting in delayed peptide loading and accumulation of cell surface MHC class II/10 kDa Ii complexes. Although cathepsin S-deficient mice have normal numbers of B and T cells and normal IgE responses, they show markedly impaired antibody class switching to IgG2a and IgG3. These results indicate cathepsin S is a major Ii-processing enzyme in splenocytes and dendritic cells. Its role in humoral immunity critically depends on how antigens access the immune system.

The ectodomain of Toll-like receptor 9 is cleaved to generate a functional receptor

Mammalian Toll-like receptors (TLRs) 3, 7, 8 and 9 initiate immune responses to infection by recognizing microbial nucleic acids; however, these responses come at the cost of potential autoimmunity owing to inappropriate recognition of self nucleic acids. The localization of TLR9 and TLR7 to intracellular compartments seems to have a role in facilitating responses to viral nucleic acids while maintaining tolerance to self nucleic acids, yet the cell biology regulating the transport and localization of these receptors remains poorly understood. Here we define the route by which TLR9 and TLR7 exit the endoplasmic reticulum and travel to endolysosomes in mouse macrophages and dendritic cells. The ectodomains of TLR9 and TLR7 are cleaved in the endolysosome, such that no full-length protein is detectable in the compartment where ligand is recognized. Notably, although both the full-length and cleaved forms of TLR9 are capable of binding ligand, only the processed form recruits MyD88 on activation, indicating that this truncated receptor, rather than the full-length form, is functional. Furthermore, conditions that prevent receptor proteolysis, including forced TLR9 surface localization, render the receptor non-functional. We propose that ectodomain cleavage represents a strategy to restrict receptor activation to endolysosomal compartments and prevent TLRs from responding to self nucleic acids.

Cystatin C deficiency in human atherosclerosis and aortic aneurysms

The pathogenesis of atherosclerosis and abdominal aortic aneurysm involves breakdown of the elastic laminae. Elastolytic cysteine proteases, including cathepsins S and K, are overexpressed at sites of arterial elastin damage, but whether endogenous local inhibitors counterbalance these proteases is unknown. We show here that, whereas cystatin C is normally expressed in vascular wall smooth muscle cells (SMCs), this cysteine protease inhibitor is severely reduced in both atherosclerotic and aneurysmal aortic lesions. Furthermore, increased abdominal aortic diameter among 122 patients screened by ultrasonography correlated inversely with serum cystatin C levels. In vitro, cytokine-stimulated vascular SMCs secrete cathepsins, whose elastolytic activity could be blocked when cystatin C secretion was induced by treatment with TGF-beta(1). The findings highlight a potentially important role for imbalance between cysteine proteases and cystatin C in arterial wall remodeling and establish that cystatin C deficiency occurs in vascular disease.

Mast cells promote atherosclerosis by releasing proinflammatory cytokines.

Mast cells contribute importantly to allergic and innate immune responses by releasing various preformed and newly synthesized mediators. Previous studies have shown mast cell accumulation in human atherosclerotic lesions. This report establishes the direct participation of mast cells in atherogenesis in low-density lipoprotein receptor–deficient (Ldlr−/−) mice. Atheromata from compound mutant Ldlr−/− KitW-sh/W-sh mice showed decreased lesion size, lipid deposition, T-cell and macrophage numbers, cell proliferation and apoptosis, but increased collagen content and fibrous cap development. In vivo, adoptive transfer of syngeneic wild-type or tumor necrosis factor (TNF)-α-deficient mast cells restored atherogenesis to Ldlr−/−KitW-sh/W-sh mice. Notably, neither interleukin (IL)-6- nor interferon (IFN)-γ-deficient mast cells did so, indicating that the inhibition of atherogenesis in Ldlr−/−KitW-sh/W-sh mice resulted from the absence of mast cells and mast cell–derived IL-6 and IFN-γ. Compared with wild-type or TNF-α-deficient mast cells, those lacking IL-6 or IFN-γ did not induce expression of proatherogenic cysteine proteinase cathepsins from vascular cells in vitro or affect cathepsin and matrix metalloproteinase activities in atherosclerotic lesions, implying that mast cell–derived IL-6 and IFN-γ promote atherogenesis by augmenting the expression of matrix-degrading proteases. These observations establish direct participation of mast cells and mast cell–derived IL-6 and IFN-γ in mouse atherogenesis and provide new mechanistic insight into the pathogenesis of this common disease.

Lysosomal Cysteine Proteases in Atherosclerosis

Atherosclerosis is an inflammatory disease characterized by extensive remodeling of the extracellular matrix architecture of the arterial wall. Although matrix metalloproteinases and serine proteases participate in these pathologic events, recent data from atherosclerotic patients and animals suggest the participation of lysosomal cysteine proteases in atherogenesis. Atherosclerotic lesions in humans overexpress the elastolytic and collagenolytic cathepsins S, K, and L but show relatively reduced expression of cystatin C, their endogenous inhibitor, suggesting a shift in the balance between cysteine proteases and their inhibitor that favors remodeling of the vascular wall. Extracts of human atheromatous tissue show greater elastolytic activity in vitro than do those from healthy donors. The cysteinyl protease inhibitor E64d limits this increased elastolysis, indicating involvement of cysteine proteases in elastin degradation during atherogenesis. Furthermore, inflammatory cytokines augment expression and secretion of active cysteine proteases from cultured monocyte-derived macrophages, vascular smooth muscle cells, and endothelial cells and increase degradation of extracellular elastin and collagen. Cathepsin S–deficient cells or those treated with E64d show significantly impaired elastolytic or collagenolytic activity. Additionally, recent in vivo studies of atherosclerosis-prone, LDL receptor–null mice lacking cathepsin S show participation of this enzyme in the initial infiltration of leukocytes, medial elastic lamina degradation, endothelial cell invasion, and neovascularization, illustrating an important role for cysteine proteases in arterial remodeling and atherogenesis.

Deficiency of cathepsin S reduces atherosclerosis in LDL receptor–deficient mice

Human atherosclerotic lesions overexpress the lysosomal cysteine protease cathepsin S (Cat S), one of the most potent mammalian elastases known. In contrast, atheromata have low levels of the endogenous Cat S inhibitor cystatin C compared with normal arteries, suggesting involvement of this protease in atherogenesis. The present study tested this hypothesis directly by crossing Cat S-deficient (CatS(-/-)) mice with LDL receptor-deficient (LDLR(-/-)) mice that develop atherosclerosis on a high-cholesterol diet. Compared with LDLR(-/-) mice, double-knockout mice (CatS(-/-)LDLR(-/-)) developed significantly less atherosclerosis, as indicated by plaque size (plaque area and intimal thickening) and stage of development. These mice also had markedly reduced content of intimal macrophages, lipids, smooth muscle cells, collagen, CD4(+) T lymphocytes, and levels of IFN-gamma. CatS(-/-)LDLR(-/-) monocytes showed impaired subendothelial basement membrane transmigration, and aortas from CatS(-/-)LDLR(-/-) mice had preserved elastic laminae. These findings establish a pivotal role for Cat S in atherogenesis.

Cathepsin S activity regulates antigen presentation and immunity.

MHC class II molecules display antigenic peptides on cell surfaces for recognition by CD4(+) T cells. Proteolysis is required in this process both for degradation of invariant chain (Ii) from class II-Ii complexes to allow subsequent binding of peptides, and for generation of the antigenic peptides. The cysteine endoprotease, cathepsin S, mediates Ii degradation in human and mouse antigen-presenting cells. Studies described here examine the functional significance of cathepsin S inhibition on antigen presentation and immunity. Specific inhibition of cathepsin S in A20 cells markedly impaired presentation of an ovalbumin epitope by interfering with class II-peptide binding, not by obstructing generation of the antigen. Administration of a cathepsin S inhibitor to mice in vivo selectively inhibited activity of cathepsin S in splenocytes, resulting in accumulation of a class II-associated Ii breakdown product, attenuation of class II-peptide complex formation, and inhibition of antigen presentation. Mice treated with inhibitor had an attenuated antibody response when immunized with ovalbumin but not the T cell-independent antigen TNP-Ficoll. In a mouse model of pulmonary hypersensitivity, treatment with the inhibitor also abrogated a rise in IgE titers and profoundly blocked eosinophilic infiltration in the lung. Thus, inhibition of cathepsin S in vivo alters Ii processing, antigen presentation, and immunity. These data identify selective inhibition of cysteine proteases as a potential therapeutic strategy for asthma and autoimmune disease processes.