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H A Chapman

Publications and source records attributed to H A Chapman.

At least 37 records · Page 2Linked to original sources
PubMed1999

A role for caveolin and the urokinase receptor in integrin-mediated adhesion and signaling.

The assembly of signaling molecules surrounding the integrin family of adhesion receptors remains poorly understood. Recently, the membrane protein caveolin was found in complexes with beta1 integrins. Caveolin binds cholesterol and several signaling molecules potentially linked to integrin function, e.g., Src family kinases, although caveolin has not been directly implicated in integrin-dependent adhesion. Here we report that depletion of caveolin by antisense methodology in kidney 293 cells disrupts the association of Src kinases with beta1 integrins resulting in loss of focal adhesion sites, ligand-induced focal adhesion kinase (FAK) phosphorylation, and adhesion. The nonintegrin urokinase receptor (uPAR) associates with and stabilizes beta1 integrin/caveolin complexes. Depletion of caveolin in uPAR-expressing 293 cells also disrupts uPAR/integrin complexes and uPAR-dependent adhesion. Further, beta1 integrin/caveolin complexes could be disassociated by uPAR-binding peptides in both uPAR-transfected 293 cells and human vascular smooth muscle cells. Disruption of complexes by peptides in intact smooth muscle cells blocks the association of Src family kinases with beta1 integrins and markedly impairs their migration on fibronectin. We conclude that ligand-induced signaling necessary for normal beta1 integrin function requires caveolin and is regulated by uPAR. Caveolin and uPAR may operate within adhesion sites to organize kinase-rich lipid domains in proximity to integrins, promoting efficient signal transduction.

Caveolin 1↗
PubMed1999

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.

Animals↗
PubMed1999

Proteases involved in MHC class II antigen presentation.

Major histocompatibility complex class II antigen presentation requires the participation of lysosomal proteases in two convergent processes. First, the antigens endocytosed by the antigen-presenting cells must be broken down into antigenic peptides. Second, class II molecules are synthesized with their peptide-binding site blocked by invariant chain (Ii), and they acquire the capacity to bind antigens only after Ii has been degraded in the compartments where peptides reside. The study of genetically modified mice deficient in single lysosomal proteases has allowed us to determine their role in these processes. Cathepsins (Cat) B and D, previously considered major players in MHC class II antigen presentation, are dispensable for degradation of Ii and for generation of several antigenic determinants. By contrast, Cat S plays an essential role in removal of Ii in B cells and dendritic cells, whereas Cat L apparently does so in thymic epithelial cells. Accordingly, the absence of Cat S and L have major consequences for the onset of humoral immune responses and for T-cell selection, respectively. It is likely that other as yet uncharacterized lysosomal enzymes also play a role in Ii degradation and in generation of antigenic determinants. Experiments involving drugs that interfere with protein traffic suggest that more than one mechanism for Ii removal, probably involving different proteases, can co-exist in the same antigen-presenting cell. These findings may allow the development of protease inhibitors with possible therapeutic applications.

Animals↗
PubMed1999

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.

Aorta↗
PubMed1999

Role of urokinase receptor and caveolin in regulation of integrin signaling.

Emerging evidence indicates a prominent role for non-integrin membrane adaptors in the dynamic regulation of integrin signaling. Two such integrin-associated proteins are the glycosylphosphatidyl-inositol (GPI)-linked urokinase receptor (u-PAR) and the cholesterol-binding protein, caveolin-1. Recent studies indicate that caveolin is required for the association of Src-family kinases with beta 1 integrins. Loss of caveolin/beta 1 integrin association results in loss of ligand-induced focal adhesion kinase (FAK) phosphorylation and impaired development of focal adhesion sites. Similarly, fibronectin-dependent fyn signaling through alpha 5/beta 1 leading to mitogen-activated protein (MAP) kinase activation requires the presence of caveolin-1. Caveolin binds Src-family kinases and such binding maintains these kinases in an inactive state. Current evidence favors a model in which ligand-induced integrin clustering, a central event in integrin activation, promotes caveolin oligomerization leading to release and/or activation of Src-family kinases and initiation of integrin signaling. The presence of u-PAR promotes these events because the extracellular domain(s) of u-PAR binds to beta 1 and beta 2 integrins and the GPI anchor of u-PAR, like that of other GPI-anchored proteins, interacts with cholesterol-rich membrane domains enriched in caveolin and tyrosine kinases. Integrins, caveolin, and u-PAR form interdependent functional complexes, promoting the association of integrins with caveolin-rich signaling domains. During states of accelerated cellular migration, such as during inflammation and tumorigenesis, expression of u-PAR may be a key facilitator of integrin signaling. Interruption of u-PAR/integrin interactions may be a strategy to regulate cellular migration in these settings.

Animals↗
PubMed1998

Human cathepsin F. Molecular cloning, functional expression, tissue localization, and enzymatic characterization.

A cDNA for a novel human papain-like cysteine protease, designated cathepsin F, has been cloned from a lambdagt10-skeletal muscle cDNA library. The nucleotide sequence encoded a polypeptide of 302 amino acids composed of an 88-residue propeptide and a 214-residue mature protein. Protein sequence comparisons revealed 58% homology with cathepsin W; about 42-43% with cathepsins L, K, S, H, and O; and 38% with cathepsin B. Sequence comparisons of the propeptides indicated that cathepsin F and cathepsin W may form a new cathepsin subgroup. Northern blot analysis showed high expression levels in heart, skeletal muscle, brain, testis, and ovary; moderate levels in prostate, placenta, liver, and colon; and no detectable expression in peripheral leukocytes and thymus. The precursor polypeptide of human recombinant cathepsin F, produced in Pichia pastoris, was processed to its active mature form autocatalytically or by incubation with pepsin. Mature cathepsin F was highly active with comparable specific activities toward synthetic substrates as reported for cathepsin L. The protease had a broad pH optimum between 5.2 and 6.8. Similar to cathepsin L, its pH stability at cytosolic pH (7.2) was short, with a half-life of approximately 2 min. This may suggest a function in an acidic cellular compartment. Transient expression of T7-tagged cathepsin F in COS-7 cells revealed a vesicular distribution of the gene product in the juxtanuclear region of the cells. However, contrary to all known cathepsins, the open reading frame of the cathepsin F cDNA did not encode a signal sequence, thus suggesting that the protease is targeted to the lysosomal compartment via an N-terminal signal peptide-independent lysosomal targeting pathway.

Amino Acid Sequence↗
PubMed1998

Fc epsilon receptor I on dendritic cells delivers IgE-bound multivalent antigens into a cathepsin S-dependent pathway of MHC class II presentation.

In this study, we elucidate the Fc epsilon RI-mediated Ag uptake and presentation mechanisms of dendritic cells (DC). We found that Fc epsilon RI-bound IgE, after polyvalent but not after monovalent ligation, is efficiently internalized into acidic, proteolytic compartments, degraded, and delivered into organelles containing MHC class II, HLA-DM, and lysosomal proteins. To follow the fate of the fragmented ligand, we sought to interfere with invariant chain (Ii) degradation, a process critical for peptide loading of nascent MHC class II molecules. We found DC to express cathepsin (Cat) S, a cysteine protease involved in Ii processing by B cells. Exposure of DC to a specific, active-site inhibitor of Cat S resulted in the loss of anti-Cat S immunoreactivity, led to the appearance of an N-terminal Ii remnant, and decreased the export of newly synthesized MHC class II to the DC surface. Furthermore, inactivation of Cat S as well as blockade of protein neosynthesis by cycloheximide strongly reduced IgE/Fc epsilon RI-mediated Ag presentation by DC. Thus, multimeric ligands of Fc epsilon RI, instead of being delivered into a recycling MHC class H pathway, are channeled efficiently into MIIC (MHC class II compartment)-like organelles of DC, in which Cat S-dependent Ii processing and peptide loading of newly synthesized MHC class II molecules occur. This IgE/Fc epsilon RI-dependent signaling pathway in DC may be a particularly effective route for immunization and a promising target for interfering with the early steps of allergen presentation.

Animals↗
PubMed1998

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.

Arteriosclerosis↗
PubMed1998

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.

Animals↗
PubMed1998

Cross-class inhibition of the cysteine proteinases cathepsins K, L, and S by the serpin squamous cell carcinoma antigen 1: a kinetic analysis.

The human squamous cell carcinoma antigens (SCCA) 1 and 2 are tandemly arrayed genes that encode two high-molecular-weight serine proteinase inhibitors (serpins). Although these proteins are 92% identical, differences in their reactive site loops suggest that they inhibit different types of proteinases. Our previous studies show that SCCA2 inhibits chymotrypsin-like serine proteinases [Schick et al. (1997) J. Biol. Chem. 272, 1849-1855]. We now show that, unlike SCCA2, SCCA1 lacks inhibitory activity against any of the more common types of serine proteinases but is a potent cross-class inhibitor of the archetypal lysosomal cysteine proteinases cathepsins K, L, and S. Kinetic analysis revealed that SCCA1 interacted with cathepsins K, L, and S at 1:1 stoichiometry and with second-order rate constants >/= 1 x 10(5) M-1 s-1. These rate constants were comparable to those obtained with the prototypical physiological cysteine proteinase inhibitor, cystatin C. Also relative to cystatin C, SCCA1 was a more potent inhibitor of cathepsin K-mediated elastolytic activity by forming longer lived inhibitor-proteinase complexes. The t1/2 of SCCA1-cathepsin S complexes was >1155 min, whereas that of cystatin C-cathepsin complexes was 55 min. Cleavage between the Gly and Ser residues of the reactive site loop and detection of a stable SCCA1-cathepsin S complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that the serpin interacted with the cysteine proteinase in a manner similar to that observed for typical serpin-serine proteinase interactions. These data suggest that, contingent upon their reactive site loop sequences, mammalian serpins, in general, utilize their dynamic tertiary structure to trap proteinases from more than one mechanistic class and that SCCA1, in particular, may be involved in a novel inhibitory pathway aimed at regulating a powerful array of lysosomal cysteine proteinases.

Amino Acid Sequence↗
PubMed1998

Endosomal proteolysis and MHC class II function.

Newly synthesized MHC class II alpha and beta chains associate with a protein chaperone, the invariant chain, which promotes the proper assembly of MHC class II complexes and their trafficking through cells and prevents their untimely loading with peptides. Efficient loading of MHC class II heterodimers with antigenic peptides requires concurrent proteolytic processing of both the invariant chain and endocytosed proteins. Recent studies have elucidated the critical roles of specific cysteine proteases, especially cathepsins S and L, in degrading the invariant chain and regulating the convergence of processed antigen and MHC class II dimers competent for peptide loading.

Amino Acid Sequence↗
PubMed1998

Genetic epidemiology of severe, early-onset chronic obstructive pulmonary disease. Risk to relatives for airflow obstruction and chronic bronchitis.

Severe alpha-1-antitrypsin deficiency is the only proven genetic risk factor for chronic obstructive pulmonary disease (COPD). We have assembled a cohort of 44 probands with severe, early-onset COPD, who do not have severe alpha-1-antitrypsin deficiency. A surprisingly high prevalence of females (79.6%) was found. Assessment of the risk to relatives of these early-onset COPD probands for airflow obstruction and chronic bronchitis was performed to determine whether significant familial aggregation for COPD, independent of alpha-1-antitrypsin deficiency, could be demonstrated. First- degree relatives of early-onset COPD probands had significantly lower FEV1 and FEV1/FVC values than control subjects (p < 0.01), despite similar pack-years of smoking. Reduced spirometric values in first-degree relatives of early-onset COPD probands were found only in current or ex-cigarette smokers. The mean FEV1 in current or ex-smoking first-degree relatives was 76.1 +/- 20.9% predicted compared to 89.2 +/- 14.4% predicted in current or ex-smoking control subjects (p < 0.01); in lifelong nonsmokers, the mean FEV1 was 93.4% predicted for both control subjects and first-degree relatives of early-onset COPD probands. Generalized estimating equations, adjusting for age and pack-years of smoking, demonstrated increased odds of reduced FEV1 and chronic bronchitis in current or ex-smoking first-degree relatives of early-onset COPD probands. Using a new method to estimate relative risk from relative odds, we estimate that the relative risks for FEV1 below 60%, FEV1 below 80%, and chronic bronchitis are each approximately three in current or ex-smoking first-degree relatives of early-onset COPD probands. The increased risk to relatives of early-onset COPD probands for reduced FEV1 and chronic bronchitis, limited to current or ex-smokers, suggests genetic risk factor(s) for COPD that are expressed in response to cigarette smoking.

Bronchitis↗
PubMed1997

Degradation of mouse invariant chain: roles of cathepsins S and D and the influence of major histocompatibility complex polymorphism.

Antigen-presenting cells (APC) degrade endocytosed antigens into peptides that are bound and presented to T cells by major histocompatibility complex (MHC) class II molecules. Class II molecules are delivered to endocytic compartments by the class II accessory molecule invariant chain (Ii), which itself must be eliminated to allow peptide binding. The cellular location of Ii degradation, as well as the enzymology of this event, are important in determining the sets of antigenic peptides that will bind to class II molecules. Here, we show that the cysteine protease cathepsin S acts in a concerted fashion with other cysteine and noncysteine proteases to degrade mouse Ii in a stepwise fashion. Inactivation of cysteine proteases results in incomplete degradation of Ii, but the extent to which peptide loading is blocked by such treatment varies widely among MHC class II allelic products. These observations suggest that, first, class II molecules associated with larger Ii remnants can be converted efficiently to class II-peptide complexes and, second, that most class II-associated peptides can still be generated in cells treated with inhibitors of cysteine proteases. Surprisingly, maturation of MHC class II in mice deficient in cathepsin D is unaffected, showing that this major aspartyl protease is not involved in degradation of Ii or in generation of the bulk of antigenic peptides.

Amino Acid Sequence↗
PubMed1997

Plasmin and plasminogen activator inhibitor type 1 promote cellular motility by regulating the interaction between the urokinase receptor and vitronectin.

The urokinase receptor (uPAR) coordinates plasmin-mediated cell-surface proteolysis and promotes cellular adhesion via a binding site for vitronectin on uPAR. Because vitronectin also binds plasminogen activator inhibitor type 1 (PAI-1), and plasmin cleavage of vitronectin reduces PAI-1 binding, we explored the effects of plasmin and PAI-1 on the interaction between uPAR and vitronectin. PAI-1 blocked cellular binding of and adhesion to vitronectin by over 80% (IC50 approximately 5 nM), promoted detachment of uPAR-bearing cells from vitronectin, and increased cellular migration on vitronectin. Limited cleavage of vitronectin by plasmin also abolished cellular binding and adhesion and induced cellular detachment. A series of peptides surrounding a plasmin cleavage site (arginine 361) near the carboxy-terminal end of vitronectin were synthesized. Two peptides spanning res 364-380 blocked binding of uPAR to vitronectin (IC50 approximately 8-25 microM) identifying this region as an important site of uPAR-vitronectin interaction. These data illuminate a complex regulatory scheme for uPAR-dependent cellular adhesion to vitronectin: Active urokinase promotes adhesion and also subsequent detachment through activation of plasmin or complex formation with PAI-1. Excess PAI-1 may also promote migration by blocking cellular adhesion and/or promoting detachment, possibly accounting in part for the strong correlation between PAI-1 expression and tumor cell metastasis.

Cell Adhesion↗
PubMed1997

Structure and chromosomal assignment of the human cathepsin K gene.

Cathepsin K is a recently identified lysosomal cysteine proteinase that is the major protease responsible for bone resorption and remodeling. Mutations in this gene cause the sclerosing osteochondrodysplasia pycnodysostosis. To assess its evolutionary relatedness to other cysteine proteases and to facilitate mutation identification in patients with pycnodysostosis, a genomic clone, 74e16, containing the cathepsin K gene was isolated from a human PAC library, and the cathepsin K genomic structure was determined. The cathepsin K gene contained eight exons and spanned approximately 9 kb. The transcription initiation site, determined by primer extension analysis, was 169 nucleotides upstream from the translation initiation site. The 5'-flanking region lacked a TATA box but contained two AP1 sites. Comparison of genomic and cDNA sequences suggested that this flanking sequence may be the major promoter in osteoclasts and macrophages. Cathepsin K was mapped to chromosome 1q21 by fluorescence in situ hybridization and found to reside within 150 kb of an evolutionarily related cysteine protease, cathepsin S. These findings expand our understanding of the papain family lysosomal cysteine proteases and should facilitate mutation analysis in pycnodysostosis.

Base Sequence↗
PubMed1997

Plasminogen activators, integrins, and the coordinated regulation of cell adhesion and migration.

Cellular migration is critically dependent on an interplay between forces of attachment and detachment. Recent studies show that the serine protease urokinase and its major inhibitor and receptor regulate the adhesive properties of integrins, at least in part through initiation of cellular signals. These new functions for an old protease system imply intricate connections between proteolysis and adhesion that operate at the cell surface to regulate migration.

Animals↗
PubMed1997

Partners' stress underestimations lead to women's distress: a study of pregnant inner-city women.

The authors examined how men's underestimation of the stress that their pregnant female partners reported influenced women's psychological distress and their sense that they were not supported. Participants included 68 pregnant inner-city women and their partners, among whom African Americans and European Americans were represented. Women who reported a greater number of stressful life events had increased depression if their partners did not report them as encountering these events. However, if their partners reported them as encountering a high number of stressful events, the otherwise negative impact of stress was buffered. Partners' stress report had no appreciable effect when women reported a low number of stressful events. This partner underestimation effect was independent of the influence of women's report of social support.

Adaptation, Psychological↗