Macrophage-Stimulating Protein (MSP) Alla Danilkovitch-Miagkova* and Edward J. Leonard Section of Immunopathology, Laboratory of Immunobiology, Division of Basic Sciences, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD 21702, USA * corresponding author: tel: (301) 846-1560, fax: (301) 846-6145, e-mail:
[email protected] DOI: 10.1006/rwcy.2001.0812. Chapter posted 5 November 2001
SUMMARY Macrophage-stimulating protein (MSP, also known as hepatocyte growth factor-like protein) was discovered as a serum factor inducing macrophage shape changes and chemotaxis. On the basis of structural homology MSP was placed in the plasminogen-related kringle domain protein family, most members of which are proteolytic enzymes. In contrast, MSP has lost its enzymatic activity and has become a growth and motility factor. MSP is constitutively secreted by liver cells and circulates in the bloodstream as a biologically inactive single-chain pro-MSP. At extravascular sites pro-MSP can be converted by trypsin-like serine proteases to active heterodimeric MSP. The MSP receptor, called RON in humans and STK in mice, is a transmembrane receptor tyrosine kinase. MSP/RON interaction activates RON, and initiates downstream intracellular signaling pathways mediating MSP biological activities. Recent data show that in addition to macrophages MSP affects a number of other targets such as osteoclasts and hepatocytes, epithelial, endothelial, and hematopoietic cells. The list of MSP biological activities includes adhesion and motility, cytokine production, angiogenesis, growth, differentiation, and cell survival. Active MSP was detected in injured tissues and in regions of inflammation, suggesting a role of MSP in regulation of
Cytokine Reference
inflammation and tissue regeneration. MSP was detected in embryonal tissues; its ectopic overexpression in Xenopus caused defects in development of the neural system. Abnormal MSP expression was detected in hepatocellular carcinoma tissues and lung cancer cell lines.
BACKGROUND
Discovery MSP was discovered as a normal serum factor that enhanced chemotaxis of mouse peritoneal resident macrophages in response to endotoxin-activated mouse serum (Leonard and Skeel, 1976, 1980). MSP was purified to homogeneity from human blood plasma (Skeel et al., 1991). A computer search of six partial sequences of MSP digests showed that MSP was not in the protein database. Two MSP fragments had 80% identity to sequences of the protein family that includes human prothrombin, plasminogen, and hepatocyte growth factor (HGF) (Skeel et al., 1991). Screening of a human genomic DNA library with a bovine cDNA probe coding for the kringle domains in prothrombin resulted in isolation of a new gene. The protein encoded by this gene contained four kringle and one serine protease-like domain. On the basis of the structural
Copyright # 2001 Academic Press
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Alla Danilkovitch-Miagkova and Edward J. Leonard
similarity with HGF, the new protein was called HGF-like protein (Han et al., 1991). Comparison of the amino acid sequence of human HGFL protein with the sequence of peptides derived from human MSP suggested that HGFL and MSP were the same protein (Bezerra et al., 1993a). Two years later MSP cDNA was cloned from a human hepatoma cell line (Yoshimura et al., 1993). A computer search showed that the MSP cDNA was encoded by the HGFL gene (Yoshimura et al., 1993). Comparison of MSP and HGFL amino acid sequences and functional activities led to the conclusion that HGFL and MSP are indeed identical proteins (Shimamoto et al., 1993).
Alternative names Hepatocyte growth factor-like protein (HGFL) (Han et al., 1991) or HLP (Shimamoto et al., 1993); RON ligand; MST1 (Yoshimura et al., 1993).
Structure MSP is a 80 kDa serum factor that belongs to a plasminogen-related kringle domain protein family (Han et al., 1991; Yoshimura et al., 1993). MSP, like other members of the family, is a heterodimeric protein consisting of and chains. The chain has an Nterminal domain corresponding to the plasminogen preactivation peptide (PAP or hairpin loop) and four kringle domains, and a segment that terminates in the cleavage site for activation. The chain has a serine-like protease domain (Han et al., 1991; Yoshimura et al., 1993). Whereas most of the proteins of the plasminogen-related family are serine proteases, MSP has lost its enzymatic activity because of catalytic triad mutations in the serine protease domain. Although MSP was transformed from a serine protease to a growth and motility factor, it retained the proteolytic mechanism of activation, which is a common feature of proteins of the family (Han et al., 1991; Yoshimura et al., 1993; Leonard and Danilkovitch, 2000). MSP is
Figure 1 The processing of biologically inactive pro-MSP to active mature MSP occurs at extravascular sites. MSP is constitutively secreted by hepatocytes. It circulates in bloodstream as a a single-chain biologically inactive pro-MSP. At extravascular sites pro-MSP can be converted by trypsin-like serine proteases to biologically active heterodimeric MSP. The cleavage site is located between Arg483 and Val484 (indicated by the red arrow). Active MSP consists of covalently linked 53 kDa and 25 kDa chains. K1±K4 repesent four kringle domains located in the chain. S-S shows the interchain disulfide bond between Cys468 and Cys577 located in the and chain respectively. Liver
m
ea
tr ds
α-chain (53 kDa)
o
Blo
K3 K1
K2
K1 K4
K3 K2
Biologically active heterodimeric MSP
K4 S S
β-chain (25 kDa) Extravascular site
Trypsin-like serine proteases K3
K1 K2
K4 S Biologically inactive S single chain pro-MSP
Macrophage-Stimulating Protein (MSP) 3 constitutively secreted by hepatocytes and circulates in the bloodstream as a biologically inactive single chain pro-MSP (Bezerra et al., 1993b; Leonard and Danilkovitch, 2000). Pro-MSP can be cleaved and activated at extravascular sites by trypsin-like proteases. Mature biologically active MSP consists of disulfide-linked 53 kDa and 25 kDa chains. A schematic representation of the structure and processing of pro-MSP to MSP is shown in Figure 1.
Rat MSP: X95096 (Ohshiro et al., 1996) Frog MSPs: Y08734 (MSPA, xenopus HPL, Xhl) (Aberger et al., 1996) and U57455 (MSPB, Livertine) (Ruiz and Thery, 1996). Human MSP gene promoter: U37055 (Waltz et al., 1996) and U43376 (Ueda and Yoshimura, 1996). Human MSP pseudogenes: AF083410, AF083411, AF083412, AF083413, AF083414, AF083415, AF083416, AF083417 (van der Drift et al., 1999).
Main activities and pathophysiological role
Sequence
MSP is a multifunctional factor mediating its biological activities via RON receptor tyrosine kinase. Main MSP targets and biological activities are shown in Figure 2. At present, the pathophysiological role of MSP is speculative.
GENE
Accession numbers GenBank: Human MSP: L11924 (Yoshimura et al., 1993)
Figure 2
Chromosome location The human MSP gene is located on chromosome 3 (3p21) (Han et al., 1991; Yoshimura et al., 1993). The human MSP pseudogenes are located on chromosome 1 (1p36) (van der Drift et al., 1999). The mouse MSP gene is located on chromosome 9 at a locus (Hgfl) that shows homology to band p21 on human chromosome 3 (Degen et al., 1992).
MSP targets and main biological activities.
Hematopoletic cells
Epithelial cells
Macrophages
Shape changes
MSP gene sequences of various species are available at GenBank (http://www.ncbi.nlm.nih.gov).
Osteoclasts
Adhesion
Vascular endothelial cells
Motility
Cytokine production
Chemotaxis Apoptosis Macropinocytosis Ingestion of EigMC3bi Inhibition of iNOS IL-6 production Up-regulation of arginase
Survival Bone resorption Growth induction
Growth inhibition Growth induction
Apoptosis
Liver progenitor cells
Angiogenesis
Shape changes Growth inhibition Growth induction Inhibition or promotion of differentiation
Scattering Morphogenesis
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Alla Danilkovitch-Miagkova and Edward J. Leonard
Relevant linkages The chromosomal location of the human MSP gene is 3p21 (Han et al., 1991; Yoshimura et al., 1993). This region has been proposed to contain one or more tumor suppressor genes since this locus is deleted in small cell lung and kidney cancer samples (Zbar et al., 1987; Brauch et al., 1987). Although, at present, there are no data showing a linkage between MSP gene deletion and human cancers, it is still possible that MSP may function as a tumor suppressor. When the MSP gene was cloned, the human MSP genomic probe showed the presence of the MSP gene sequences on chromosome 1 in addition to chromosome 3 (Yoshimura et al., 1993). Recently, multiple MSP pseudogenes in a local repeat cluster on p36.2 were cloned (van der Drift et al., 1999). It seems that all cloned genes have nucleotide deletions or substitutions that lead to coding nonfunctional proteins (van der Drift et al., 1999). It is possible that during evolution a copy of the MSP ancestral gene located on chromosome 3 was swept into the cluster of tRNA and snRNA genes on 1p36.2 (van der Drift et al., 1999). The recombination process that caused the repetitive structure of this cluster has led to MSP amplification and a number of polymorphisms. The biological significance of these MSP pseudogenes is unclear, but the relevance of genomic instability in 1p and human malignancies (Schwab et al., 1996) suggests a possible role of multiple MSP repeats located on 1p36.2 in human disease.
Regulatory sites and corresponding transcription factors The human MSP gene promoter contains a number of potential regulatory elements suggesting that the expression of the gene can be regulated by a variety of cytokines and steroid hormones (Waltz et al., 1996). For instance, IL-6 increases MSP expression (Zhu and Paddock, 1999), whereas retinoic acid is a repressor of the gene (Muraoka et al., 1999b). Analysis of the 50 -flanking region of the human MSP gene showed the importance of the ÿ135 to ÿ105 promoter region for the liver-specific transcriptional activation of the MSP gene (Waltz et al., 1996). This region contains a positive regulatory site interacting with HNF-4 transcriptional factor (Waltz et al., 1996). Another investigation of the 50 flanking region of the human MSP gene showed that the transcription of the gene can be regulated by both positive and negative regulatory elements. The positive regulatory elements are essential for maximal
transcription of the MSP gene, and the negative regulatory elements appear to be responsible for tissue-specific expression of the gene (Ueda and Yoshimura, 1996). Two proteins interact with this DNA region of the MSP promoter; NF-Y was identified as one of them (Ueda et al., 1998). A subunit of NF-Y directly interacts with HNF-4, and this interaction might be important for the upregulation of MSP gene expression over the basal level driven by HNF-4 alone (Ueda et al., 1998).
Cells and tissues that express the gene MSP is predominantly expressed in embryonal and adult liver, and in hepatocyte cell lines (Han et al., 1991; Yoshimura et al., 1993). The presence of multiple sized MSP mRNA in various cells and tissues reflects alternative splicing of the MSP gene (Yoshimura et al., 1993). Data showing MSP expression are summarized in Table 1.
PROTEIN
Accession numbers Human MSP precursor: GenBank A47136, SwissProt P26927 (Yoshimura et al., 1993) Rat MSP precursor: GenBank JC5061, embl locus RNMACETIM X95096.1 (Ohshiro et al., 1996) Mouse MSP precursor: GenBank A40332 (Degen et al., 1991).
Sequence MSP amino acid sequences of various species are available at GenBank and Protein Data Bank (http:// www.ncbi.nlm.nih.gov).
Description of protein Full-length human MSP is an 80 kDa protein containing 711 amino acids and three N-linked glycosylation sites (Han et al., 1991; Yoshimura et al., 1993). Cleavage of the single-chain molecule between Arg483 and Val484 converts it to two-chain heterodimeric MSP. In MSP the large chain (residues 1±483) is covalently linked to the smaller chain (residues 484±711) via the disulfide bridge between Cys468 and Cys588 located in and chain
Macrophage-Stimulating Protein (MSP) 5 respectively. Several domains were identified in MSP showing MSP structural similarity to plasminogenrelated proteins. Features of the chain include a signal peptide, an N-terminal domain corresponding to the plasminogen preactivating peptide (known as PAP or hairpin loop), four kringle domains, and a segment that terminates in the cleavage site for activation. Kringle domains were first identified in bovine prothrombin as an internal duplication of a triple-disulfide-bounded structure consisting of approximately 80 amino acids (Magnusson et al.,
1975). In addition to prothrombin, kringle domains were found in plasminogen (Sottrup-Jensen et al., 1975), urokinase (Park and Tulinsky, 1986) and HGF (Wun et al., 1982). The chain of MSP is the serine protease-like domain that is devoid of enzymatic activity because of catalytic triad mutations. Instead of His, Asp, and Ser, which are necessary for protein catalytic activity, MSP has Gln522, Gln568, and Tyr661. However, MSP has retained the proteolytic mechanism of activation, a common feature of other
Table 1 Cells and tissues that express MSP Cell line/tissue
Origin
Detection methods
References
HepG2
Hepatocellular carcinoma, human
N
Han et al., 1991; Degen et al., 1991; Yoshimura et al., 1993; Zhu and Paddock, 1999
RT-PCR
Willett et al., 1997
Hep3B
Hepatocellular carcinoma, human
N
Zhu and Paddock, 1999
H60
Small cell lung cancer, human
RT-PCR
Willett et al., 1997
H146
Small cell lung cancer human
RT-PCR
Willett et al., 1997
H187
Small cell lung cancer human
RT-PCR
Willett et al., 1997
Human
N
Degen et al., 1991; Yoshimura et al., 1993; Harrison et al., 1994; Zhu and Paddock, 1999
Mouse
N
Degen et al., 1991; Han et al., 1991
N, W
Bezerra et al., 1998
N
Degen et al., 1991; Han et al., 1991
N, ISH
Bezerra et al., 1994
Cell lines
Normal adult tissues Liver
Rat
N
Ohshiro et al., 1996
Kidney
Human
N
Yoshimura et al., 1993
Testis
Rat
N, ISH
Ohshiro et al., 1996
Mice
N
Degen et al., 1991; Han et al., 1991
Rat
N
Degen et al., 1991; Han et al., 1991
Lung
Rat
N
Degen et al., 1991
Adrenal
Rat
N
Degen et al., 1991
Placenta
Rat maternal tissue
N
Degen et al., 1991
Nervous System
Chicken
INS
Thery et al., 1995
Xenopus
N, ISH
Nakamura et al., 1996; Aberger et al., 1996
Embryonal tissue Liver
ISH, in situ hybridization; N, northern blotting; RT-PCR, reverse transcriptase polymerase chain reaction; W, western blotting.
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Alla Danilkovitch-Miagkova and Edward J. Leonard
members of the plasminogen-related protein family. These proteins are secreted as single-chain biologically inactive precursors, which can be activated by specific serine proteases cleaving an Arg-X bond, in which X is most frequently Val (Francis and Marden, 1990). The protease cleavage site in MSP is Arg483-Val484 (Yoshikawa et al., 1999). It is interesting to note that elimination of that site via substitution of Arg483 by Glu did not prevent MSP processing and did not affect MSP activity. These data suggest an additional proteolytic cleavage site that produces biologically active MSP (Waltz et al., 1997). A schematic representation of human MSP domain structure is shown in Figure 3. MSP molecules of other species have a high similarity to human MSP. Structural features of ecombinant MSP Development of an expression system in CHO cells provided recombinant pro-MSP for structural and
Figure 3
biological investigations (Wahl et al., 1997; Yoshikawa et al., 1999). It was noticed that MSP derived from recombinant pro-MSP had weak biological activity (Wahl et al., 1997; Yoshikawa et al., 1999). SDS-PAGE under nonreducing conditions showed that recombinant MSP is heterogeneous: in addition to the expected heterodimer there are free and chains (Wahl et al., 1997; Yoshikawa et al., 1999). The presence of free and chains indicated that MSP subunits were not disulfide linked. Molecular modeling of the serine proteinase domain of MSP based on homology to human trypsin showed that MSP has an additional Cys residue at position 672 (Wahl et al., 1997). This Cys672 interferes with intersubunit disulfide bridge formation between Cys468 and Cys578, leading to appearance of free biologically inactive MSP chains. Substitution of Cys672 by Ala resulted in fully disulfide-linked MSP and restored MSP biological activity (Wahl et al., 1997).
Structural domains of the human MSP molecule. Arg483-Val484
CHO NH2 SP
PAP
CHO K1
K2
K3
chain (1-483 amino acids)
SP
PAP
K1
CHO X K4
X COOH
chain (484-711 amino acids)
Signal peptide Preactivation peptide of plasminogen (hairpin loop)
Kringle domain (K1-1st, K2-2nd, K3-3d and K4-4th kringles) Serine protease-like domain (corresponding whole b chain) Amino acids representing the catalytic triad (Gln522, Gln568 and Tyr661)
X
CHO Arg483-Val484
Cys468 and Cys588, forms the interchain disulfide bond
N-linked glycosylation sites (located at Asn 72, 296 and 615)
Proteolytic cleavage site
Macrophage-Stimulating Protein (MSP) 7
Discussion of crystal structure At present, MSP crystal structures are unavailable.
Important homologies MSP is most closely related to hepatocyte growth factor/scatter factor (HGF/SF), with which it has 45% sequence similarity (Han et al., 1991; Yoshimura et al., 1993). Comparison of MSP kringle domains revealed 33±66% identity to all other kringle-containing proteins and 30±45% identity of the serine protease-like domain to numerous serine proteases (Han et al., 1991). There is relatively low sequence similarity between MSP of various species: 58% identity between chick and mouse and 76% identity between mouse and human MSP (Ruiz and Thery, 1996). This opens the possibility that there might be a subfamily of MSP-related genes. Isolation of two closely related, but not identical MSP genes from Xenopus called xenopus HLP, Xhl (Aberger et al., 1996) and Livertine (Ruiz and Thery, 1996) supports this hypothesis. The data that disruption of the mouse MSP receptor is lethal (Muraoka et al., 1999a), whereas MSP (the only known ligand) knockout mice are viable (Bezerra et al., 1998), also suggest the existence of other vital MSP-related molecules that use the receptor. However, the presence of two MSP-related molecules in Xenopus might represent polymorphisms, and at present, MSP-related proteins have not been identified in mammals.
Posttranslational modifications The main MSP posttranslational modifications include glycosylation, signal peptide processing and proteolytic cleavage that converts inactive pro-MSP to active MSP. Glycosylation MSP has three potential N-linked glycosylation sites (Han et al., 1991; Yoshimura et al., 1993). Investigation of purified MSP subunits showed that two glycosylation sites are located in the chain at Asn72 and Asn296, and one is in the chain at Asn 615 (Yoshikawa et al., 1999). The N-linked oligosaccharides at the three Asn loci are heterogeneous: 11 different sugars were identified, all being sialylated fucosyl biantennary structures. Four different oligosaccharidies are bound to Asn72, six to Asn296 and eight to Asn615 (Yoshikawa et al., 1999).
Signal peptide processing A series of leucines near the N-terminus of MSP suggested the presence of a signal peptide for this secreted protein (Yoshimura et al., 1993). A cleavage site between Gly18 and Gln19 fits with the frequencies of the last six residues in signal peptides described by Heijne (Andreadis et al., 1987). Further analysis showed that the chain of MSP indeed begins with Gln19 (Yoshikawa et al., 1999). Proteolytic cleavage of single-chain inactive pro-MSP to two-chain active heterodimeric MSP Proteolytic activation of pro-MSP is similar to activation of other members of the kringle protein family, most of which are proteolytic enzyme precursors that become activated after cleavage of an Arg-X bond. Although MSP is not a serine protease, it has retained the same posttranslational activation mechanism. Data concerning the enzymes that cleave pro-MSP are summarized in Table 2. Conversion of pro-MSP to MSP by blood coagulation proteins Investigation of proteolytic cleavage of pro-MSP by various purified blood coagulation enzymes showed that kallikrein and factor XIa completely cleaved pro-MSP to MSP (Wang et al., 1994b). However pro-MSP is not cleaved by these enzymes when blood is shed, probably because it is not a preferred substrate for the coagulation enzymes, which have a very short half-life in vivo. Conversion of pro-MSP to MSP by members of the Kallikrein family The finding that serum kallikrein cleaves pro-MSP to MSP (Wang et al., 1994b) suggested that other proteases having a kallikrein-like structure may also serve as pro-MSP-converting enzymes. The suggestion was supported by experimental data showing that two members of the kallikrein family nerve growth factor and epidermal growth factor-binding protein cleave and activate pro-MSP to heterodimeric MSP (Wang et al., 1994a). Proteolytic Activation of Pro-MSP by Resident Peritoneal Macrophage Membrane Proteases It has been found that in addition to enzymes of the coagulation cascade, incubation of pro-MSP with murine peritoneal macrophages may cause its conversion to MSP (Wang et al., 1996). Macrophage membranes have nonspecific and specific pro-MSP
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Alla Danilkovitch-Miagkova and Edward J. Leonard
Table 2 Proteases that cleave pro-MSP Protease
Type of cleavage
References
Clotting factor XIIa
Conversion to MSP
Wang et al., 1994a,1994b
Serum kallikrein
Conversion to MSP
Wang et al., 1994a,1994b
Clotting factor XIa
Conversion to MSP
Wang et al., 1994a,1994b
Plasmin
Degradation
Wang et al., 1994a,1994b
Unidentified pro-MSP convertase(s)
Conversion to MSP
Wang et al., 1996
Unidentified proteases
Degradation
1. From the coagulation cascade
2. Macrophage-membrane associated
3. Wound fluid proteases Unidentified pro-MSP convertase(s)
Conversion to MSP
Nanney et al., 1998
Conversion to MSP
Wang et al., 1994a
Conversion to MSP
Wang et al., 1994a
Degradation
Wang et al., 1994a
4. Kallikrein family proteases Nerve growth factor Epidermal growth factor Binding protein 5. Other trypsin-like proteases Trypsin
proteolytic activities, which were not present in the macrophage culture medium (Wang et al., 1996). Incubation of pro-MSP with murine macrophages caused its proteolytic cleavage to inactive fragments. Addition of soybean trypsin inhibitor to macrophages cultured with pro-MSP inhibited nonspecific cleavage and revealed a macrophage activity that cleaved proMSP to MSP. These data suggest that macrophage membrane-associated proteases and serum protease inhibitors are components of an MSP regulatory system. At sites of inflammation or injury pro-MSP may diffuse from the circulation into extravascular sites where a macrophage convertase can cleave proMSP to MSP. At the same site MSP then can act on its targets, including macrophages themselves.
exudate pro-MSP to MSP (Nanney et al., 1998). A partially purified pro-MSP convertase from wound fluid is not inhibited by 2-macroglobulin or C1-inhibitor, which are inhibitors of coagulation system proteases (Skeel and Leonard, unpublished data). It suggests that this convertase is distinct from serum kallikrein, factor XIIa or factor XIa. The above data are the first in vivo evidence that pro-MSP is cleaved to MSP following tissue injury. MSP can act on macrophages, keratinocytes and capillaries, the components of the wound healing response. The presence of both active MSP and its receptor in the wound areas suggest a possible important role of MSP in wound healing processes.
Conversion of pro-MSP to MSP by a wound fluid enzyme
CELLULAR SOURCES AND TISSUE EXPRESSION
The investigation of RON (the MSP receptor) expression and MSP status in wounds showed that active MSP was present in wound exudates in the concentration range for optimal activity (Nanney et al., 1998). At the same time RON expression was upregulated in burn wound epidermis and accessory structures. The presence of MSP was attributed to trypsin-like serine proteases capable of cleaving
Cellular sources that produce MSP is constitutively secreted into the circulation by liver cells (Bezerra et al., 1993b). Although the MSP message was detected in several other tissues (Yoshimura et al., 1993; Ohshiro et al., 1996), the liver is the main source of MSP in vivo. MSP
Macrophage-Stimulating Protein (MSP) 9 expression in cell lines, embryonal and adult tissues is summarized in Table 1.
Eliciting and inhibitory stimuli, including exogenous and endogenous modulators Pro-MSP production by cells can be regulated by various stimuli at different levels (DNA, RNA or protein), whereas the level of active MSP is regulated at extravascular sites by serine-like proteases. These proteases can be divided into two groups: specific pro-MSP convertases that cleave pro-MSP to MSP, and non-specific proteases that can cleave both proMSP and MSP to inactive fragments. Regulation of MSP Gene Transcription The promoter region of the human MSP gene contains a number of potential regulatory sequences, suggesting that MSP gene expression can be regulated by a number of cytokines and hormones (Waltz et al., 1997). Treatment of HepG2 cells with retinoic acid, thyroid hormone, dexamethasone, progesterone or estrogen caused a decrease in the expression of MSP mRNA (Muraoka et al., 1999b). The proposed mechanism of MSP gene suppression is due to
inhibition of the interaction between HNF-4 and CBP (cyclic adenosine 30 ,50 -monophosphate response element (CREB)-binding protein) (Muraoka et al., 1999b). Nuclear receptors for retinoic, thyroid hormone, glucocorticoid, estrogen and progesterone in the presence of their unique ligands can directly interact with CBP (Janknecht and Hunter, 1996; Kamei et al., 1996; Teng et al., 1996), which interrupts HNF-4 binding to CBP and causes transcriptional repression of the MSP gene. In contrast to the above agents, IL-6 enhances MSP gene transcription (Zhu and Paddock, 1999) possibly via activation of putative IL-6 response elements identified in the MSP promoter (Waltz et al., 1997). Regulation of Tissue Concentrations of MSP Inactive pro-MSP is constitutively secreted by hepatocytes into the circulation (Bezerra et al., 1993b; Leonard and Danilkovitch, 2000), and it also is degraded by the liver (Leonard and Skeel, 1996). Conversion of pro-MSP to MSP is thought to occur at extravascular sites where MSP target cells are located. Pro-MSP convertases and their inhibitors (see Tables 2 and 3) are components of a pro-MSP/ MSP regulatory system, in which the concentration of components may play a critical role. At present, three sources of pro-MSP-converting enzymes are known:
Table 3 Inhibitors of pro-MSP proteases Inhibitor
Proteases inhibited
References
Leupeptin
Serum pro-MSP convertases
Wang et al., 1994b
Wound fluid pro-MSP convertase
Nanney et al., 1998
Serum pro-MSP convertases
Wang et al., 1994b
Wound fluid pro-MSP convertase
Nanney et al., 1998
Serum pro-MSP convertases
Wang et al., 1994b
Macrophage membrane-associated pro-MSP degrading proteases
Wang et al., 1996
AEBSF
Macrophage membrane-associated pro-MSP degrading proteases
Wang et al., 1996
C1-inhibitor
Serum kallikrein
Wang et al., 1994b
Nerve growth factor
Wang et al., 1994a
Macrophage membrane-associated pro-MSP degrading proteases
Skeel and Leonard, 2001
Macrophage membrane-associated pro-MSP convertases
Skeel and Leonard, 2001
Macrophage membrane-associated pro-MSP degrading proteases
Skeel and Leonard, 2001
Aprotinin Soybean trypsin inhibitor
1-Antichymotrypsin, high concentrations (7 mM as in human plasma)
low concentrations (0.4 mM as expected in extracellular fluid)
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Alla Danilkovitch-Miagkova and Edward J. Leonard
proteases of the coagulation cascade (Wang et al., 1994b), enzymes associated with the macrophage membrane (Wang et al., 1996) and a protease from wound fluid (Nanney et al., 1998). These proteases have the potential to regulate tissue levels of MSP. Murine resident peritoneal macrophages have two cell surface proteolytic activities that cleave pro-MSP to MSP or to inactive fragments (Wang et al., 1996). The degrading protease activity is inhibited by low concentrations of blood plasma, which allows the specific convertase to cleave pro-MSP to MSP (Wang et al., 1996). The blood plasma inhibitor was identified as 1-antichymotrypsin (Skeel and Leonard, 2001). At the expected concentration range (approximately 0.4 mM) in extracellular fluid under non-inflammatory conditions, 1-antichymotrypsin preferentially inhibits the pro-MSP-degrading enzyme, which allows the macrophage convertase to cleave pro-MSP to MSP. In contrast, in inflammatory exudates, 1-antichymotrypsin approaches its 7 mM concentration in blood plasma, and at these levels it inhibits both macrophage enzymes (Skeel and Leonard, 2001). Thus, the MSP detected in inflammatory exudates is probably generated by fluid phase enzymes that can cleave pro-MSP to MSP.
RECEPTOR UTILIZATION MSP is a ligand for RON. The first investigation of MSP domains that are involved in RON binding and activation showed that the first two N-terminal kringle domains of the MSP chain stimulate RON tyrosine phosphorylation in a dose-dependent manner (Gaudino et al., 1994). The minimal portion of the MSP molecule involved in RON binding is located within these first two Nterminal kringles. Binding assays of radioiodinated MSP or its chains to RON-expressing cells showed that MSP or its chain specifically bound to RON. The calculated Kd is 0.6±0.8 nM for MSP heterodimer and 1.4 nM for its chain (Wang et al., 1997). Despite high-affinity binding of the chain to RON, functional studies indicated that only the MSP chain heterodimer induces RON tyrosine phosphorylation and biological responses, such as macrophage shape changes and epithelial cell migration and proliferation (Waltz et al., 1997; Wang et al., 1997). To initiate signal transduction from the cell surface to the cell interior, ligand binding has to cause receptor dimerization, followed by receptor autophosphorylation and activation (Ullrich and Schlessinger, 1990). Ligand-induced receptor dimerization is believed to be the key mechanism for initiation of signal transduction cascades. Although the MSP
chain binds to RON, it does not induce cellular responses. Only the MSP chain heterodimer induces biological activity and presumably receptor dimerization. It has been recently suggested that MSP induces RON dimerization by a mechanism comparable to growth hormone-induced dimerization of its receptor (Wells, 1996), in which a bivalent ligand engages binding sites of two receptors (Miller and Leonard, 1998). Detection of chain binding to RON (Danilkovitch et al., 1999) suggests that MSP has two independent binding sites with high and low affinities located in and chain respectively. This fact supports the hypothesis that two MSP sites together mediate RON dimerization and subsequent activation. Mapping of the binding site on the MSP chain showed the importance of the S1 substrate specificity pocket within the serine protease domain (Danilkovitch et al., 1999). Location of the lowaffinity binding site within the MSP chain remains unknown. Investigations of mechanisms of MSP/RON interactions revealed that both high- and low-affinity MSP binding sites are located within the SEMA domain of RON (corresponding the extracellular part of RON, amino acid residues 57±508 in human RON) (Angeloni et al., unpublished data).
IN VITRO ACTIVITIES
In vitro findings MSP has a number of biological activities that were detected predominantly by in vitro assays (Figure 2; see also Table 1 in the chapter on RON receptor). MSP activities can be grouped into several broad categories including adhesion and motility, mediator production, growth, differentiation and survival.
Regulatory molecules: Inhibitors and enhancers The level of biologically active MSP can be regulated by a variety of trypsin-like proteases (Tables 2 and 3, and the section Eliciting and inhibitory stimuli, including exogenous and endogenous modulators).
Bioassays used MSP has a number of biological activities that can be detected by a variety of in vitro tests (see Table 1 in
Macrophage-Stimulating Protein (MSP) 11 the chapter on RON receptor). The presence of biologically active MSP in samples can be detected by the macrophage shape change assay (Leonard and Skeel, 1976; Leonard and Danilkovitch, 2000). In human wound exudates, this assay can be considered as MSP-specific, because MSP-like effects on macrophage shape are eliminated by passage of exudate down an anti-MSP antibody column.
IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS
Normal physiological roles Investigations of MSP biological activities in vivo showed that MSP plays an important role in embryonal development, tissue regeneration and inflammation. Increased MSP mRNA expression was detected in vivo in rats during liver regeneration induced by 70% partial hepatectomy or carbon tetrachloride treatment of rats and during inflammation induced by administration of thioglycollate medium or turpentine (Bezerra et al., 1994). These data support a potential role of MSP in the early phase of liver regeneration and as an inflammatory mediator (Bezerra et al., 1994). A role of MSP in inflammation and tissue regeneration was shown in RON /ÿ mice. The concentration of nitrate in serum of mice that received intravenous endotoxin was higher in RON /ÿ than in serum of endotoxin-treated RON / mice (Correll et al., 1997; Muraoka et al., 1999a) and was associated with a higher lethality of endotoxin in the RON-deficient animals. These in vivo findings reflect the fact that endotoxininduced upregulation of NO synthase in murine macrophages is inhibited by MSP, and suggests that this downregulation is defective in animals with diminished MSP receptor expression. A role of MSP in embryonal development was shown in Xenopus models. Ectopic expression of MSP in Xenopus embryos affected neural morphogenesis (Aberger et al., 1996; Ruiz and Thery, 1996; Nakamura et al., 1996). In chicken embryos MSP expression was detected in developing somites, in the notochord and in the prospective floor plate region that suggest a role of MSP in mesodermal development and in development of the neural tube (Thery et al., 1995). Data about MSP expression in embryonal tissues are summarized in Table 1.
Species differences The main difference between species is the role of MSP in embryonal development. MSP is important in development of embryonic neural tissues in Xenopus (Aberger et al., 1996; Ruiz and Thery, 1996; Nakamura et al., 1996) whereas MSP knockout mice (described below) developed normally (Bezerra et al., 1998).
Knockout mouse phenotypes Disruption of the MSP gene did not prevent normal embryogenesis (Bezerra et al., 1998). MSPÿ/ÿ mice are fertile, and grow to adulthood without visible phenotypic abnormalities, except for the development of lipid-containing cytoplasmic vacuoles in hepatocytes and delayed macrophage activation (Bezerra et al., 1998). MSP-deficient mice can be used as experimental in vivo models for investigation of the role of MSP in hepatic and systemic responses to inflammation and infectious agents.
Transgenic expression At present, there are no data available.
Pharmacological effects The effects of administration of exogenous MSP remain to be investigated. Iodinated pro-MSP intravenously injected into normal mice underwent rapid hepatic proteolysis (Leonard and Skeel, 1996). The liver is the major focus of radioactivity 10± 20 minutes after the injection of radioactive proMSP. Calculated pro-MSP half-life is approximately 100 minutes (Leonard and Skeel, 1996).
Interactions with cytokine network Preliminary data indicate that MSP induces IL-6 production by a number of cell types (unpublished observation).
Endogenous inhibitors and enhancers At present, there are no data showing that effects of MSP inhibitors and enhancers in vitro also take place in vivo.
12
Alla Danilkovitch-Miagkova and Edward J. Leonard
PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY
Normal levels and effects The mean concentration of pro-MSP in human plasma is 4 nM (Nanney et al., 1998), which is in the range for optimal biological activity. To act on target cells pro-MSP must diffuse into tissues and be cleaved by pro-MSP convertase to active MSP.
Role in experiments of nature and disease states A role of MSP in human diseases remains to be investigated. At present, only limited data demonstrating MSP status in human pathology are available. Increased MSP mRNA was detected in a sample of subacute fulminant hepatic necrosis (Zhu and Paddock, 1999), whereas the level of MSP in patients with fulminant hepatic failure was reduced (Harrison et al., 1994). Elevated MSP was detected in sputum of patients with bronchiectasis (Takano et al., 2000). MSP is expressed in non-small and small cell lung tumors (Willett et al., 1997, 1998) and hepatocellular carcinomas (Zhu and Paddock, 1999), suggesting possible MSP involvement in the pathogenesis of these tumors.
IN THERAPY At present, MSP is not used for therapy.
ACKNOWLEDGEMENTS The authors thank Dr J. Oppenheim for critical reading and discussion of this manuscript.
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LICENSED PRODUCTS Human recombinant MSP (RON ligand) (Cat# 352-MS-010 or 050, R&D Systems, USA)
The source of this recombinant protein is the NSO mouse myeloma cell line. The protein can be used for ELISA, for binding assays, and for activation of mouse and human RON.
Anti-human recombinant MSP goat polyclonal antibodies (Cat#AF-352-PB, R&D Systems, USA)
These antibodies were developed against human recombinant MSP produced by the NSO mouse myeloma cell line. Antibodies can be used for ELISA.
Anti-human MSP goat polyclonal antibodies (HGFL (N-19), Cat# sc-6088, Santa Cruz Biotechnol., Santa Cruz, CA, USA)
These antibodies were developed against a peptide mapping at the amino terminus of the MSP precursor (pro-MSP) of human origin. Antibodies can be used for Western blotting and ELISA; non-crossreactive with HGF.
Anti-mouse MSP goat polyclonal antibodies (HGFL (T-19), Cat# sc-6090, Santa Cruz Biotechnol., Santa Cruz, CA, USA)
These antibodies were developed against a peptide mapping at the N-terminus of the MSP precursor (pro-MSP) of mouse origin. They react with MSP of mouse and rat origin. Antibodies can be used for western blotting and ELISA; non-crossreactive with HGF.
