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H A Chapman

Publications and source records attributed to H A Chapman.

At least 55 records · Page 3Linked to original sources
PubMed1997

Emerging roles for cysteine proteases in human biology.

Cysteine proteases have traditionally been viewed as lysosomal mediators of terminal protein degradation. However, recent findings refute this limited view and suggest a more expanded role for cysteine proteases in human biology. Several newly discovered members of this enzyme class are regulated proteases with limited tissue expression, which implies specific roles in cellular physiology. These roles appear to include apoptosis, MHC class II immune responses, prohormone processing, and extracellular matrix remodeling important to bone development. The ability of macrophages and other cells to mobilize elastolytic cysteine proteases to their surfaces under specialized conditions may also lead to accelerated collagen and elastin degradation at sites of inflammation in diseases such as atherosclerosis and emphysema. The development of inhibitors of specific cysteine proteases promises to provide new drugs for modifying immunity, osteoporosis, and chronic inflammation.

Amino Acid Sequence↗
PubMed1996

Mac-1 (CD11b/CD18) and the urokinase receptor (CD87) form a functional unit on monocytic cells.

The leukocyte integrin Mac-1 (CD11b/CD18) and the urokinase receptor (uPAR, CD87) mediate complementary functions in myelomonocytic cells. Both receptors promote degradation of fibrin(ogen) and also confer adhesive properties on cells because Mac-1 and uPAR bind fibrin and vitronectin, respectively. Staining of lung biopsy specimens from patients with acute lung injury indicated that fibrin and vitronectin colocalize at exudative sites in which macrophages bearing these receptors accumulate. Because of the parallel roles and physical proximity of Mac-1 and uPAR, the capacity of these receptors to functionally interact was explored. Induction of Mac-1 and uPAR expression on monocytic cell lines by transforming growth factor- beta 1 and 1.25-(OH)2 vitamin D3 conferred urokinase and uPAR-dependent adhesion to vitronectin, which was further promoted by engagement of Mac-1. Vitronectin attachment promoted subsequent Mac-1-mediated fibrinogen degradation threefold to fourfold. In contrast, enhancement of uPAR occupancy by exogenous urokinase or receptor binding fragments thereof inhibited Mac-1 function. Addition of urokinase progressively inhibited Mac-1-mediated fibrinogen binding and degradation (maximal inhibition, 91% +/- 14% and 72% +/- 15%, respectively). Saturation of uPAR with urokinase also inhibited binding of the procoagulant Mac-1 ligand, Factor X. These inhibitory effects of uPAR were reproduced in fresh monocytes, cultured monocytic cells, and in Chinese hamster ovary (CHO) cells transfected with both human Mac-1 and human uPAR. These data show that the procoagulant and fibrinolytic potential of monocytic cells is co-ordinately regulated by ligand binding to both Mac-1 and uPAR and identify uPAR as a regulator of integrin function. Vitronectin-enhanced fibrin(ogen) turnover by Mac-1 may operate as a salvage pathway in the setting of urokinase and plasmin inhibitors to promote clearance of the provisional matrix and subsequent healing.

Animals↗
PubMed1996

Regulation of integrin function by the urokinase receptor.

Integrin function is central to inflammation, immunity, and tumor progression. The urokinase-type plasminogen activator receptor (uPAR) and integrins formed stable complexes that both inhibited native integrin adhesive function and promoted adhesion to vitronectin via a ligand binding site on uPAR. Interaction of soluble uPAR with the active conformer of integrins mimicked the inhibitory effects of membrane uPAR. Both uPAR-mediated adhesion and altered integrin function were blocked by a peptide that bound to uPAR and disrupted complexes. These data provide a paradigm for regulation of integrins in which a nonintegrin membrane receptor interacts with and modifies the function of activated integrins.

Amino Acid Sequence↗
PubMed1996

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.

Amino Acid Sequence↗
PubMed1996

Essential role for cathepsin S in MHC class II-associated invariant chain processing and peptide loading.

Destruction of li by proteolysis is required for MHC class II molecules to bind antigenic peptides, and for transport of the resulting complexes to the cell surface. The cysteine protease cathepsin S is highly expressed in spleen, lymphocytes, monocytes, and other class II-positive cells, and is inducible with interferon-gamma. Specific inhibition of cathepsin S in B lymphoblastoid cells prevented complete proteolysis of li, resulting in accumulation of a class II-associated 13 kDa li fragment in vivo. Consequently, the formation of SDS-stable complexes was markedly reduced. Purified cathepsin S, but not cathepsin B, H, or D, specifically digested li from alpha beta li trimers, generating alpha beta-CLIP complexes capable of binding exogenously added peptide in vitro. Thus, cathepsin S is essential in B cells for effective li proteolysis necessary to render class II molecules competent for binding peptides.

Antigen Presentation↗
PubMed1996

Mechanisms of pertussis toxin-induced myelomonocytic cell adhesion: role of Mac-1(CD11b/CD18) and urokinase receptor (CD87).

Stimulation of monoblastic U937 cells with transforming growth factor beta 1 and 1,25-(OH)2 vitamin D3 (TGF-beta 1/D3) upregulates urokinase receptor (uPAR) and confers urokinase-dependent adhesiveness to the cells for serum- or vitronectin-coated surfaces. Recent studies show that uPAR itself is a high-affinity adhesion receptor for vitronectin and that urokinase (uPA) is an activator of this adhesive function. In the course of exploring possible G-protein involvement in this adhesion it was observed that TGF-beta 1/D3-primed U937 cells became adhesive to vitronectin in an uPAR-dependent manner when exposed to pertussis toxin (PTX). The adherent response is concentration- and time-dependent, and was not due to the ADP-ribosyltransferase activity of the toxin because the purified B-subunit of PTX was equally effective. Although promoting adhesion to serum- or vitronectin-coated surfaces, PTX blocked spontaneous cell adhesion to fibrinogen, an endogenous ligand for the Mac-1 receptor (CD11b/CD18). Flow cytometry study showed that expression of the alpha-subunit of Mac-1 (CD11b) on primed cells was increased by nearly threefold. Monoclonal antibody to CD11b abolished the PTX-induced cell adhesion and the binding of the primed cells to PTX-coated plates. Activation of Mac-1 receptor by its endogenous ligand fibrinogen induced cell adherent response similar to PTX. PTX, but not uPA, triggered a rapid rise in [Ca2+]i in primed U937 cells, and PTX-induced adhesion was significantly attenuated by 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxy-methyl ester (BAPTA/AM), a selective membrane-permeant [Ca2+]i chelator. PTX-induced cell adhesion was also prevented by antibodies to uPAR and by conditioned medium containing soluble uPAR. Together these data indicate that PTX B-subunit may bind to Mac-1 integrin, which leads to a rapid rise in [Ca2+]i and subsequent activation of uPAR for adherence to vitronectin, suggesting a functional link between Mac-1 and activation of uPAR important to cellular trafficking and host defence in response to Bordetella pertussis infection.

Antibodies, Monoclonal↗
PubMed1995

Lysosomal processing of amyloid precursor protein to A beta peptides: a distinct role for cathepsin S.

To investigate the potential contribution of the lysosomal compartment in the processing of amyloid precursor protein (APP) to amyloid beta-peptides (A beta s), we stably overexpressed a series of lysosomal proteases (the cysteine proteases, cathepsins B, L and S, and the aspartic protease, cathepsin D) in a human kidney epithelial cell line (293) transfected to express high levels of beta APP. Preliminary experiments indicated that 293 cells endogenously synthesize cathepsins B, L and D, but not cathepsin S. A beta secretion was assessed by immunoprecipitation and ELISA and found to be increased approximately 2-fold following cathepsin S expression, but to be unchanged (cathepsins B, L) or decreased (cathepsin D) in the other double transfectants. E-64d, an inhibitor of lysosomal cysteine proteases, significantly reduced A beta secretion by the cathepsin S transfectants, but had no effect on cells expressing the other proteases. Radiosequencing of A beta secreted by cathepsin S-expressing cells revealed that a previously unreported variant beginning at Met -1 (relative to the most common A beta N-terminus, Asp -1) accounted for most of the increase in A beta secretion. Immunostaining of human brain sections revealed cathepsin S in cortical neurons and glia in samples of brain from patients with Alzheimer's disease. These results provide evidence in living cells for a pathway in which cathepsin S generates A beta from amyloidogenic fragments of beta APP in the endosomal/lysosomal compartment. This pathway appears to be inducible, distinct from a constitutive pathway used by 293 and other cells to generate A beta, and may be relevant to the pathogenesis of Alzheimer's disease.

Alzheimer Disease↗
PubMed1995

Molecular cloning of human cathepsin O, a novel endoproteinase and homologue of rabbit OC2.

A 1670-bp cDNA coding for a novel human cysteine protease has been isolated from a monocyte-derived macrophage cDNA library. This cDNA predicts a 329-amino acid preprocathepsin with more than 50% identity to both human cathepsin S and cathepsin L and 94% identity to a rabbit cDNA, termed OC2, recently isolated from osteoclasts. Based on its high homology to OC2, we have named the human enzyme cathepsin O. Cathepsin O mRNA was identified as a single approximately 1.7 kb transcript in cultures of 15-day-old monocyte-derived macrophages, but was not expressed in human monocytes or alveolar macrophages. When transfected into COS-7 cells, cathepsin O displayed potent endoprotease activity against fibrinogen at acid pH. This novel endoprotease may play an important role in extracellular matrix degradation.

Amino Acid Sequence↗
PubMed1995

Urokinase receptor is a multifunctional protein: influence of receptor occupancy on macrophage gene expression.

Binding of urokinase to the glycolipid-anchored urokinase receptor (uPAR) has been implicated in macrophage differentiation. However, no biochemical markers of differentiation have yet been directly linked to uPAR occupancy. As extensive changes in proteolytic profile characterize monocytic differentiation, we have examined the role of uPAR occupancy on protease expression by differentiating phagocytes. Antibodies to either urokinase or to uPAR that prevent receptor binding inhibited induction of cathepsin B in cultured monocytes and both cathepsin B and 92-kD gelatinase mRNA and protein in phorbol diester-stimulated myeloid cells. Mannosamine, an inhibitor of glycolipid anchor assembly, also blocked protease expression. Anti-catalytic urokinase antibodies, excess inactive urokinase, or aprotinin had no effect, indicating that receptor occupancy per se regulated protease expression. Antibodies to the integrins CD11a and CD29 or to the glycolipid-anchored proteins CD14 and CD55 also had no effect. Protease induction was independent of matrix attachment. Antibodies to urokinase or uPAR affected neither the decrease in cathepsin G nor the increase in tumor necrosis factor-alpha in phorbol ester-stimulated cells. These data establish that uPAR is a multifunctional receptor, not only promoting pericellular proteolysis and matrix attachment, but also effecting cysteine- and metallo-protease expression during macrophage differentiation.

Cathepsin B↗
PubMed1995

The lysosomal cysteine protease, cathepsin S, is increased in Alzheimer's disease and Down syndrome brain. An immunocytochemical study.

Expression of cathepsin (cat) S, a lysosomal cysteine protease, has recently been shown to cause an increase in production of amyloid beta-peptides in transfected human cells. In this study, we examined the presence and localization of cat S by immunocytochemistry in 21 control, 24 Alzheimer's disease (AD), and 10 Down syndrome (DS) postmortem brains. An antiserum to a human cat S fusion protein was affinity purified and its specificity confirmed by abolition of immunoreactivity after adsorption with cat S but not cat L fusion protein. A small minority of control cases showed light, focal staining of scattered cortical neurons. Many control cases, as well as most AD and DS cases, showed prominent staining of vascular smooth muscle cells, particularly in leptomeningeal vessels. Both AD and DS brain tissue showed increased immunoreactivity in a subset of neocortical and hippocampal neurons and glia. Cat S immunoreactivity occurred in a granular, cytoplasmic pattern in some neurons or in a more dense staining pattern in certain neurofibrillary tangle-bearing neurons. Cat S-positive neurons were also present in amygdala and basal forebrain in AD brains. A subset of astrocytes were immunoreactive with the cat S antibody in AD and DS but not in control brains. In rare AD cases, cat S immunostaining was observed in astrocytes in the periphery of amyloid-beta-containing plaques. These results suggest that cat S is up-regulated in AD and DS brain. The association of cat S immunoreactivity with tangle-bearing neurons, astrocytes, and rare senile plaques implies a role for altered cat S activity in the pathogenesis of AD.

Adult↗
PubMed1994

Identification of the urokinase receptor as an adhesion receptor for vitronectin.

Urokinase receptors, expressed on surfaces of many cell types, focus to the pericellular space plasminogen-dependent proteolysis important in matrix remodeling and cell movement. We now report that the urokinase receptor (uPAR) is also a high affinity (Kd < 30 nM) receptor for vitronectin. Recombinant uPAR binds vitronectin in the absence of urokinase, but vitronectin binding is promoted by concurrent receptor binding of either urokinase or fragments thereof containing its uPAR binding domain. Stable epithelial cell transfectants expressing membrane-anchored uPAR, but not cells expressing soluble uPAR, become strongly adhesive with altered morphology in the absence of urokinase. These observations identify a new class of vitronectin receptor and imply a duality in function for the receptor that intrinsically links matrix adhesion to regulation of protease activity. Increases in urokinase receptor expression known to be associated with cellular activation and malignant transformation could modulate cellular trafficking and function by promoting attachment to vitronectin.

Adsorption↗
PubMed1994

Reversible cellular adhesion to vitronectin linked to urokinase receptor occupancy.

Urokinase receptors are distributed on surfaces of many cell types where they are thought to focus plasminogen-dependent proteolysis important to migration and tissue remodeling to the immediate pericellular space. In addition to its well characterized role in proteolysis, urokinase receptor binding per se promotes the adhesiveness of leukemic cell lines exposed to differentiating cytokines in vitro. We sought to determine if a serum or matrix component is involved in urokinase-dependent adhesion. We now report that cytokine-stimulated human myelomonocytic cells express a divalent cation- and Arg-Gly-Asp-independent high affinity receptor for urea-purified vitronectin (Kd < 10 nM). Soluble native vitronectin does not effectively bind to the receptor, while cellular adhesion was noted to both urea-purified and native vitronectin when adsorbed to plastic. The activity of this receptor is tightly coupled to urokinase receptor occupancy. Urokinase receptor binding thus induces selective and reversible cellular adhesion to the matrix form of vitronectin. Because transfer of vitronectin-bound plasminogen activator inhibitor type 1 to urokinase promotes rapid turnover of receptor-bound enzyme, these results illuminate a novel binding cycle by which urokinase receptor occupancy coordinately regulates cellular adhesiveness and pericellular proteolysis.

Adenocarcinoma↗
PubMed1994

Human cathepsin S: chromosomal localization, gene structure, and tissue distribution.

The human lysosomal cysteine proteinases, cathepsins H, L, and B, have been mapped to chromosomes 15, 9, and 8, respectively, and the genomic structures of cathepsins L and B have been determined. We report here the chromosomal localization and partial gene structure for a recently sequenced human cysteine proteinase, cathepsin S. A 20-kilobase pair genomic clone of the human cathepsin S gene was isolated from a human fibroblast genomic library and used to map the human cathepsin S gene to chromosome 1q21 by fluorescence in situ hybridization. This clone contains exons 1 through 5, introns 1 through 4, part of intron 5, and > 7 kilobase pairs of the 5'-flanking sequence. The gene structure of human cathepsin S is similar to that of cathepsin L through the first 5 exons, except that cathepsin S introns are substantially larger. Sequencing of the 5'-flanking region revealed, similar to human cathepsin B, no classical TATA or CAAT box. In contrast to cathepsin B, cathepsin S contains only two SP1 and at least 18 AP1 binding sites that potentially could be involved in regulation of the gene. This 5'-flanking region also contains CA microsatellites. The presence of AP1 sites and CA microsatellites suggest that cathepsin S can be specifically regulated. Results of Northern blotting using probes for human cathepsins B, L, and S are consistent with this hypothesis; only cathepsin S shows a restricted tissue distribution, with highest levels in spleen, heart, and lung. In addition, immunostaining of lung tissue demonstrated detectable cathepsin S only in lung macrophages. The high level of expression in the spleen and in phagocytes suggests that cathepsin S may have a specific function in immunity, perhaps related to antigen processing.

Animals↗
PubMed1994

The role of thiol proteases in tissue injury and remodeling.

Human lung macrophages express all four of the known lysosomal thiol proteases: cathepsins B, H, L, and S. These enzymes share a similar size and targeting mechanism for lysosomal accumulation and all have relatively indiscriminate substrate specificity in comparison with such highly selective serine proteases as urokinase or thrombin. These enzymes do have distinctive properties: only cathepsin B has C-terminal dipeptidase activity, only cathepsin H has potent aminopeptidase activity, and only cathepsin L and S are elastolytic. Cathepsin S is unique in that it is stable at neutral pH; indeed, at neutral pH it has elastolytic activity roughly comparable with that of neutrophil elastase. Recent studies of the differential expression of these cathepsins suggest they not only cooperate in terminal degradation of endocytized protein but also have specific functions such as proenzyme activation, antigen processing, and tissue remodeling, especially bone matrix resorption. Lysates of lung macrophages degrade elastin at neutral pH, suggesting that necrosis of macrophages at sites of macrophage accumulation, e.g., caseation necrosis, could contribute to tissue destruction. Tissue destruction and remodeling by thiol proteases expressed by live macrophages, however, is limited by tight compartmentalization of cathepsins to lysosomes. Nonetheless, macrophages accumulate at sites of known injury in cigarette smokers. Because these cells contain potent elastases, and because lysosomal enzyme release and cell surface acidification are regulated events, dysregulation of thiol protease expression in stimulated macrophages may contribute to the injury observed in cigarette smokers with non-alpha-1-protease inhibitor-type emphysema.

Amino Acid Sequence↗
PubMed1993

Cytokines induce urokinase-dependent adhesion of human myeloid cells. A regulatory role for plasminogen activator inhibitors.

Differentiation of monocytic precursors often results in adhesive properties thought to be important in migration. In this study, the influence of cytokines, known to induce macrophage differentiation, on the adhesiveness of the monocytic cell line U937 was examined in vitro. Despite development of a macrophage morphology, < 5% of cytokine-stimulated U937 cells were adherent at 24 h. Addition of 1-10 nM urokinase-type plasminogen activator (uPA) induced adherence in the presence of transforming growth factor type beta-1, 1,25-(OH)2 vitamin D3, granulocyte macrophage colony-stimulating factor, or tumor necrosis factor alpha. uPA-dependent adhesiveness was reversible after 24 h of stimulation with cytokines and uPA as adherence was prevented by the subsequent addition of anti-uPA antibodies. Adherence induced by diisopropylfluorophosphate-inactivated uPA was severalfold greater than that seen with active uPA. This difference was largely due to cell-surface turnover of active uPA complexed with plasminogen activator inhibitor (PAI). These data indicate that cytokines prime monocyte progenitors for uPA receptor-mediated signals leading to adherence, continued uPA receptor occupancy is required for adherence, and PAI decreases adherence by promoting clearance of uPA/PAI complexes. Thus the interaction of uPA and PAI at the cell surface, known to affect extracellular matrix proteolysis and hence myeloid cell migration, also regulates adhesion. The coordinated regulation of these two uPA functions by PAI may enhance the migratory potential of monocytic cells.

Cell Adhesion↗
PubMed1993

Human alveolar macrophages have 15-lipoxygenase and generate 15(S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid and lipoxins.

Eicosanoids derived from lipoxygenase (LO)-catalyzed reactions play important roles in pulmonary inflammation. Here, we examined formation of LO-derived products by human alveolar macrophages (HAM). HAM converted [1-14C]-arachidonic acid to a product carrying 14C-radiolabel that was identified as 15(S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid (15-HETE) by physical methods. 15-LO mRNA was demonstrated in HAM by reverse transcription-polymerase chain reaction. Incubation of HAM for 3 d with interleukin 4(IL-4) before exposure to [1-14C]arachidonic acid led to both increased mRNA for 15-LO and a 4-fold increase in 15-HETE formation. In contrast, 5(S)-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid generation was not significantly altered by prior exposure to IL-4. Additionally, lipoxins (LXA4 and LXB4) were detected from endogenous substrate, albeit in lower levels than leukotriene B4 (LTB4), in electrochemical detection/high performance liquid chromatography profiles from HAM incubated in the presence and absence of the chemotactic peptide (FMLP) or the calcium ionophore (A23187). Exposure of HAM to leukotriene A4 (LTA4) resulted in a 2-fold increase in LXA4 and 10-fold increase in LXB4. These results demonstrate the presence of 15-LO mRNA and enzyme activity in HAM and the production of LXA4 and LXB4 by these cells. Along with 5-LO-derived products, the biosynthesis of 15-LO-derived eicosanoids by HAM may also be relevant in modulating inflammatory responses in the lung.

Arachidonate 15-Lipoxygenase↗
PubMed1992

Molecular cloning and expression of human alveolar macrophage cathepsin S, an elastinolytic cysteine protease.

Human alveolar macrophages (HAM) express an elastase activity of acidic pH optimum inhibitable by cysteine protease inhibitors. Recent studies indicate that the only known eukaryotic elastinolytic cysteine protease, cathepsin L, cannot completely account for this activity. In order to search for additional cysteine proteases with elastinolytic activity, low degeneracy oligonucleotide primers based on regions of strong homology among the known cysteine proteases were used to screen reverse-transcribed HAM RNA for cysteine proteases by the polymerase chain reaction. Among the cDNA sequences generated was a 493-base pair product highly homologous to bovine cathepsin S. Screening of a HAM cDNA eukaryotic expression library with this cDNA yielded a 1.7-kilobase full-length cDNA highly homologous to bovine cathepsin S (approximately 85% identical). This cDNA predicts a 331-amino acid preprocathepsin. Expression of this cDNA in COS cells revealed the active enzyme to be a single chain 28-kDa protease, as judged by active site labeling with a novel iodinated analogue of N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucylamido-(4-gua nido)butane (E-64). The recombinant enzyme was found to be elastinolytic toward 3H-labeled elastin (bovine ligamentum nuchae) at pH 5.5 but with 25% of this activity retained at pH 7.0. Labeling of HAM with the active site probe revealed these cells express a 28-kDa cysteine protease, and Northern blot analysis revealed the presence of a approximately 1.7-kilobase cathepsin S mRNA. These data establish that human macrophages express at least two cysteine proteases with elastinolytic activity. The relatively broad pH range of human cathepsin S activity suggests this enzyme may contribute to the contact-dependent elastase activity of live human alveolar macrophages.

Amino Acid Sequence↗