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1.
Mol Cell Biol ; 21(17): 5857-68, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11486025

ABSTRACT

beta-Catenin is an oncogenic protein involved in regulation of cell-cell adhesion and gene expression. Accumulation of cellular beta-catenin occurs in many types of human cancers. Four mechanisms are known to cause increases in beta-catenin: mutations of beta-catenin, adenomatous polyposis coli, or axin genes and activation of Wnt signaling. We report a new cause of beta-catenin accumulation involving oncogenic mutants of RON and MET receptor tyrosine kinases (RTKs). Cells transfected with oncogenic RON or MET were characterized by beta-catenin tyrosine phosphorylation and accumulation; constitutive activation of a Tcf transcriptional factor; and increased levels of beta-catenin/Tcf target oncogene proteins c-myc and cyclin D1. Interference with the beta-catenin pathway reduced the transforming potential of mutated RON and MET. Activation of beta-catenin by oncogenic RON and MET constitutes a new pathway, which might lead to cell transformation by these and other mutant growth factor RTKs.


Subject(s)
Cytoskeletal Proteins/metabolism , Proto-Oncogene Proteins c-met/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Cell Surface/metabolism , Repressor Proteins , Signal Transduction , Trans-Activators , 3T3 Cells , Animals , Axin Protein , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cell Line , Cell Transformation, Neoplastic , Cyclin D1/biosynthesis , Dogs , Glycogen Synthase Kinase 3 , Mice , Mutagenesis, Site-Directed , Phosphorylation , Proteins/metabolism , Proto-Oncogene Proteins c-met/genetics , Proto-Oncogene Proteins c-myc/biosynthesis , Proto-Oncogene Proteins c-myc/genetics , Receptor Protein-Tyrosine Kinases/genetics , Receptors, Cell Surface/genetics , TCF Transcription Factors , Transcription Factor 7-Like 2 Protein , Transcription Factors/genetics , Transcriptional Activation , Tyrosine/metabolism , beta Catenin
2.
J Biol Chem ; 276(24): 21932-7, 2001 Jun 15.
Article in English | MEDLINE | ID: mdl-11274154

ABSTRACT

Macrophage stimulating protein (MSP) is secreted as 78-kDa single chain pro-MSP, which is converted to biologically active, disulfide-linked alphabeta chain MSP by cleavage at Arg(483)-Val(484). Murine resident peritoneal macrophages have two cell surface proteolytic activities that cleave pro-MSP. One is a pro-MSP convertase, which cleaves pro-MSP to active MSP; the other degrades pro-MSP. The degrading protease is inhibited by soybean trypsin inhibitor or by low concentrations of blood plasma, which allows the convertase to cleave pro-MSP to MSP. Using pro-MSP cleavage as the assay, we purified the inhibitor from human plasma. The bulk of the plasma protein was removed by salting out and by isoelectric precipitation of albumin. Highly purified inhibitor was then obtained in three steps: dye-ligand binding and elution, ion exchange chromatography, and high performance liquid chromatography gel filtration. After SDS-polyacrylamide gel electrophoresis and transfer to a polyvinylidene membrane, N-terminal sequencing of the product identified it as alpha(1)-antichymotrypsin. The mean concentration of alpha(1)-antichymotrypsin in human plasma is 7 micrometer. At this concentration, alpha(1)-antichymotrypsin inhibits both macrophage enzymes. A concentration of 0.4 micrometer, which is in the expected concentration range in extracellular fluid, preferentially inhibits the degrading enzyme, which allows for cleavage to active MSP by the pro-MSP convertase.


Subject(s)
Endopeptidases/metabolism , Growth Substances/metabolism , Macrophages, Peritoneal/enzymology , Protein Precursors/metabolism , alpha 1-Antichymotrypsin/blood , alpha 1-Antichymotrypsin/pharmacology , Animals , Enzyme Precursors/metabolism , Humans , Male , Mice , Mice, Inbred C3H , Protein Processing, Post-Translational , Serine Proteinase Inhibitors/blood , Serine Proteinase Inhibitors/pharmacology , alpha 1-Antichymotrypsin/isolation & purification
3.
J Biol Chem ; 275(20): 14783-6, 2000 May 19.
Article in English | MEDLINE | ID: mdl-10747844

ABSTRACT

Cooperation between integrins and growth factor receptors plays an important role in the regulation of cell growth, differentiation, and survival. The function of growth factor receptor tyrosine kinases (RTKs) can be regulated by cell adhesion to extracellular matrix (ECM) even in the absence of ligand. We investigated the pathway involved in integrin-mediated RTK activation, using RON, the receptor for macrophage-stimulating protein. Adhesion of RON-expressing epithelial cells to ECM caused phosphorylation of RON, which depended on the kinase activity of both RON itself and c-Src. This conclusion is based on these observations: 1) ECM-induced RON phosphorylation was inhibited in cells expressing kinase-inactive c-Src; 2) active c-Src could phosphorylate immunoprecipitated RON from ECM-stimulated cells but not from unstimulated cells; and 3) ECM did not cause RON phosphorylation in cells expressing kinase-dead RON, nor could active c-Src phosphorylate RON immunoprecipitated from these cells. The data fit a pathway in which ECM-induced integrin aggregation causes both c-Src activation and RON oligomerization followed by RON kinase-dependent autophosphorylation; this results in RON becoming a target for activated c-Src, which phosphorylates additional tyrosines on RON. Integrin-induced epidermal growth factor receptor (EGFR) phosphorylation also depended on both EGFR and c-Src kinase activities. This sequence appears to be a general pathway for integrin-dependent growth factor RTK activation.


Subject(s)
Integrins/metabolism , Macrophages/physiology , Proto-Oncogene Proteins pp60(c-src)/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Cell Surface/metabolism , Animals , Cell Adhesion , Cell Line , Collagen/physiology , Epithelial Cells/physiology , ErbB Receptors/metabolism , Humans , Mutagenesis, Site-Directed , Phosphorylation , Receptor Protein-Tyrosine Kinases/genetics , Receptors, Cell Surface/genetics , Recombinant Proteins/metabolism , Transfection
4.
Mol Cell Biol ; 20(6): 2218-27, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10688668

ABSTRACT

In addition to its effects on macrophage function, macrophage-stimulating protein (MSP) is a growth and motility factor for epithelial cells. The growth and survival of epithelial cells generally require two signals, one generated by interaction with extracellular matrix via integrins, the other initiated by a growth factor. Therefore we investigated the effect of MSP on epithelial cell survival. Survival of epithelial cells cultured overnight in serum-free medium was promoted by adhesion, which activated both the phosphatidylinositol 3'-kinase (PI3-K)/AKT and mitogen-activated protein kinase (MAPK) pathways, operating independently of one another. The number of apoptotic cells resulting from inhibition of either pathway alone was approximately doubled by simultaneous inhibition of both pathways. This shows that each pathway made a partial contribution to the prevention of apoptosis. In the presence of an inhibitor of either pathway, MSP increased the activity of the other pathway so that the single uninhibited pathway alone was sufficient to prevent apoptosis. In contrast to the results with adherent cells, although MSP also prevented apoptosis of cells in suspension (anoikis), its effect was mediated only by the PI3-K/AKT pathway. Despite activation of MAPK by MSP, anoikis was not prevented in suspended cells with a blocked PI3-K/AKT pathway. Thus, activation of MAPK alone is not sufficient to mediate MSP antiapoptotic effects. Cell adhesion generates an additional signal, which is essential for MSP to use MAPK in an antiapoptotic pathway. This may involve translocation of MSP-activated MAPK from the cytoplasm into the nucleus, which occurs only in adherent cells. Our results suggest that there is cross talk between cell matrix adhesion and growth factors in the regulation of cell survival via the MAPK pathway. Growth factors induce MAPK activation, and adhesion mediates MAPK translocation from the cytoplasm into the nucleus.


Subject(s)
Epithelial Cells/physiology , Growth Substances/physiology , Hepatocyte Growth Factor , Proto-Oncogene Proteins , Signal Transduction/physiology , Animals , Apoptosis/physiology , Cell Line , Dogs , Epithelial Cells/pathology , MAP Kinase Signaling System , Phosphatidylinositol 3-Kinases/physiology , Protein Precursors/physiology
5.
Exp Cell Res ; 248(2): 575-82, 1999 May 01.
Article in English | MEDLINE | ID: mdl-10222149

ABSTRACT

Macrophage stimulating protein (MSP) is a growth and motility factor that mediates its activity via the RON/STK receptor tyrosine kinase. MSP promotes integrin-dependent epithelial cell migration, which suggests that MSP may regulate integrin receptor functions. Integrins are cell surface receptors for extracellular matrix. Epithelial cell adhesion and motility are mediated by integrins. We studied the enhancement by MSP of cell adhesion and the molecular mechanisms mediating this effect. MSP decreased the time required for adhesion of 293 and RE7 epithelial cells to substrates coated with collagen or fibronectin. Prevention of adhesion by an RGD-containing peptide showed that the cell-substrate interaction was mediated by integrins. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), blocked MSP-dependent adhesion, which shows that PI3-K is in the MSP-induced adhesion pathway. MSP also affected focal adhesion kinase (FAK) which is important for some types of cell adhesion and motility. Although MSP caused PI3-K-independent tyrosine phosphorylation and activation of FAK, experiments with dominant-negative FAK constructs showed that FAK does not mediate the effects of MSP on cell adhesion or motility. Thus PI3-K, but not FAK, mediates MSP-induced integrin-dependent adhesion of epithelial cells. Also, we found ligand-independent association between RON and beta1 integrin, which is additional evidence for a relationship between these two receptor systems.


Subject(s)
Cell Adhesion Molecules/metabolism , Cell Adhesion , Epithelial Cells/drug effects , Growth Substances/pharmacology , Hepatocyte Growth Factor , Phosphatidylinositol 3-Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins , Cell Adhesion Molecules/genetics , Cell Line , Cell Movement , Focal Adhesion Protein-Tyrosine Kinases , Integrin beta1 , Integrins/metabolism , Kidney/cytology , Mutation , Phosphorylation , Protein Binding , Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Cell Surface/metabolism
6.
Arch Biochem Biophys ; 363(2): 356-60, 1999 Mar 15.
Article in English | MEDLINE | ID: mdl-10068459

ABSTRACT

Human serum macrophage-stimulating protein (MSP) induces motile activity of murine resident peritoneal macrophages and is a growth and motility factor for epithelial cells. It belongs to the plasminogen-related family of kringle proteins, and is secreted as a single-chain, 78-kDa, biologically inactive pro-MSP. Proteolytic cleavage of pro-MSP at a single site yields active MSP, a disulfide-linked alphabeta-chain heterodimer. However cleavage of recombinant pro-MSP yielded not only the disulfide-linked heterodimer, but also free alpha- and beta-chains, indicating that some of the recombinant molecules lacked an alphabeta-chain disulfide. We purified the free chains for characterization. The beta-chain of MSP has three extra cysteines, Cys527, Cys562, and Cys672, which are not found in the plasminogen beta-chain. Disulfide bond analysis showed a Cys527-Cys562, but also a Cys588-Cys672. Coopting Cys588 by Cys672 prevented the expected formation of a disulfide between alpha-chain Cys468 and beta-chain Cys588. Concomitant studies determined structures of oligosaccharides at the three Asn-linked glycosylation sites of MSP. The oligosaccharides at the three Asn loci are heterogeneous; 11 different sugars were identified, all being sialylated fucosyl biantennary structures. We also located the pro-MSP signal peptide cleavage site at Gly18-Gln19 and the scissile bond for formation of mature MSP at Arg483-Val484.


Subject(s)
Growth Substances/chemistry , Hepatocyte Growth Factor , Proto-Oncogene Proteins , Recombinant Proteins/chemistry , Amino Acids/analysis , Animals , CHO Cells , Carbohydrate Metabolism , Chromatography, Ion Exchange , Cricetinae , Cysteine/metabolism , Electrophoresis, Polyacrylamide Gel , Gas Chromatography-Mass Spectrometry , Growth Substances/isolation & purification , Humans , Kallikreins/metabolism , Molecular Sequence Data , Molecular Weight , Protein Precursors/metabolism , Protein Structure, Tertiary , Recombinant Proteins/isolation & purification
7.
J Invest Dermatol ; 111(4): 573-81, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9764835

ABSTRACT

Macrophage stimulating protein (MSP) exists in blood as inactive pro-MSP. Cleavage yields active MSP, the ligand for a membrane receptor (RON) that is expressed on keratinocytes as well as macrophages. Because both cells have roles in tissue injury, we looked for active MSP and expressed RON in wounds. Concentration of pro-MSP + MSP in wound exudates was in the range for optimal activity. Western blot showed that MSP comprised about half the total, in contrast to less than 10% of the total in blood plasma. The presence of MSP was attributed to an exudate pro-MSP convertase that had an inhibitor profile consistent with a trypsin-like serine protease. Exudate evoked morphologic changes in macrophages in vitro like that of MSP. Removal of this activity by an anti-MSP column shows that exudate stimulation of macrophages is due to MSP. RON was infrequently detected in normal skin. RON protein was markedly upregulated in burn wound epidermis and accessory structures, in proliferating cells or differentiated cells, or both. RON was also detected on macrophages and capillaries. Tissue injury leads to cleavage of pro-MSP to MSP, which has potential to act on keratinocytes, macrophages, and capillaries, all components of the wound healing response.


Subject(s)
Growth Substances/metabolism , Hepatocyte Growth Factor , Proto-Oncogene Proteins , Receptor Protein-Tyrosine Kinases/physiology , Receptors, Cell Surface/physiology , Wounds and Injuries/metabolism , Animals , Burns/metabolism , Cell Movement , Exudates and Transudates/enzymology , Humans , Keratinocytes/cytology , Mice , Mice, Inbred C3H , Protein Precursors/blood , Skin/chemistry , Up-Regulation , Wounds and Injuries/physiopathology
8.
J Leukoc Biol ; 60(4): 453-8, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8864128

ABSTRACT

We injected 125I-pro-macrophage-stimulating protein (pro-MSP) intravenously into normal mice to determine its clearance from the circulation and to test for conversion of pro-MSP to the biologically active heterodimer in the absence of inflammation or tissue injury. Pro-MSP was cleared from the circulation with a half-life of approximately 100 min. This rapid clearance was not peculiar to 125I-pro-MSP, since clearance rates of unlabeled pro-MSP and of 125I-bovine serum albumin were comparable. The liver was the major locus of radioactivity 10-20 min after the intravenous injection of 125I-pro-MSP. By 90 min, over 60% of total recovered radioactivity was in the small intestine. Reflecting gastrointestinal transit, counts decreased in the small intestine and appeared in the colon by 180 min. Essentially all counts in urine and feces obtained at later times were soluble in trichloracetic acid. These findings reflected rapid hepatic proteolysis of pro-MSP to fragments undetectable by antibody to pro-MSP; within 20 min after intravenous administration, immunoprecipitable counts were only 22% of the total liver extract radioactivity. Comparison of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and radioautography data for immunoprecipitated plasma and liver extract revealed no evidence for hepatic conversion of pro-MSP to MSP. Thus, the hepatic catabolic pathway of pro-MSP is degradative and does not yield mature MSP. The results support our view that MSP is not released into the circulation but is generated at specific extravascular loci by pro-MSP convertases.


Subject(s)
Growth Substances/metabolism , Hepatocyte Growth Factor , Liver/metabolism , Proto-Oncogene Proteins , Animals , Blood Proteins/metabolism , Kringles , Male , Metabolic Clearance Rate , Mice , Mice, Inbred C3H , Protein Binding , Protein Precursors/metabolism , Recombinant Proteins , Tissue Distribution
9.
Exp Cell Res ; 226(1): 39-46, 1996 Jul 10.
Article in English | MEDLINE | ID: mdl-8660937

ABSTRACT

Macrophage stimulating protein (MSP) is a chemotactic factor for murine peritoneal macrophages. The receptor for human MSP was recently identified as the ron gene product, a transmembrane protein tyrosine kinase cloned from a human keratinocyte cDNA library. Here we report that MSP induced proliferation of murine primary keratinocytes and established keratinocyte cell lines in a concentration-dependent manner. The growth efficacy of MSP was comparable to that of epidermal growth factor and keratinocyte growth factor. In three of four cell lines tested in a chemotaxis chamber, MSP also stimulated migration of keratinocytes on a collagen type IV substratum. The action of MSP was mediated by specific binding of MSP to the STK gene product, a murine homologue of the RON MSP receptor. Binding of MSP to keratinocyte STK induced phosphorylation of the 150 kDa STK beta chain. Herbimycin A, a protein tyrosine kinase inhibitor, blocked MSP-mediated phosphorylation of the STK receptor as well as proliferation of keratinocytes, suggesting the importance of tyrosine kinase activity for transduction of the message delivered by MSP. Previously, the only known target cell for MSP was the resident peritoneal macrophage. These studies establish the keratinocyte as a new target cell for MSP. The action of MSP on keratinocytes may have implications for tissue repair, wound healing, and tumor growth.


Subject(s)
Growth Substances/pharmacology , Hepatocyte Growth Factor , Keratinocytes/cytology , Proto-Oncogene Proteins , Animals , Animals, Newborn , Cell Division/drug effects , Cell Line/chemistry , Cell Line/cytology , Cell Line/drug effects , Cell Movement/drug effects , Gene Expression/drug effects , Humans , Iodine Radioisotopes , Keratinocytes/chemistry , Keratinocytes/drug effects , Mice , Mice, Inbred BALB C , Mitogens/pharmacology , Phosphorylation , Protein Precursors/pharmacology , Receptor Protein-Tyrosine Kinases/drug effects , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Cell Surface/drug effects , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Tyrosine/metabolism
10.
J Clin Invest ; 97(3): 720-7, 1996 Feb 01.
Article in English | MEDLINE | ID: mdl-8609228

ABSTRACT

Macrophage stimulating protein (MSP), which is secreted as biologically inactive pro-MSP, is activated to MSP by cleavage at a single peptide bond. Our objectives were to determine the form of MSP in circulating blood and to study proteolytic activation of pro-MSP by its target cell. Western blot of immunoaffinity-purified serum MSP showed that all the protein was pro-MSP, without detectable MSP. The circulating form of the protein is therefore pro-MSP, and conversion to MSP does not occur when blood is shed. Incubation of radiolabeled pro-MSP with murine peritoneal macrophages caused proteolytic cleavage to predominantly inactive fragments. Among several protease inhibitors, soybean trypsin inhibitor was one of two that inhibited nonspecific cleavage and revealed a macrophage proteolysis of pro-MSP, and certain concentrations enhanced cleavage to mature MSP. Macrophage membranes had nonspecific and specific pro-MSP proteolytic activity, which was not present in macrophage culture fluids. The results suggest that control of MSP activity can occur at the level of the target cell by proteolytic cleavage of pro-MSP to mature MSP or to inactive fragments.


Subject(s)
Cell Membrane/enzymology , Endopeptidases/metabolism , Growth Substances/metabolism , Hepatocyte Growth Factor , Macrophages, Peritoneal/enzymology , Protein Precursors/metabolism , Proto-Oncogene Proteins , Animals , Cell Compartmentation , Cells, Cultured , Culture Media, Conditioned , Dose-Response Relationship, Drug , Growth Substances/blood , Growth Substances/pharmacology , Humans , Mice , Mice, Inbred C3H , Protein Precursors/blood , Serpins/pharmacology
11.
Proc Natl Acad Sci U S A ; 92(9): 3933-7, 1995 Apr 25.
Article in English | MEDLINE | ID: mdl-7732008

ABSTRACT

Macrophage-stimulating protein (MSP) was originally identified as an inducer of murine resident peritoneal macrophage responsiveness to chemoattractants. We recently showed that the product of RON, a protein tyrosine kinase cloned from a human keratinocyte library, is the receptor for MSP. Similarity of murine stk to RON led us to determine if the stk gene product is the murine receptor for MSP. Radiolabeled MSP could bind to NIH 3T3 cells transfected with murine stk cDNA (3T3/stk). Binding was saturable and was inhibited by unlabeled MSP but not by structurally related proteins, including hepatocyte growth factor and plasminogen. Specific binding to STK was demonstrated by cross-linking of 125I-labeled MSP to membrane proteins of 3T3/stk cells, which resulted in a protein complex with a molecular mass of 220 kDa. This radiolabeled complex comprised 125I-MSP and STK, since it could be immunoprecipitated by antibodies to the STK beta chain. Binding of MSP to stk cDNA-transfected cells induced tyrosine phosphorylation of the 150-kDa STK beta chain within 1 min and caused increased motile activity. These results establish the murine stk gene product as a specific transmembrane protein tyrosine kinase receptor for MSP. Inasmuch as the stk cDNA was cloned from a hematopoietic stem cell, our data suggest that in addition to macrophages and keratinocytes, a cell in the hematopoietic lineage may also be a target for MSP.


Subject(s)
Growth Substances/metabolism , Hepatocyte Growth Factor , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins , Receptors, Growth Factor/metabolism , Receptors, Transforming Growth Factor beta , 3T3 Cells , Animals , Humans , Iodine Radioisotopes , Keratinocytes/metabolism , Kinetics , Mice , Protein Precursors/metabolism , Protein Serine-Threonine Kinases/metabolism , Radioligand Assay , Receptors, Growth Factor/biosynthesis , Transfection
12.
Science ; 266(5182): 117-9, 1994 Oct 07.
Article in English | MEDLINE | ID: mdl-7939629

ABSTRACT

Macrophage-stimulating protein (MSP) is a member of the hepatocyte growth factor-scatter factor (HGF-SF) family. Labeled MSP bound to Madin-Darby canine kidney (MDCK) cells transfected with complementary DNA encoding Ron, a cell membrane protein tyrosine kinase. Cross-linking of 125I-labeled MSP to transfected cells (MDCK-RE7 cells) and immunoprecipitation by antibodies to Ron revealed a 220-kilodalton complex, a size consistent with that of MSP (80 kilodaltons) cross-linked to the beta chain of Ron (150 kilodaltons). The binding of 125I-labeled MSP to MDCK-RE7 cells was inhibited by unlabeled MSP, but not by HGF-SF. MSP caused phosphorylation of the beta chain of Ron and induced migration of MDCK-RE7 cells. These results establish the ron gene product as a specific cell-surface receptor for MSP.


Subject(s)
Growth Substances/metabolism , Proto-Oncogene Proteins , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Cell Surface/metabolism , Animals , Binding Sites , Binding, Competitive , Cell Line , Cell Movement/drug effects , Cross-Linking Reagents , Dogs , Growth Substances/pharmacology , Hepatocyte Growth Factor/metabolism , Humans , Phosphorylation , Plasminogen/metabolism , Receptor Protein-Tyrosine Kinases/genetics , Receptors, Cell Surface/genetics , Transfection
13.
J Biol Chem ; 269(19): 14027-31, 1994 May 13.
Article in English | MEDLINE | ID: mdl-7514598

ABSTRACT

Human serum macrophage-stimulating protein (MSP) is a disulfide-linked heterodimer that induces motile and phagocytic activity of mouse resident peritoneal macrophages. In this work, we found that MSP blocked the increase in macrophage nitric oxide synthase mRNA, as well as the associated increase in nitric oxide production, that occurred in response to several stimuli. These included bacterial products and mammalian cytokines: endotoxin, and interferon-gamma plus endotoxin, interleukin-2, or tumor necrosis factor-alpha. The inhibition by MSP of induction of nitric oxide synthase mRNA and nitric oxide secretion was concentration-dependent. The concentration of MSP that caused maximal inhibition of nitric oxide production was comparable with the optimum for stimulation of macrophage motile and phagocytic activity. Time course studies showed that nitrite was first detected in culture fluid about 8 h after endotoxin stimulation, and it accumulated at a linear rate during the ensuing 16 h. Inhibition by MSP occurred during the 8-h lipopolysaccharide (LPS) induction period; inhibition was maximal when MSP and LPS were added together and decreased progressively to no inhibition as the interval between LPS and MSP addition increased to 11 h.


Subject(s)
Growth Substances/physiology , Hepatocyte Growth Factor , Macrophages, Peritoneal/metabolism , Nitric Oxide/biosynthesis , Proto-Oncogene Proteins , Amino Acid Oxidoreductases/genetics , Animals , Cytokines/physiology , Endotoxins/physiology , Humans , Kinetics , Macrophage Activation , Mice , Mice, Inbred C3H , Nitric Oxide/antagonists & inhibitors , Nitric Oxide Synthase , RNA, Messenger/biosynthesis
14.
J Immunol ; 152(9): 4618-23, 1994 May 01.
Article in English | MEDLINE | ID: mdl-8157975

ABSTRACT

Macrophage-stimulating protein (MSP) induces mouse resident peritoneal macrophages to become responsive to the chemoattractant C5a and to ingest C3bi-coated erythrocytes. We now show that MSP action is not limited to complement-induced responses, because it also induced responsiveness to the noncomplement chemoattractant casein. In addition to stimulating responsiveness to attractants, MSP functioned alone as a chemoattractant for resident peritoneal macrophages, with an optimal concentration of approximately 0.2 nM. A critical difference between MSP and C5a is that resident macrophages did not migrate to C5a without an additional stimulus such as MSP in the cell suspension, whereas macrophages suspended in medium alone migrated to MSP in the attractant well. Thus, in contrast to C5a, MSP seems capable of a dual role, both activator and attractant. MSP had no effect on responsiveness of mouse peritoneal exudate macrophages to C5a; nor could it attract exudate macrophages or human blood monocytes. Absorption studies showed that resident macrophages have a receptor for MSP, but exudate macrophages do not. In view of these findings, it seems that the biological role of MSP is not as a recruiter of blood monocytes to sites of inflammation, but as an activator of mature macrophages. The MSP-induced activated state for responsiveness to C5a or C3bi was transient, and decayed at a first order rate with a t 1/2 of approximately 1 h. This is a new example of the transience of activation induced in macrophages by proinflammatory stimuli.


Subject(s)
Growth Substances/pharmacology , Hepatocyte Growth Factor , Macrophages, Peritoneal/drug effects , Proto-Oncogene Proteins , Animals , Cell Movement/drug effects , Cell Movement/immunology , Complement C5a/immunology , Erythrocytes/immunology , Humans , In Vitro Techniques , Macrophages, Peritoneal/immunology , Macrophages, Peritoneal/physiology , Mice , Phagocytosis , Sheep
15.
J Biol Chem ; 269(19): 13806-10, 1994 May 13.
Article in English | MEDLINE | ID: mdl-8188657

ABSTRACT

Promacrophage-stimulating protein (MSP) is an 80-kDa protein that acquires biological activity after cleavage at an Arg-Val bond to a disulfide-linked alpha beta heterodimer by serine proteases of the intrinsic coagulation cascade. These proteases, which include serum kallikrein, factor XIIa and factor XIa, are members of the trypsin family of serine proteases. We now report that two other members of the family, nerve growth factor-gamma (NGF-gamma) and epidermal growth factor-binding protein (EGF-BP), cleave and activate pro-MSP to the disulfide-linked alpha beta heterodimer. Cleavage of 1.5 nM pro-MSP by 1 nM NGF-gamma or EGF-BP at 37 degrees C was almost complete within 30 min. These concentrations of enzyme are about 2 orders of magnitude less than is required for cleavage by serum kallikrein or factor XIIa. Cleavage of pro-MSP to MSP was associated with a conformational change in the protein, because the cleaved product, but not pro-MSP, was detected by a sandwich enzyme-linked immunoassay. Cleavage caused the appearance of biological activity, as measured by chemotactic activity of MSP for resident peritoneal macrophages, by MSP-induced macrophage shape change, and by stimulation of macrophage ingestion of C3bi-coated erythrocytes. These findings suggest the possibility of cooperative interactions between NGF-gamma or EGF-BP and pro-MSP in inflammation and wound healing.


Subject(s)
Endopeptidases/metabolism , Growth Substances/metabolism , Hepatocyte Growth Factor , Kallikreins/metabolism , Nerve Growth Factors/metabolism , Protein Precursors/metabolism , Proto-Oncogene Proteins , Serine Endopeptidases/metabolism , Animals , CHO Cells , Cricetinae , Growth Substances/genetics , Humans , Hydrolysis , Kallikreins/genetics , Protein Precursors/genetics
16.
J Biol Chem ; 269(5): 3436-40, 1994 Feb 04.
Article in English | MEDLINE | ID: mdl-7508914

ABSTRACT

Human serum macrophage stimulating protein (MSP) is a disulfide-linked heterodimer that induces motile and phagocytic activity of mouse resident peritoneal macrophages. It is a member of the family of kringle proteins, which typically exist in extracellular fluid as single chain precursors that are activated by proteolytic cleavage. In this work, we expressed [35S]cysteine-labeled recombinant pro-MSP in MSP cDNA-transfected Chinese hamster ovary cells and studied proteolytic processing of pro-MSP and the requirement of cleavage for biological activity. In media containing heat-inactivated fetal bovine serum, the protein was secreted as single chain pro-MSP, which was cleaved over a period of hours to the mature heterodimer. Cleavage was prevented by serine protease inhibitors such as leupeptin or aprotinin; it did not occur if cells were cultured in serum-free medium. Nanomolar concentrations of coagulation proteases kallikrein, factor XIIa or factor XIa cleaved pro-MSP to MSP within 30 min. Pro-MSP had no biological activity. After cleavage by kallikrein, biological activity was quantitatively comparable to that of natural MSP isolated from human plasma. These results support our hypothesis that MSP circulates as the biologically inactive precursor and can be activated by enzymes of the intrinsic coagulation cascade.


Subject(s)
Blood Coagulation Factors/metabolism , Growth Substances/biosynthesis , Hepatocyte Growth Factor , Macrophages/metabolism , Macrophages/physiology , Protein Precursors/biosynthesis , Protein Precursors/metabolism , Proto-Oncogene Proteins , Animals , Aprotinin/pharmacology , CHO Cells , Cells, Cultured , Chemotaxis/drug effects , Cloning, Molecular , Cricetinae , Cysteine/metabolism , Enzyme-Linked Immunosorbent Assay , Factor XIIa/metabolism , Factor XIa/metabolism , Growth Substances/isolation & purification , Growth Substances/pharmacology , Humans , Kallikreins/metabolism , Kinetics , Leupeptins/pharmacology , Macromolecular Substances , Macrophages/cytology , Macrophages/drug effects , Mice , Molecular Weight , Protein Precursors/isolation & purification , Protein Precursors/pharmacology , Protein Processing, Post-Translational/drug effects , Protein Processing, Post-Translational/physiology , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Serine Proteinase Inhibitors/pharmacology , Transfection
17.
J Leukoc Biol ; 54(4): 289-95, 1993 Oct.
Article in English | MEDLINE | ID: mdl-7691976

ABSTRACT

Macrophage stimulating protein (MSP) is a member of a family of proteins characterized by a triple disulfide loop structure (kringle). We developed antibodies to human MSP for detection in Western blots, quantification in biological fluids, and neutralization of activity. Immunogens included native MSP, reduced and alkylated alpha and beta chains, and peptides of MSP regions with minimal sequence similarity to other kringle proteins. We found three antibody categories based on interaction with the following types of epitope: primary sequence, discontinuous (dependent on disulfide bonds), and cryptic (not exposed in native MSP). None of the antibodies reacted with related kringle proteins. A specific sandwich ELISA was developed for measuring human MSP. The mean serum concentration was 4 nM. Serum MSP did not increase over a 24-h period in response to intravenous lipopolysaccharide, indicating that MSP is not an acute phase protein. These findings are consistent with the hypothesis that regulation of MSP activity is by conversion of pro-MSP to MSP rather than by rapid changes in rates of synthesis.


Subject(s)
Antibodies, Monoclonal , Epitopes/analysis , Growth Substances/blood , Growth Substances/immunology , Hepatocyte Growth Factor , Proto-Oncogene Proteins , Amino Acid Sequence , Animals , Antibody Specificity , Blotting, Western , Carcinoma, Hepatocellular , Cattle , Enzyme-Linked Immunosorbent Assay , Growth Substances/analysis , Humans , Liver Neoplasms , Macromolecular Substances , Mice , Molecular Sequence Data , Molecular Weight , Rats , Sequence Homology, Amino Acid , Sheep , Tumor Cells, Cultured
18.
J Biol Chem ; 268(21): 15461-8, 1993 Jul 25.
Article in English | MEDLINE | ID: mdl-8393443

ABSTRACT

A human hepatoma (HepG2) cell line library was screened with an oligonucleotide probe for macrophage stimulating protein (MSP) to clone an MSP cDNA. Deduced sequences of isolated clones were compared with peptide fragment sequences of MSP. MSP9 cDNA encoded most of the known sequence of MSP except for a small segment of the 5' end of the open reading frame. Consequently, a hybrid 2300-base pair cDNA that encoded the complete MSP amino acid sequence was constructed from 2 clones. Culture fluid from COS-7 cells transfected with this full-length MSP cDNA had MSP biological activity, and the expressed MSP was detected by immunoprecipitation with antibody against native MSP. The deduced amino acid sequence of MSP includes 4 kringle domains, which have been found in hepatocyte growth factor and several proteins of the blood coagulation system. Among them, MSP has the highest sequence similarity to hepatocyte growth factor (45% identity). The MSP cDNA hybridized strongly to mRNA from liver, and to a lesser extent to mRNA from kidney and pancreas, suggesting that a cell type in the liver is the source of MSP. Several cloned and sequenced MSP cDNAs had insertions or deletions, suggesting that alternatively spliced MSP mRNAs may occur. This was reflected in Northern blots probed with an MSP cDNA, which showed more than one mRNA species. Furthermore, although the gene coding for MSP is on chromosome 3, the sequence of one of the cDNAs was identical with a unique sequence in chromosome 1, indicating that there may be a family of MSP genes, located on chromosomes 3 and 1.


Subject(s)
Chromosomes, Human, Pair 3 , Growth Substances/genetics , Hepatocyte Growth Factor , Macrophages/immunology , Peptide Fragments/genetics , Proto-Oncogene Proteins , Amino Acid Sequence , Animals , Base Sequence , Cells, Cultured , Chromosome Mapping , Cloning, Molecular , DNA , Growth Substances/physiology , Humans , Mice , Molecular Sequence Data , Nucleic Acid Hybridization , Sequence Homology, Amino Acid , Sheep , Tumor Cells, Cultured
19.
Adv Exp Med Biol ; 351: 55-64, 1993.
Article in English | MEDLINE | ID: mdl-7942299

ABSTRACT

Concentrations of MCP-1 and NAP-1 in culture fluids of human leukocytes were measured by sandwich ELISA. PPD caused PBMC's from tuberculin-sensitive subjects to secrete MCP-1 and NAP-1. PPD did not stimulate secretion by cells from a tuberculin-negative subject. Since the amounts secreted were more than could be produced by the few PPD-sensitized lymphocytes in the culture, we postulate that other cells were stimulated to secrete these chemoattractants. This study evaluated secretory capacity of one of the cell types in the PBMC culture. Unstimulated monocytes did not secrete MCP-1 or NAP-1. In order of increasing effect, IL-2 + IFN gamma, IL-1 alpha, and LPS caused monocyte secretion of MCP-1. The rank order for NAP-1 secretion was the same. TNF alpha did not cause secretion of MCP-1, but caused about the same amount of NAP-1 secretion as IL-2 + IFN gamma. Composition of the culture medium was especially critical for LPS-induced secretion of MCP-1, which was greatly enhanced by FCS and by Iscove's DMEM compared to RPMI 1640. IL-4 inhibited LPS-induced secretion of both MCP-1 and NAP-1. Secretory patterns were also a function of mononuclear phagocyte phenotype. LPS-induced secretion of MCP-1 was much greater for monocytes cultured several days in CSF-1 than for freshly isolated monocytes. LPS stimulation of bronchoalveolar macrophages caused NAP-1 secretion, but no secretion of MCP-1 above a relatively low baseline level.


Subject(s)
Chemotactic Factors/metabolism , Leukocytes, Mononuclear/physiology , Phagocytes/physiology , Chemokine CCL2 , Chemotactic Factors/pharmacology , Cytokines/pharmacology , Humans , Interleukin-8/metabolism , Interleukin-8/pharmacology , Leukocytes, Mononuclear/immunology , Lipopolysaccharides/pharmacology , Macrophages/physiology , Monocytes/physiology , Tuberculin/immunology
20.
J Exp Med ; 173(5): 1227-34, 1991 May 01.
Article in English | MEDLINE | ID: mdl-1827141

ABSTRACT

Macrophage stimulating protein (MSP) was purified to homogeneity from human blood plasma by selection of biologically active fractions obtained by sequential immunoaffinity and high pressure liquid ion exchange chromatography. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis the molecular mass of MSP was 70 kilodaltons (kD); under reducing conditions two gel bands were seen, at 47 and 22 kD. The disulfide-linked two-chain structure of MSP was confirmed by separation of reduced and alkylated MSP chains. A computer search comparison of six partial sequences of MSP digests showed that MSP has not been recorded in data banks of protein sequences. Two MSP fragments had greater than 80% identity in overlaps of 12-16 residues to sequences in the protein family that includes human prothrombin, plasminogen, and hepatocyte growth factor. The concentration of purified MSP required for half-maximal biological activity was the order of 10(-10) M. In addition to making mouse resident peritoneal macrophages responses to chemoattractants, MSP caused the appearance of long cytoplasmic processes and pinocytic vesicles in freshly plated macrophages. MSP also caused phagocytosis via the C3b receptor, CR1. Whereas resident peritoneal macrophages bind but do not ingest sheep erythrocytes opsonized with IgM anti-Forssman antibody and mouse C3b, addition of MSP caused ingestion. Thus, MSP causes direct or indirect activation of two receptors of the mouse resident peritoneal macrophage, CR1 and the C5a receptor.


Subject(s)
Growth Substances/isolation & purification , Macrophage Colony-Stimulating Factor/isolation & purification , Macrophages , Amino Acid Sequence , Amino Acids/analysis , Animals , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Growth Substances/analysis , Growth Substances/physiology , Humans , Macrophage Colony-Stimulating Factor/analysis , Macrophage Colony-Stimulating Factor/physiology , Macrophages/drug effects , Macrophages/physiology , Macrophages/ultrastructure , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Molecular Sequence Data , Phagocytosis/drug effects , Phagocytosis/physiology , Receptors, Complement/physiology , Receptors, Complement 3b
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