ABSTRACT
Thrombin is a pleiotropic enzyme best known for its contribution to fibrin formation and platelet aggregation during vascular hemostasis. There is increasing evidence to suggest a role for thrombin in the development of interstitial fibrosis, but interstitial thrombin has not been demonstrated by the direct determination of activity. Rather its presence is inferred by products of thrombin action such as fibrin and activated fibroblasts. This review will focus on possible mechanisms of thrombin formation in the interstitial space, the possible actions of thrombin, processes regulating thrombin activity in the interstitial space, and evidence supporting a role for thrombin in fibrosis.
Subject(s)
Extracellular Space/metabolism , Thrombin/metabolism , Animals , Blood Coagulation , Extracellular Matrix/metabolism , Fibrin/metabolism , Fibrinogen/metabolism , Fibroblasts/metabolism , Fibrosis , Hemostasis , Humans , Liver Cirrhosis/physiopathology , Mice , Platelet Aggregation , Prothrombin/metabolism , Pulmonary Fibrosis/physiopathology , Signal TransductionABSTRACT
Receptor-recognized forms of α2 -macroglobulin (α2 M) bind to cell surface-associated GRP78 and initiate pro-proliferative and anti-apoptotic signaling. Ligation of GRP78 with α2 M also upregulates TFII-I, which binds to the GRP78 promoter and enhances GRP78 synthesis. In addition to its transcriptional functions, cytosolic TFII-I regulates agonist-induced Ca(2+) entry. In this study we show that down regulation of TFII-I gene expression by RNAi profoundly impairs its cell surface expression and anti-apoptotic signaling as measured by significant reduction of GRP78, Bcl-2, and cyclin D1 in 1-Ln and DU-145 human prostate cancer cells stimulated with α2 M. In contrast, this treatment significantly increases levels of the pro-apoptotic proteins p53, p27, Bax, and Bak and causes DNA fragmentation. Furthermore, down regulation of TFII-I expression activates agonist-induced Ca(2+) entry. In plasma membrane lysates p-PLCγ1, TRPC3, GRP78, MTJ1, and caveolin co-immunoprecipitate with TFII-I suggesting multimeric complexes of these proteins. Consistent with this hypothesis, down regulating TFII-I, MTJ1, or GRP78 expression by RNAi greatly attenuates cell surface expression of TFII-I. In conclusion, we demonstrate that not only does cell surface GRP78 regulate apoptosis, but it also regulates Ca(2+) homeostasis by controlling cell surface localization of TFII-I.
Subject(s)
Apoptosis/drug effects , Prostatic Neoplasms/metabolism , Transcription Factors, TFII/metabolism , alpha-Macroglobulins/pharmacology , Apoptosis/genetics , Blotting, Western , Caveolin 1/genetics , Caveolin 1/metabolism , Cell Line, Tumor , Cell Membrane/metabolism , Endoplasmic Reticulum Chaperone BiP , Flow Cytometry , HSP40 Heat-Shock Proteins/genetics , HSP40 Heat-Shock Proteins/metabolism , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Humans , Immunoprecipitation , Male , Membrane Proteins/genetics , Membrane Proteins/metabolism , Prostatic Neoplasms/genetics , Protein Binding , RNA Interference , TRPC Cation Channels/genetics , TRPC Cation Channels/metabolism , Transcription Factors, TFII/geneticsABSTRACT
B-cell chronic lymphocytic leukemia (CLL) is characterized by slow accumulation of malignant cells, which are supported in the microenvironment by cell-cell interactions and soluble cytokines such as tumor necrosis factor (TNF). We evaluated the effect of the small molecule TNF inhibitor LMP-420 on primary CLL cells. The mean concentration of LMP-420 required to induce 50% cytotoxicity (ED50) at 72 h was 245 n. LMP-420-induced time- and dose-dependent apoptosis, as shown by annexin V staining, caspase activation and DNA fragmentation. These changes were associated with decreased expression of anti-apoptotic proteins Mcl-1, Bcl-xL and Bcl-2. CLL cells from patients with poor prognostic indicators showed LMP-420 sensitivity equal to that for cells from patients with favorable characteristics. In addition, LMP-420 potentiated the cytotoxic effect of fludarabine and inhibited in vitro proliferation of stimulated CLL cells. Gene expression profiling indicated that the mechanism of action of LMP-420 may involve suppression of nuclear factor-kappaB and immune response pathways in CLL cells. LMP-420 had minimal effects on normal peripheral blood mononuclear cell, B- and T-cell function, and hematopoietic colony formation. Our data suggest that LMP-420 may be a useful treatment for CLL with negligible hematologic toxicities.
Subject(s)
Antineoplastic Agents/pharmacology , Boron Compounds/pharmacology , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Purines/pharmacology , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Antineoplastic Agents/toxicity , Apoptosis/drug effects , Boron Compounds/toxicity , Cell Line, Tumor , Dose-Response Relationship, Drug , Drug Synergism , Female , Flow Cytometry , Gene Expression Profiling , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Male , Prognosis , Purines/toxicity , Vidarabine/analogs & derivatives , Vidarabine/pharmacologyABSTRACT
Receptor-recognized forms of alpha(2)-macroglobulin (alpha(2)M*) bind to cancer cell surface GRP78, which functions as a signaling receptor promoting proliferation and survival. Patients with prostate, ovary, and skin cancer may develop auto-antibodies to the alpha(2)M* binding site which are receptor agonists whose presence indicates a poor prognosis. By contrast, antibodies directed against the COOH-terminal domain of GPR78 (anti-CTD antibody), are antagonists which down regulate pro-proliferative signaling and upregulate p53. Unfolded protein response (UPR) signaling plays an important role in cell survival and proliferation as well as apoptosis. We, therefore, studied the effect of anti-CTD antibody on UPR signaling in 1-LN and DU-145 prostate cancer cells. Treatment of these cells, which express GRP78 on their cell surface, with this antibody significantly downregulated IRE1-alpha, PERK, and ATF6alpha-dependent UPR signaling. By contrast, the pro-apoptotic protein GADD153 was elevated. Anti-CTD antibody treatment also elevated apoptotic components, cleaved PARP-1, and Erdj5. In general, a two to threefold effect was observed for the parameters which were studied. These studies suggest that anti-CTD antibody induces growth inhibitory and pro-apoptotic effects by modulating UPR signaling in human prostate cancer cells.
Subject(s)
Antibodies/immunology , Antibodies/pharmacology , Heat-Shock Proteins/chemistry , Heat-Shock Proteins/immunology , Prostatic Neoplasms/metabolism , Unfolded Protein Response/drug effects , Apoptosis Regulatory Proteins/metabolism , Bcl-2-Like Protein 11 , Caspases/metabolism , Cell Line, Tumor , Down-Regulation/drug effects , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/pathology , Endoplasmic Reticulum Chaperone BiP , Enzyme Activation/drug effects , Eukaryotic Initiation Factor-2/metabolism , Humans , Immunoblotting , Male , Membrane Proteins/metabolism , Neoplasm Proteins/metabolism , Poly(ADP-ribose) Polymerases/metabolism , Prostatic Neoplasms/enzymology , Prostatic Neoplasms/pathology , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Transcription Factor CHOP/metabolism , Up-Regulation/drug effects , eIF-2 Kinase/metabolismABSTRACT
Ligation of cancer cell surface GRP78 by activated alpha2-macroglobulin (alpha2M*) triggers pro-proliferative and anti-apoptotic signaling pathways. Cancer patients who develop autoantibodies to the alpha2M* binding site in GRP78 have a poor prognosis since these antibodies are receptor agonists. The NF-kappaB family of transcription factors induces expression of genes affecting cell growth and differentiation. NF-kappaB1 plays a major regulatory role in controlling innate immunity and inflammation, whereas NF-kappaB2 plays a greater role in cancer cell proliferation. Here we report that treatment of prostate cancer cells with antibody directed against the carboxyl terminal domain of GRP78 inhibits alpha2M*-induced activation of NF-kappaB2 by approximately 50% while exerting a lesser effect of approximately 20% on NF-kappaB1 activation. Treatment of these cells nearly abolished alpha2M*-induced activation of IKKalpha involved in the activation of NF-kappaB2. This antibody also suppressed alpha2M*-induced phosphorylation of IKKalpha, IKKalpha/beta, IkappaBalpha, and IkappaBbeta as well as levels of NIK. Antibody treatment of cancer cells elevated pro-apoptotic p21WAF and p27kip while reducing cyclin D1 levels. These studies demonstrate that antibody directed against the carboxyl terminal domain of GRP78 inhibits the pro-proliferative NF-kappaB signaling cascade in cancer cells.
Subject(s)
Antibodies, Neoplasm/pharmacology , Autoantibodies/pharmacology , Heat-Shock Proteins/immunology , NF-kappa B p50 Subunit/antagonists & inhibitors , NF-kappa B p52 Subunit/antagonists & inhibitors , Prostatic Neoplasms/metabolism , Cell Line, Tumor , Cell Proliferation , Cyclin D1/metabolism , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Endoplasmic Reticulum Chaperone BiP , Heat-Shock Proteins/metabolism , Humans , Male , NF-kappa B p50 Subunit/agonists , NF-kappa B p52 Subunit/agonists , Prostatic Neoplasms/pathology , Protein Structure, Tertiary , Tumor Suppressor Protein p53/metabolism , alpha-Macroglobulins/immunology , alpha-Macroglobulins/metabolismABSTRACT
Antibodies against the COOH-terminal domain of cell surface GRP78 induce apoptosis in cancer cell lines via activation of p53 signaling. We now have studied the effects of PFT-alpha, an inhibitor of p53-mediated apoptotic pathways, on anti-GRP78 antibody-induced activation of p53 and pro-apoptotic signaling in 1-LN prostate cancer cells. Pretreatment of 1-LN cancer cells with this agent significantly inhibited antibody or doxorubicin-induced upregulation of p53. Concomitantly, PFT-alpha treatment prevented down regulation of ERK1/2 activation by either antibody or doxorubicin. Likewise, PFT-alpha prevented increases in the pro-apoptotic proteins BAD, BAK, BAX, PUMA, and NOXA as well as activation of caspases-3, -7, and -9. We conclude that antibody-induced apoptosis in prostate cancer cells is mediated predominantly by p53 using the mitochondrial pathway of apoptosis.
Subject(s)
Apoptosis/drug effects , Benzothiazoles/pharmacology , Prostatic Neoplasms/metabolism , Toluene/analogs & derivatives , Tumor Suppressor Protein p53/antagonists & inhibitors , Antibodies, Neoplasm/immunology , Apoptosis Regulatory Proteins/antagonists & inhibitors , Apoptosis Regulatory Proteins/metabolism , Autoantibodies/immunology , Caspases/metabolism , Cell Line, Tumor , Endoplasmic Reticulum Chaperone BiP , Heat-Shock Proteins , Humans , Male , Prostatic Neoplasms/immunology , Prostatic Neoplasms/pathology , Toluene/pharmacology , Tumor Suppressor Protein p53/metabolismABSTRACT
Receptor-recognized forms of alpha(2)-macroglobulin (alpha(2)M*) bind to cell surface-associated GRP78 and induce proliferative and survival signaling in prostate cancer cells. As part of the cellular response to alpha(2)M*, GRP78 expression is itself upregulated. In response to other stimuli, the transcription factor TFII-I upregulates GRP78 by binding to its gene promoter. We have, therefore, studied the role of TFII-I in transcriptional upregulation of GRP78 in 1-LN human prostate cancer cells stimulated with alpha(2)M*. This treatment caused a two- to threefold increase in TFII-I and GRP78 synthesis from [(35)S]-labeled precursor amino acids. Synthesis of both TFII-I and GRP78 were significantly reduced by silencing TFII-I gene expression or pretreatment of cells with genistein or actinomycin D. Confocal microscopy was employed to demonstrate relocation of TFII-I to the nucleus. In alpha(2)M*-stimulated cells, moreover, TFII-I bound to the GRP78 promoter as determined by CHIP assay. We also demonstrate binding of TFII-I to the c-fos promoter, consistent with its role in upregulating c-fos gene expression. In non-lymphoid cells, phosphorylated c-Src is an activator of TFII-I. Ligation of GRP78 on 1-LN cells with alpha(2)M* was followed by tyrosine phosphorylation of c-Src as well as TFII-I. We conclude that alpha(2)M*-induced increase in GRP78 synthesis is caused by transcriptional upregulation of TFII-I which binds to the GRP78 promoter and thus potentiates its cell survival and antipoptotic functions in 1-LN prostate cancer cells.
Subject(s)
Gene Expression Regulation, Neoplastic/genetics , Heat-Shock Proteins/genetics , Molecular Chaperones/genetics , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Transcription Factors, TFII/metabolism , Transcription, Genetic/genetics , Up-Regulation/genetics , CSK Tyrosine-Protein Kinase , Cell Line, Tumor , Endoplasmic Reticulum Chaperone BiP , Gene Expression Regulation, Neoplastic/drug effects , Heat-Shock Proteins/biosynthesis , Humans , Male , Molecular Chaperones/biosynthesis , Phosphorylation , Phosphotyrosine/metabolism , Prostatic Neoplasms/metabolism , Protein Binding , Protein Transport , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-fos/metabolism , RNA Interference , Transcription Factors, TFII/genetics , Transcription, Genetic/drug effects , Up-Regulation/drug effects , alpha-Macroglobulins/pharmacology , src-Family KinasesABSTRACT
Amyloid precursor protein (APP) and amyloid beta-peptide (Abeta) have been implicated in a variety of physiological and pathological processes underlying nervous system functions. APP shares many features with adhesion molecules in that it is involved in neurite outgrowth, neuronal survival and synaptic plasticity. It is, thus, of interest to identify binding partners of APP that influence its functions. Using biochemical cross-linking techniques we have identified ATP synthase subunit alpha as a binding partner of the extracellular domain of APP and Abeta. APP and ATP synthase colocalize at the cell surface of cultured hippocampal neurons and astrocytes. ATP synthase subunit alpha reaches the cell surface via the secretory pathway and is N-glycosylated during this process. Transfection of APP-deficient neuroblastoma cells with APP results in increased surface localization of ATP synthase subunit alpha. The extracellular domain of APP and Abeta partially inhibit the extracellular generation of ATP by the ATP synthase complex. Interestingly, the binding sequence of APP and Abeta is similar in structure to the ATP synthase-binding sequence of the inhibitor of F1 (IF(1)), a naturally occurring inhibitor of the ATP synthase complex in mitochondria. In hippocampal slices, Abeta and IF(1) similarly impair both short- and long-term potentiation via a mechanism that could be suppressed by blockade of GABAergic transmission. These observations indicate that APP and Abeta regulate extracellular ATP levels in the brain, thus suggesting a novel mechanism in Abeta-mediated Alzheimer's disease pathology.
Subject(s)
Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/metabolism , Mitochondrial Proton-Translocating ATPases/metabolism , Adenosine Triphosphate/biosynthesis , Amyloid beta-Protein Precursor/genetics , Animals , Biotinylation/methods , Brain/ultrastructure , Cells, Cultured , Dose-Response Relationship, Drug , Female , GABA Antagonists/pharmacology , Heart/drug effects , Hippocampus/cytology , Humans , Immunoprecipitation/methods , Long-Term Potentiation/drug effects , Long-Term Potentiation/physiology , Male , Mice , Mice, Inbred C57BL , Mitochondria/drug effects , Mitochondria/metabolism , Mitochondrial Proton-Translocating ATPases/pharmacokinetics , Neuroblastoma , Neurons/drug effects , Neurons/metabolism , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/pharmacology , Picrotoxin/pharmacology , Protein Binding/drug effects , Protein Binding/physiology , Protein Transport/physiology , Rats , Transfection/methodsABSTRACT
OBJECTIVES: Lipoteichoic acid (LTA), induces some of the clinical symptoms of Behçet's disease (BD) in a rat animal model. These results led to the hypothesis that LTA may also trigger BD in humans. We investigated the humoral and cellular immune response against LTA and lipopolysaccharide (LPS) in patients with BD, and compared these responses with those of patients with active chronic oral ulcers (OU) and normal controls. METHODS: Samples were obtained from 12 active BD, 12 inactive BD, 12 active OU and 12 normal controls. Anti-LTA, anti-LPS antibodies levels and the capacity of immune complexes anti-LTA IgG-LTA to activate complement were studied. Exposed mannose residues in anti-LTA IgG were analyzed in the four groups. The interleukin-8 (IL-8) production by peripheral blood mononuclear cells cultures after LTA and LPS stimulation was also studied in all groups. RESULTS: The capacity to bind mannan binding protein (MBP) of anti-LTA IgGs was significantly higher in BD and active OU patients relative to normal controls (p < 0.001). However, only active BD patients generated significantly higher levels of C5a than controls (p < 0.0001). The IgGs purified from the sera of BD patients showed a high specificity for LTA from Streptococcus sanguis or Streptococcus faecalis. LTA also stimulates the secretion of IL-8 in peripheral blood mononuclear cells isolated from active BD patients. Anti-LPS IgA and IgG titers were significantly higher only in active OU patients relative to normal controls (p < 0.0018). CONCLUSION: These results suggest a mechanism involving LTA from streptococci in the pathogenesis of BD.
Subject(s)
Behcet Syndrome/immunology , Immunoglobulin G/blood , Interleukin-8/blood , Leukocytes, Mononuclear/immunology , Lipopolysaccharides/immunology , Teichoic Acids/immunology , Adult , Antigen-Antibody Complex/immunology , Antigen-Antibody Complex/metabolism , Antigen-Antibody Complex/pharmacology , Behcet Syndrome/metabolism , Cells, Cultured , Complement Activation/drug effects , Complement C5/immunology , Complement C5/metabolism , Female , Glycosylation , Humans , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Male , Mannose-Binding Lectin/immunology , Mannose-Binding Lectin/metabolismABSTRACT
The alpha2-macroglobulin signalling receptor is upregulated in highly metastatic 1-LN prostate cancer cells. Stimulation of 1-LN cells with activated alpha2-macroglobulin (alpha2M*) caused a two- to threefold increase in [3H]thymidine uptake and cell number. These events require the Ras-dependent MAPK and PI 3-kinase/Akt signalling cascades. Incubation of 1-LN cells with alpha2M* induced Grb2, shc, sos and Raf-1 expression, as well as phosphorylation of MEK 1/2, ERK 1/2, p38 MAPK and JNK. This treatment also increased PI 3-kinase activation, PDK1 expression, Akt phosphorylation and p70s6k phosphorylation. Levels of the early gene products c-fos protein and thymidylate synthase were comparably increased. Exposure of 1-LN cells to alpha2M* significantly raised the levels of phosphorylated CREB by about 15-20 min and phosphorylated p53 by about 60-90 min of incubation. We conclude that the growth regulatory effects of ligating the alpha2M* signalling receptor on 1-LN cells are exerted via the onset and crosstalk between the Ras-dependent MAPK and PI 3-kinase/Akt signalling cascades.
Subject(s)
Cell Division/drug effects , Low Density Lipoprotein Receptor-Related Protein-1/metabolism , MAP Kinase Kinase Kinase 1 , MAP Kinase Signaling System/physiology , Phosphatidylinositol 3-Kinases/metabolism , alpha-Macroglobulins/pharmacology , 3-Phosphoinositide-Dependent Protein Kinases , Cyclic AMP Response Element-Binding Protein/metabolism , Enzyme Activation/drug effects , Humans , MAP Kinase Kinase Kinases/metabolism , Male , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation/drug effects , Prostatic Neoplasms/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-fos/metabolism , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , Thymidylate Synthase/metabolism , Tumor Cells, Cultured , Tumor Suppressor Protein p53/metabolism , p38 Mitogen-Activated Protein KinasesABSTRACT
The low density lipoprotein receptor-related protein (LRP) consists of two subunits, M(r) approximately 515,000 and 85,000. LRP is a receptor for activated alpha2-macrogobulin (alpha2M*), Pseudomonas exotoxin A, and many other proteins. We now report that ubiquitinylation of the LRP heavy chain occurred when either Pseudomonas exotoxin A or alpha2M* bound to LRP on macrophages. Ubiquitinylation was dose-dependent and maximal about 30 min after ligation of the receptor. Addition of the proteosome inhibitor MG-132 sustained the level of ubiquitin-LRP for longer time intervals in macrophages treated with either alpha2M* or Pseudomonas exotoxin A. By contrast, when receptor associated protein (RAP) bound to LRP, ubiquitinylation did not occur. While RAP is not found in the extracellular environment it binds to LRP and is believed to function as an intracellular chaperone. The presence of RAP within the cell may, therefore, contribute to the recycling of intact LRP which has ligated and internalized its ligands.
Subject(s)
ADP Ribose Transferases , LDL-Receptor Related Protein-Associated Protein/pharmacology , LDL-Receptor Related Proteins/metabolism , Protein Processing, Post-Translational/drug effects , Ubiquitins/metabolism , Virulence Factors , Animals , Bacterial Proteins/metabolism , Bacterial Toxins/metabolism , Exotoxins/metabolism , Ligands , Macrophages, Peritoneal/metabolism , Mice , Mice, Inbred C57BL , Protein Kinase Inhibitors , alpha-Macroglobulins/metabolism , Pseudomonas aeruginosa Exotoxin AABSTRACT
Apolipoprotein E (ApoE) is a 34-kDa cholesterol transport protein that also possesses immunomodulatory properties. In this study, we demonstrate that ApoE initiates a signaling cascade in murine peritoneal macrophages that leads to increased production of inositol triphosphate with mobilization of intracellular Ca(2+) stores. This cascade is inhibited by pretreatment with receptor-associated protein and Ni(2+), and it is mediated by a pertussis toxin-sensitive G protein. These properties are characteristic of signal transduction induced via ligand binding to the cellular receptor, lipoprotein receptor-related protein. A peptide derived from the receptor-binding region of ApoE also initiates signal transduction in a manner similar to that of the intact protein, suggesting that this isolated region is sufficient for signal transduction. The ApoE-mimetic peptide competed for binding with the intact protein, confirming that they both interact with the same site. ApoE-dependent signal transduction might play a role in mediating the functional properties of this lipoprotein.
Subject(s)
Apolipoproteins E/pharmacology , Calcium Signaling , Macrophages/immunology , Animals , Binding, Competitive , Cells, Cultured , Inositol 1,4,5-Trisphosphate/biosynthesis , Kinetics , Low Density Lipoprotein Receptor-Related Protein-1 , Macrophages/drug effects , Mice , Nickel/pharmacology , Peptides/pharmacology , Pertussis Toxin , Receptors, Immunologic/physiology , Virulence Factors, Bordetella/pharmacologyABSTRACT
A safe, effective, more potent adjuvant than currently available would be beneficial in developing new therapeutics and diagnostic reagents. We report here a technique for the rapid, efficient incorporation of non-proteolytic antigens into alpha(2)-macroglobulin (alpha(2)M; tradename, SynerVax), allowing us to covalently couple much larger subunit antigens to alpha(2)M than previously possible. Our goal was to determine if incorporation of HB, the monomeric form of Hepatitis B virus (HBV) surface antigen (HBsAg), into alpha(2)M would result in increased immune reactivity. Earlier attempts to immunize animals using HB did not generate significant levels of antibodies. Using HB complexes prepared with alpha(2)M we now report dramatically-increased immunogenicity of HB in BALB/c mice. Combining these soluble complexes with a depot-generating agent (alum), titers>1:1,000,000 are obtained with a single injection. This novel adjuvant technology should provide a valuable tool for the development of either prophylactic and therapeutic vaccines, or monoclonal antibodies against hitherto poorly-immunogenic subunit antigens.
Subject(s)
Hepatitis B Vaccines/administration & dosage , alpha-Macroglobulins/administration & dosage , Animals , Enzyme-Linked Immunosorbent Assay , Female , Hepatitis B Antibodies/blood , Hepatitis B Surface Antigens/immunology , Hepatitis B Vaccines/chemistry , Hepatitis B Vaccines/immunology , Immunization , Mice , Mice, Inbred BALB C , Vaccines, Subunit/immunologyABSTRACT
We have studied the induction of cyclooxygenase-2 (COX-2) in macrophages consequent to ligating the alpha(2)-macroglobulin (alpha(2)M) signalling receptor (alpha(2)MSR) with receptor-recognized forms of alpha(2)M (alpha(2)M*). Macrophage stimulation with alpha(2)M* increased total cellular and nuclear COX-2 two- to threefold. The maximal increase in COX-2 occurred at a ligand concentration of 50-100 pM and after 2 h. Modulation of intracellular Ca(2+) levels or incubation of [35S] methionine-labelled macrophages with actinomycin D, prior to treatment with alpha(2)M*, markedly reduced the induction of total cellular and nuclear COX-2. Protein kinase C (PKC) or phospholipase A(2) (PLA(2)) inhibition in alpha(2)M*-stimulated macrophages or inhibition of the p21(ras)-dependent mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI 3-kinase) signalling pathways also significantly reduced alpha(2)M*-induced total cellular and nuclear COX-2 expression. Thus, COX-2 induction is dependent on cPLA(2) activity, Ca(2+) mobilization, and PKC activity and requires participation of both the p21(ras)-dependent MAPK and PI 3-kinase signalling pathways. COX-2 activation may mediate alpha(2)M*-induced mitogenesis, which we have previously observed in this and other cell types.
Subject(s)
Isoenzymes/biosynthesis , Low Density Lipoprotein Receptor-Related Protein-1/physiology , Macrophages/enzymology , Prostaglandin-Endoperoxide Synthases/biosynthesis , Animals , Calcium/physiology , Cell Nucleus/metabolism , Cells, Cultured , Cyclooxygenase 2 , Dactinomycin/pharmacology , Enzyme Inhibitors/pharmacology , MAP Kinase Signaling System , Mice , Nucleic Acid Synthesis Inhibitors/pharmacology , Phosphatidylinositol Diacylglycerol-Lyase , Phosphoinositide-3 Kinase Inhibitors , Phospholipases A/antagonists & inhibitors , Protein Kinase C/antagonists & inhibitors , Proto-Oncogene Proteins p21(ras)/physiology , Type C Phospholipases/antagonists & inhibitors , alpha-Macroglobulins/antagonists & inhibitors , alpha-Macroglobulins/pharmacologyABSTRACT
Thioglycollate (TG)-elicited murine, peritoneal macrophages express two receptors for activated forms of the proteinase inhibitor alpha2-macroglobulin (alpha2M*)--namely, the low density lipoprotein receptor-related protein (LRP) and the alpha2M signaling receptor (alpha2MSR). We now report that resident peritoneal macrophages express only 400+/-50 alpha2MSR receptors/cell compared to 5000+/-500 receptor/TG-elicited macrophage. By contrast, LRP expression is only 2-2.5-fold greater on elicited cells. The low level of alpha2MSR expression by resident cells is insufficient to trigger signal transduction in contrast to TG-elicited cells which when exposed to alpha2M* demonstrate a rapid rise in inositol 1,4,5-trisphosphate and a concomitant increase in cytosolic free Ca2+. We then studied a variety of preparations injected subcutaneously for their ability to upregulate alpha2MSR. Macroaggregated bovine serum albumin (macroBSA) injection upregulated alpha2MSR and triggered signaling responses by splenic macrophages. Nonaggregated BSA injection alone or in the presence of alum, by contrast, did not alter alpha2MSR expression. Recombivax (hepatitis B antigen adsorbed to alum) injection also upregulated alpha2MSR on splenic macrophages while the alum carrier had no effect. We conclude that macrophage alpha2M* receptors are inducible and their expression may be regulated, in part, by potential antigens.
Subject(s)
Gene Expression Regulation/immunology , Low Density Lipoprotein Receptor-Related Protein-1/biosynthesis , Macrophages/metabolism , Second Messenger Systems/physiology , Adjuvants, Immunologic , Albumins/immunology , Alum Compounds/pharmacology , Animals , Calcium Signaling/drug effects , Cattle , Hepatitis B Surface Antigens/immunology , Hepatitis B Vaccines/immunology , Inositol 1,4,5-Trisphosphate/physiology , Low Density Lipoprotein Receptor-Related Protein-1/genetics , Macrophages/immunology , Macrophages, Peritoneal/immunology , Macrophages, Peritoneal/metabolism , Mice , Mice, Inbred C57BL , Peritonitis/chemically induced , Peritonitis/immunology , Serum Albumin, Bovine/immunology , Specific Pathogen-Free Organisms , Spleen/cytology , Thioglycolates/toxicity , Vaccines, Synthetic/immunology , alpha-Macroglobulins/pharmacologyABSTRACT
We have previously shown that antigens complexed to the receptor-recognized form of alpha(2)-macroglobulin (alpha(2)M*) demonstrate enhanced immune responsiveness mediated by the low density lipoprotein receptor-related protein LRP/CD91. Recently, we developed a proteinase-independent method to covalently bind antigens to alpha(2)M*. Given the potential applications of this chemistry, we analyzed the kinetics, thermodynamics, and pH dependence of this reaction. The incorporation of lysozyme into alpha(2)M* was a mixed bimolecular second-order reaction with a specific rate constant of 91.0 +/- 6.9 m(-1) s(-1), 50.0 degrees C, pH 7.4. The activation energy, activation entropy, and Gibbs' free energy at 50.0 degrees C were 156 kJ mol(-1), 266 J mol(-1) K(-1), and 70 kJ mol(-1), respectively. The rate of incorporation increased as a function of pH from pH 5.0 to 7.0 and was unchanged thereafter. Furthermore, the reaction between alpha(2)M* and lysozyme was irreversible. The data are consistent with a two-step mechanism. In the first step, alpha(2)M* reforms its thiol ester bond, entering a reactive state that mimics the proteolytically induced "nascent state." In the rate-limiting second step, the reformed bond quickly undergoes nucleophilic attack by lysozyme. The kinetic equations derived in this study are the basis for optimizing the formation of stable alpha(2)M*.antigen complexes.
Subject(s)
alpha-Macroglobulins/metabolism , Humans , Hydrogen-Ion Concentration , Kinetics , Ligands , Muramidase/metabolism , TemperatureABSTRACT
A hallmark of inflammation is the release of oxidants, proteinases, and cytokines, all important mediators of the inflammatory cascade. alpha(2)-Macroglobulin (alpha(2)M) is a high-affinity, broad-specificity proteinase inhibitor that also binds and regulates the biological activities of a number of cytokines. We demonstrated recently that hypochlorite-oxidized alpha(2)M has decreased ability to inhibit proteinases and regulate cytokines in vitro. The role of oxidation in regulating alpha(2)M functions in vivo is largely unknown. To determine the extent and biological consequence of in vivo alpha(2)M oxidation, we measured the degree of oxidative alpha(2)M modification from rheumatoid arthritis (RA) synovial fluid and compared this with osteoarthritis (OA) as noninflammatory controls. We found that RA synovial fluid alpha(2)M is significantly more oxidized than that from OA. RA synovial fluid also contains a twofold higher median alpha(2)M level than OA, while having only half the alpha(2)M-proteinase inhibitory activity. Detailed biochemical analysis demonstrates proteolytically degraded alpha(2)M in RA greater than in OA synovial fluid. Additionally, the hypochlorite-mediated oxidation product, chlorotyrosine, is present in RA more than in OA or plasma alpha(2)M samples. Taken together, these findings confirm a role for oxidative regulation of inflammation by altering the functions of extracellular mediators such as alpha(2)M.
Subject(s)
Arthritis, Rheumatoid/metabolism , Synovial Fluid/metabolism , Tyrosine/analogs & derivatives , alpha-Macroglobulins/metabolism , Humans , Hypochlorous Acid/metabolism , Mass Spectrometry , Osteoarthritis/metabolism , Oxidation-Reduction , Peptide Hydrolases/metabolism , Tyrosine/analysis , alpha-Macroglobulins/analysisABSTRACT
Angiostatin blocks tumor angiogenesis in vivo, almost certainly through its demonstrated ability to block endothelial cell migration and proliferation. Although the mechanism of angiostatin action remains unknown, identification of F(1)-F(O) ATP synthase as the major angiostatin-binding site on the endothelial cell surface suggests that ATP metabolism may play a role in the angiostatin response. Previous studies noting the presence of F(1) ATP synthase subunits on endothelial cells and certain cancer cells did not determine whether this enzyme was functional in ATP synthesis. We now demonstrate that all components of the F(1) ATP synthase catalytic core are present on the endothelial cell surface, where they colocalize into discrete punctate structures. The surface-associated enzyme is active in ATP synthesis as shown by dual-label TLC and bioluminescence assays. Both ATP synthase and ATPase activities of the enzyme are inhibited by angiostatin as well as by antibodies directed against the alpha- and beta-subunits of ATP synthase in cell-based and biochemical assays. Our data suggest that angiostatin inhibits vascularization by suppression of endothelial-surface ATP metabolism, which, in turn, may regulate vascular physiology by established mechanisms. We now have shown that antibodies directed against subunits of ATP synthase exhibit endothelial cell-inhibitory activities comparable to that of angiostatin, indicating that these antibodies function as angiostatin mimetics.
Subject(s)
Adenosine Triphosphate/biosynthesis , Endothelium, Vascular/enzymology , Enzyme Inhibitors/pharmacology , Peptide Fragments/pharmacology , Plasminogen/pharmacology , Proton-Translocating ATPases/physiology , Angiostatins , Animals , Catalysis , Cattle , Cell Division/drug effects , Endothelium, Vascular/cytology , Humans , Protein Conformation , Protein Subunits , Proton-Translocating ATPases/antagonists & inhibitors , Proton-Translocating ATPases/chemistryABSTRACT
The fibroblast growth factor (FGF) family has an important role in processes such as angiogenesis, wound healing, and development in which precise control of proteinase activity is important. The human plasma proteinase inhibitor alpha(2)-macroglobulin (alpha(2)M) regulates cellular growth by binding and modulating the activity of many cytokines and growth factors. These studies investigate the ability of native and activated alpha(2)M (alpha(2)M*) to bind to members of the FGF family. Both alpha(2)M and alpha(2)M* bind specifically and saturably to FGF-1, -2, -4, and -6, although the binding to alpha(2)M* is of significantly higher affinity. Neither alpha(2)M nor alpha(2)M* bind to FGF-5, -7, -9, or -10. FGF-2 was chosen for more extensive study in view of its important role in angiogenesis. It was demonstrated that FGF-2 binds to the previously identified TGF-beta binding site. The alpha(2)M* inhibits FGF-2-dependent fetal bovine heart endothelial cell proliferation in a dose-dependent manner. Unexpectedly, alpha(2)M* does not affect FGF-2-induced vascular tubule formation on Matrigel basement membrane matrix or collagen gels. Further studies demonstrate that FGF-2 partitions between fluid-phase alpha(2)M* and solid-phase Matrigel or collagen. These studies suggest that the ability of alpha(2)M* to modulate the activity of FGF-2 is dependent on an interplay with extracellular matrix components. (Blood. 2001;97:3450-3457)
Subject(s)
Fibroblast Growth Factor 2/pharmacology , Neovascularization, Physiologic/drug effects , alpha-Macroglobulins/pharmacology , Animals , Binding Sites , Binding, Competitive , Cattle , Cell Division/drug effects , Collagen , Drug Combinations , Endothelium, Vascular/cytology , Fibroblast Growth Factor 2/antagonists & inhibitors , Fibroblast Growth Factor 2/metabolism , Humans , Laminin , Proteoglycans , Transforming Growth Factor beta/metabolism , alpha-Macroglobulins/metabolismABSTRACT
We have studied the regulation of cytosolic phospholipase A2 (cPLA2) synthesis in macrophages stimulated with receptor-recognized forms of alpha2-macroglobulin (alpha2M*). [35S]methionine-labeled cells were stimulated with alpha2M* and [35S]cPLA2 was immunoprecipitated with a monoclonal antibody directed against cPLA2. The precipitates were electrophoresed, immunoblotted, cPLA2 detected by Enhanced Chemifluorescence, and its radioactivity determined. Stimulation of cells with alpha2M* caused a two- to threefold increase in cPLA2 synthesis compared to buffer-treated cells which was consistently maximal at 200 pM of alpha2M*. Actinomycin D or cycloheximide treatment of cells drastically reduced alpha2M*-induced cPLA2 synthesis. Likewise, inhibition of protein kinase C with chelerythrin, farnesyl transferase with manumycin A, MEK kinase with U0126, Erk1/2 kinases with PD98059, p38MAPK with SB203580, PI 3-kinase with wortmannin or LY294002, p70s6k with rapamycin, or depletion of [Ca2+]i with either BAPTA/AM or EGTA drastically reduced alpha2M* induction of cPLA2. Inhibition of NFKB activation with BAY11-7182 or PGA1 also abolished alpha2M* induction of cPLA2. We conclude that alpha2M*-induced cPLA2 synthesis is controlled by [Ca2+]i levels, tyrosine kinase activity, the p21ras-dependent MAPK and PI 3-kinase downstream signaling pathways, and regulation of NFkappaB.