Macrophage-Stimulating Protein

14 Pages·0.227 MB·English

by  Danilkovitch-Miagkova A., Leonard E.J.

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: [phone], fax: [phone], e-mail: [email protected]
DOI: 10.1006/rwcy.2001.0812.
Chapter posted 5 November 2001
SUMMARY inflammation and tissue regeneration. MSP was
detected in embryonal tissues; its ectopic over-
Macrophage-stimulating protein (MSP, also known expression in Xenopus caused defects in development
as hepatocyte growth factor-like protein) was of the neural system. Abnormal MSP expression was
discovered as a serum factor inducing macrophage detected in hepatocellular carcinoma tissues and lung
shape changes and chemotaxis. On the basis of cancer cell lines.
structural homology MSP was placed in the
plasminogen-related kringle domain protein family,
most members of which are proteolytic enzymes. In
BACKGROUND
contrast, MSP has lost its enzymatic activity and has
become a growth and motility factor. MSP is con-
Discovery
stitutively secreted by liver cells and circulates in the
bloodstream as a biologically inactive single-chain
pro-MSP. At extravascular sites pro-MSP can be MSP was discovered as a normal serum factor that
convertedbytrypsin-likeserineproteasestoactive(cid:11)(cid:12) enhanced chemotaxis of mouse peritoneal resident
heterodimeric MSP. The MSP receptor, called macrophages in response to endotoxin-activated
RON in humans and STK in mice, is a transmem- mouse serum (Leonard and Skeel, 1976, 1980).
brane receptor tyrosine kinase. MSP/RON inter- MSP was purified to homogeneity from human
action activates RON, and initiates downstream blood plasma (Skeel et al., 1991). A computer search
intracellular signaling pathways mediating MSP of six partial sequences of MSP digests showed that
biological activities. MSP was not in the protein database. Two MSP
Recent data show that in addition to macrophages fragments had 80% identity to sequences of the
MSP affects a number of other targets such as protein family that includes human prothrombin,
osteoclasts and hepatocytes, epithelial, endothelial, plasminogen, and hepatocyte growth factor (HGF)
and hematopoietic cells. The list of MSP biological (Skeel et al., 1991). Screening of a human genomic
activities includes adhesion and motility, cyto- DNA library with a bovine cDNA probe coding
kine production, angiogenesis, growth, differentia- for the kringle domains in prothrombin resulted in
tion, and cell survival. Active MSP was detected isolation of a new gene. The protein encoded by
in injured tissues and in regions of inflamma- this gene contained four kringle and one serine
tion, suggesting a role of MSP in regulation of protease-like domain. On the basis of the structural
CytokineReference Copyright#2001AcademicPress
2 Alla Danilkovitch-Miagkova and Edward J. Leonard
similarity with HGF, the new protein was called Structure
HGF-like protein (Han et al., 1991). Comparison of
the amino acid sequence of human HGFL protein
MSP is a 80kDa serum factor that belongs to a
with the sequence of peptides derived from human
plasminogen-related kringle domain protein family
MSP suggested that HGFL and MSP were the
(Han et al., 1991; Yoshimura et al., 1993). MSP, like
same protein (Bezerra et al., 1993a). Two years later
othermembersofthefamily,isaheterodimericprotein
MSP cDNA was cloned from a human hepatoma
consisting of (cid:11) and (cid:12) chains. The (cid:11) chain has an N-
cell line (Yoshimura et al., 1993). A computer
terminal domain corresponding to the plasminogen
search showed that the MSP cDNA was encoded by
preactivation peptide (PAP or hairpin loop) and four
theHGFLgene(Yoshimuraetal.,1993).Comparison
kringledomains,andasegmentthatterminatesinthe
of MSP and HGFL amino acid sequences and
cleavagesiteforactivation.The(cid:12)chainhasaserine-like
functional activities led to the conclusion that HGFL
protease domain (Han et al., 1991; Yoshimura et al.,
and MSP are indeed identical proteins (Shimamoto
1993). Whereas most of the proteins of the plasmino-
et al., 1993).
gen-related family are serine proteases, MSP has lost
its enzymatic activity because of catalytic triad
mutations in the serine protease domain. Although
Alternative names
MSP was transformed from a serine protease to a
growth and motility factor, it retained the proteolytic
Hepatocyte growth factor-like protein (HGFL) (Han mechanismofactivation,whichisacommonfeatureof
et al., 1991) or HLP (Shimamoto et al., 1993); RON proteins of the family (Han et al., 1991; Yoshimura
ligand; MST1 (Yoshimura et al., 1993). etal.,1993;LeonardandDanilkovitch,2000).MSPis
Figure1 Theprocessingofbiologicallyinactivepro-MSPtoactivemature
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 53kDa (cid:11) and 25kDa
(cid:12) chains. K1–K4 repesent four kringle domains located in the (cid:11) chain. S-S
shows the interchain disulfide bond between Cys468 and Cys577 located in
the (cid:11) and (cid:12) chain respectively.
Liver
m
Blo o d stre a α-chain (53 kDa)
K1 K3
K2 K4
K 3 K 4 S
Biologically active
K 1 K 2 heterodimeric MSP S
β-chain (25 kDa)
Extravascular
site Trypsin-like
serine
proteases
K1 K3
K2 K4
S
Biologically inactive
S
single chain pro-MSP
Macrophage-Stimulating Protein (MSP) 3
constitutively secreted by hepatocytes and circulates Rat MSP: X95096 (Ohshiro et al., 1996)
in the bloodstream as a biologically inactive single Frog MSPs: Y08734 (MSPA, xenopus HPL, Xhl)
chain pro-MSP (Bezerra et al., 1993b; Leonard and (Aberger et al., 1996) and U57455 (MSPB, Livertine)
Danilkovitch, 2000). Pro-MSP can be cleaved and (Ruiz and Thery, 1996).
activated at extravascular sites by trypsin-like pro- Human MSP gene promoter: U37055 (Waltz et al.,
teases. Mature biologically active MSP consists 1996) and U43376 (Ueda and Yoshimura, 1996).
of disulfide-linked 53kDa (cid:11) and 25kDa (cid:12) chains. A Human MSP pseudogenes: AF083410, AF083411,
schematic representation of the structure and pro- AF083412, AF083413, AF083414, AF083415,
cessingofpro-MSPtoMSPisshowninFigure1. AF083416, AF083417 (van der Drift et al., 1999).
Main activities and
Sequence
pathophysiological role
MSP gene sequences of various species are available
MSP is a multifunctional factor mediating its biol-
at GenBank (http://www.ncbi.nlm.nih.gov).
ogical activities via RON receptor tyrosine kinase.
MainMSPtargetsandbiologicalactivitiesareshown
inFigure2.Atpresent,thepathophysiologicalroleof
Chromosome location
MSP is speculative.
The human MSP gene is located on chromosome 3
GENE
(3p21)(Hanetal.,1991;Yoshimuraetal.,1993).The
humanMSPpseudogenesarelocatedonchromosome
Accession numbers
1 (1p36) (van der Drift et al., 1999).
The mouse MSP gene is located on chromosome 9
GenBank: atalocus(Hgfl)thatshowshomologytobandp21on
Human MSP: L11924 (Yoshimura et al., 1993) human chromosome 3 (Degen et al., 1992).
Figure 2 MSP targets and main biological activities.
Epithelial Hematopoletic
Macrophages cells cells
Vascular Liver
Osteoclasts endothelial progenitor
Shape Adhesion
changes cells Apoptosis cells
Motility
Chemotaxis Cytokine
production
Apoptosis
Macro-
pinocytosis Survival Shape
changes
Bone Angio- Scattering
Ingestion resorption Growth genesis
of EigMC3bi inhibition Growth Morpho-
inhibition
Growth genesis
Inhibition induction Growth
Growth
of iNOS induction
induction
IL-6
production Inhibition or
promotion of
differentiation
Up-regulation
of arginase
4 Alla Danilkovitch-Miagkova and Edward J. Leonard
Relevant linkages transcription of the MSP gene, and the negative
regulatory elements appear to be responsible for
tissue-specific expression of the gene (Ueda and
ThechromosomallocationofthehumanMSPgeneis
Yoshimura, 1996). Two proteins interact with this
3p21 (Han et al., 1991; Yoshimura et al., 1993). This
DNA region of the MSP promoter; NF-Y was
region has been proposed to contain one or more
identifiedasoneofthem(Uedaetal.,1998).Asubunit
tumor suppressor genes since this locus is deleted in
of NF-Y directly interacts with HNF-4, and this
smallcelllungandkidneycancersamples(Zbaretal.,
interaction might be important for the upregulation
1987;Brauchetal.,1987).Although,atpresent,there
ofMSPgeneexpressionoverthebasalleveldrivenby
are no data showing a linkage between MSP gene
HNF-4 alone (Ueda et al., 1998).
deletion and human cancers, it is still possible that
MSP may function as a tumor suppressor.
When the MSP gene was cloned, the human
Cells and tissues that express
MSP genomic probe showed the presence of the
the gene
MSPgenesequencesonchromosome1inadditionto
chromosome 3 (Yoshimura et al., 1993). Recently,
multipleMSPpseudogenesinalocalrepeatclusteron MSP is predominantly expressed in embryonal and
p36.2werecloned(vanderDriftetal.,1999).Itseems adult liver, and in hepatocyte cell lines (Han et al.,
that all cloned genes have nucleotide deletions or 1991; Yoshimura et al., 1993). The presence of
substitutions that lead to coding nonfunctional multiple sized MSP mRNA in various cells and
proteins (van der Drift et al., 1999). It is possible tissues reflects alternative splicing of the MSP
that during evolution a copy of the MSP ancestral gene (Yoshimura et al., 1993). Data showing MSP
gene located on chromosome 3 was swept into the expression are summarized in Table 1.
cluster of tRNA and snRNA genes on 1p36.2 (van
derDriftetal.,1999).Therecombinationprocessthat
causedtherepetitivestructureofthisclusterhasledto PROTEIN
MSP amplification and a number of polymorphisms.
ThebiologicalsignificanceoftheseMSPpseudogenes Accession numbers
is unclear, but the relevance of genomic instability
in 1p and human malignancies (Schwab et al., 1996)
HumanMSPprecursor:GenBankA47136,SwissProt
suggests a possible role of multiple MSP repeats
P26927 (Yoshimura et al., 1993)
located on 1p36.2 in human disease.
Rat MSP precursor: GenBank JC5061, embl locus
RNMACETIM X95096.1 (Ohshiro et al., 1996)
Regulatory sites and corresponding Mouse MSP precursor: GenBank A40332 (Degen
et al., 1991).
transcription factors
Sequence
ThehumanMSPgenepromotercontainsanumberof
potential regulatory elements suggesting that the
expressionofthegenecanberegulatedbyavarietyof MSP amino acid sequences of various species are
cytokines and steroid hormones (Waltz et al., 1996). availableatGenBankandProteinDataBank(http://
For instance, IL-6 increases MSP expression (Zhu www.ncbi.nlm.nih.gov).
and Paddock, 1999), whereas retinoic acid is a
repressor of the gene (Muraoka et al., 1999b).
Description of protein
Analysis of the 50-flanking region of the human
MSP gene showed the importance of the (cid:255)135 to
(cid:255)105 promoter region for the liver-specific transcrip- Full-length human MSP is an 80kDa protein
tionalactivationoftheMSPgene(Waltzetal.,1996). containing 711 amino acids and three N-linked
This region contains a positive regulatory site glycosylation sites (Han et al., 1991; Yoshimura
interacting with HNF-4 transcriptional factor et al., 1993). Cleavage of the single-chain molecule
(Waltz et al., 1996). Another investigation of the 50- between Arg483 and Val484 converts it to two-chain
flanking region of the human MSP gene showed that heterodimeric MSP. In MSP the large (cid:11) chain
thetranscriptionofthegenecanberegulatedbyboth (residues 1–483) is covalently linked to the smaller (cid:12)
positive and negative regulatory elements. The chain (residues 484–711) via the disulfide bridge
positiveregulatoryelementsareessentialformaximal betweenCys468andCys588locatedin(cid:11)and(cid:12) chain
Macrophage-Stimulating Protein (MSP) 5
respectively. Several domains were identified in MSP 1975). In addition to prothrombin, kringle domains
showing MSP structural similarity to plasminogen- were found in plasminogen (Sottrup-Jensen et al.,
related proteins. Features of the (cid:11) chain include a 1975), urokinase (Park and Tulinsky, 1986) and
signal peptide, an N-terminal domain corresponding HGF (Wun et al., 1982).
to the plasminogen preactivating peptide (known as The (cid:12) chain of MSP is the serine protease-like
PAP or hairpin loop), four kringle domains, and a domain that is devoid of enzymatic activity because
segment that terminates in the cleavage site for of catalytic triad mutations. Instead of His, Asp,
activation. Kringle domains were first identified in and Ser, which are necessary for protein catalytic
bovine prothrombin as an internal duplication of a activity, MSP has Gln522, Gln568, and Tyr661.
triple-disulfide-bounded structure consisting of However, MSP has retained the proteolytic mechan-
approximately 80 amino acids (Magnusson et al., ism of activation, a common feature of other
Table 1 Cells and tissues that express MSP
Cell line/tissue Origin Detection References
methods
Cell lines
HepG2 Hepatocellular carcinoma, N Han et al., 1991; Degen et al.,
human 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
Normal adult tissues
Liver 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
Rat N Degen et al., 1991; Han et al., 1991
N, ISH Bezerra et al., 1994
N Ohshiro et al., 1996
Kidney Human N Yoshimura et al., 1993
Testis Rat N, ISH Ohshiro et al., 1996
Embryonal tissue
Liver 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
ISH,insituhybridization;N,northernblotting;RT-PCR,reversetranscriptasepolymerasechainreaction;W,westernblotting.
6 Alla Danilkovitch-Miagkova and Edward J. Leonard
members of the plasminogen-related protein family. biological investigations (Wahl et al., 1997;
These proteins are secreted as single-chain biologic- Yoshikawa et al., 1999). It was noticed that MSP
ally inactive precursors, which can be activated by derived from recombinant pro-MSP had weak
specific serine proteases cleaving an Arg-X bond, in biological activity (Wahl et al., 1997; Yoshikawa
which X is most frequently Val (Francis and et al., 1999). SDS-PAGE under nonreducing condi-
Marden, 1990). The protease cleavage site in MSP tions showed that recombinant MSP is heteroge-
is Arg483-Val484 (Yoshikawa et al., 1999). It is neous: in addition to the expected (cid:11)(cid:12) heterodimer
interesting to note that elimination of that site via there are free (cid:11) and (cid:12) chains (Wahl et al., 1997;
substitution of Arg483 by Glu did not prevent MSP Yoshikawa et al., 1999). The presence of free (cid:11) and
processing and did not affect MSP activity. These (cid:12) chains indicated that MSP subunits were not
data suggest an additional proteolytic cleavage site disulfide linked. Molecular modeling of the serine
that produces biologically active MSP (Waltz et al., proteinase domain of MSP based on homology
1997). A schematic representation of human MSP to human trypsin showed that MSP has an
domain structure is shown in Figure 3. MSP additional Cys residue at position 672 (Wahl et al.,
molecules of other species have a high similarity to 1997). This Cys672 interferes with intersubunit
human MSP. disulfide bridge formation between Cys468 and
Cys578, leading to appearance of free biologic-
ally inactive MSP chains. Substitution of Cys672
Structural features of ecombinant MSP
by Ala resulted in fully disulfide-linked MSP
Development of an expression system in CHO cells and restored MSP biological activity (Wahl et al.,
provided recombinant pro-MSP for structural and 1997).
Figure 3 Structural domains of the human MSP molecule.
Arg483-Val484
CHO CHO CHO
X X
NH2 SP PAP K1 K2 K3 K4 COOH
chain chain
(1-483 amino acids) (484-711 amino acids)
SP Signal peptide
PAP Preactivation peptide of plasminogen (hairpin loop)
K1 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 Cys468 and Cys588, forms the interchain disulfide bond
CHO N-linked glycosylation sites (located at Asn 72, 296 and 615)
Arg483-Val484
Proteolytic cleavage site
Macrophage-Stimulating Protein (MSP) 7
Discussion of crystal structure Signal peptide processing
A series of leucines near the N-terminus of MSP
At present, MSP crystal structures are unavailable. 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
Important homologies
frequencies of the last six residues in signal peptides
described by Heijne (Andreadis et al., 1987). Further
MSP is most closely related to hepatocyte growth analysis showed that the (cid:11) chain of MSP indeed
factor/scatter factor (HGF/SF), with which it has begins with Gln19 (Yoshikawa et al., 1999).
45%sequencesimilarity(Hanetal.,1991;Yoshimura
et al., 1993). Comparison of MSP kringle domains
Proteolytic cleavage of single-chain inactive
revealed 33–66% identity to all other kringle-contain-
pro-MSP to two-chain active heterodimeric MSP
ing proteins and 30–45% identity of the serine
protease-like domain to numerous serine proteases Proteolytic activation of pro-MSP is similar to
(Han et al., 1991). There is relatively low sequence activation of other members of the kringle protein
similarity between MSP of various species: 58% family, most of which are proteolytic enzyme
identity between chick and mouse and 76% identity precursors that become activated after cleavage of
between mouse and human MSP (Ruiz and Thery, an Arg-X bond. Although MSP is not a serine
1996).Thisopensthepossibilitythattheremightbea protease, it has retained the same posttranslational
subfamily of MSP-related genes. Isolation of two activation mechanism. Data concerning the enzymes
closely related, but not identical MSP genes from that cleave pro-MSP are summarized in Table 2.
Xenopus called xenopus HLP, Xhl (Aberger et al.,
1996) and Livertine (Ruiz and Thery, 1996) supports
Conversion of pro-MSP to MSP by blood
this hypothesis. The data that disruption of the
coagulation proteins
mouseMSPreceptorislethal(Muraokaetal.,1999a),
whereas MSP (the only known ligand) knockout Investigation of proteolytic cleavage of pro-MSP
mice are viable (Bezerra et al., 1998), also suggest by various purified blood coagulation enzymes
the existence of other vital MSP-related molecules showed that kallikrein and factor XIa completely
that use the receptor. However, the presence of two cleaved pro-MSP to MSP (Wang et al., 1994b).
MSP-related molecules in Xenopus might represent However pro-MSP is not cleaved by these enzymes
polymorphisms,andatpresent,MSP-relatedproteins when blood is shed, probably because it is not a
have not been identified in mammals. preferred substrate for the coagulation enzymes,
which have a very short half-life in vivo.
Posttranslational modifications
Conversion of pro-MSP to MSP by members of
the Kallikrein family
The main MSP posttranslational modifications
The findingthat serumkallikreincleavespro-MSP to
include glycosylation, signal peptide processing and
MSP (Wang et al., 1994b) suggested that other
proteolytic cleavage that converts inactive pro-MSP
proteases having a kallikrein-like structure may also
to active MSP.
serve as pro-MSP-converting enzymes. The sugges-
tion was supported by experimental data showing
Glycosylation
that two members of the kallikrein family nerve
MSP has three potential N-linked glycosylation sites growth factor (cid:13) and epidermal growth factor-binding
(Han et al., 1991; Yoshimura et al., 1993). protein cleave and activate pro-MSP to (cid:11)(cid:12) hetero-
Investigation of purified MSP subunits showed that dimeric MSP (Wang et al., 1994a).
two glycosylation sites are located in the (cid:11) chain at
Asn72 and Asn296, and one is in the (cid:12) chain at
Proteolytic Activation of Pro-MSP by Resident
Asn 615 (Yoshikawa et al., 1999). The N-linked
Peritoneal Macrophage Membrane Proteases
oligosaccharides at the three Asn loci are hetero-
geneous: 11 different sugars were identified, all being It has been found that in addition to enzymes of the
sialylated fucosyl biantennary structures. Four coagulation cascade, incubation of pro-MSP with
different oligosaccharidies are bound to Asn72, six murine peritoneal macrophages may cause its con-
to Asn296 and eight to Asn615 (Yoshikawa et al., version to MSP (Wang et al., 1996). Macrophage
1999). membranes have nonspecific and specific pro-MSP
8 Alla Danilkovitch-Miagkova and Edward J. Leonard
Table 2 Proteases that cleave pro-MSP
Protease Type of cleavage References
1. From the coagulation cascade
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
2. Macrophage-membrane associated
Unidentified pro-MSP convertase(s) Conversion to MSP Wang et al., 1996
Unidentified proteases Degradation
3. Wound fluid proteases
Unidentified pro-MSP convertase(s) Conversion to MSP Nanney et al., 1998
4. Kallikrein family proteases
Nerve growth factor (cid:13) Conversion to MSP Wang et al., 1994a
Epidermal growth factor
Binding protein Conversion to MSP Wang et al., 1994a
5. Other trypsin-like proteases
Trypsin Degradation Wang et al., 1994a
proteolytic activities, which were not present in the exudate pro-MSP to MSP (Nanney et al., 1998).
macrophage culture medium (Wang et al., 1996). A partially purified pro-MSP convertase from
Incubation of pro-MSP with murine macrophages wound fluid is not inhibited by (cid:11) -macroglobulin or
2
caused its proteolytic cleavage to inactive fragments. C1-inhibitor, which are inhibitors of coagulation
Additionofsoybeantrypsininhibitortomacrophages system proteases (Skeel and Leonard, unpublished
culturedwithpro-MSPinhibitednonspecificcleavage data). It suggests that this convertase is distinct
and revealed a macrophage activity that cleaved pro- fromserumkallikrein,factorXIIaorfactorXIa.The
MSP to MSP. These data suggest that macrophage abovedataarethefirstinvivoevidencethatpro-MSP
membrane-associated proteases and serum protease is cleaved to MSP following tissue injury. MSP can
inhibitors are components of an MSP regulatory act on macrophages, keratinocytes and capillaries,
system. At sites of inflammation or injury pro-MSP the components of the wound healing response. The
may diffuse from the circulation into extravascular presence of both active MSP and its receptor in the
sites where a macrophage convertase can cleave pro- wound areas suggest a possible important role of
MSP to MSP. At the same site MSP then can act on MSP in wound healing processes.
its targets, including macrophages themselves.
CELLULAR SOURCES AND
Conversion of pro-MSP to MSP by a
wound fluid enzyme TISSUE EXPRESSION
The investigation of RON (the MSP receptor)
Cellular sources that produce
expression and MSP status in wounds showed that
active MSP was present in wound exudates in the
concentration range for optimal activity (Nanney MSP is constitutively secreted into the circulation by
et al., 1998). At the same time RON expression was liver cells (Bezerra et al., 1993b). Although the MSP
upregulated in burn wound epidermis and accessory message was detected in several other tissues
structures. The presence of MSP was attributed to (Yoshimura et al., 1993; Ohshiro et al., 1996), the
trypsin-like serine proteases capable of cleaving liver is the main source of MSP in vivo. MSP
Macrophage-Stimulating Protein (MSP) 9
expression in cell lines, embryonal and adult tissues inhibitionoftheinteractionbetweenHNF-4andCBP
is summarized in Table 1. (cyclic adenosine 30,50-monophosphate response ele-
ment (CREB)-binding protein) (Muraoka et al.,
1999b). Nuclear receptors for retinoic, thyroid
Eliciting and inhibitory stimuli,
hormone, glucocorticoid, estrogen and progesterone
including exogenous and in the presence of their unique ligands can directly
interact with CBP (Janknecht and Hunter, 1996;
endogenous modulators
Kameietal.,1996;Tengetal.,1996),whichinterrupts
HNF-4 binding to CBP and causes transcriptional
Pro-MSP production by cells can be regulated by repression of the MSP gene.
various stimuli at different levels (DNA, RNA or In contrast to the above agents, IL-6 enhances
protein), whereas the level of active MSP is regulated MSP gene transcription (Zhu and Paddock, 1999)
at extravascular sites by serine-like proteases. These possibly via activation of putative IL-6 response
proteases can be divided into two groups: specific elementsidentifiedintheMSPpromoter(Waltzetal.,
pro-MSP convertases that cleave pro-MSP to MSP, 1997).
and non-specific proteases that can cleave both pro-
MSP and MSP to inactive fragments.
Regulation of Tissue Concentrations of MSP
Inactive pro-MSP is constitutively secreted by
Regulation of MSP Gene Transcription
hepatocytes into the circulation (Bezerra et al.,
The promoter region of the human MSP gene 1993b; Leonard and Danilkovitch, 2000), and it also
contains a number of potential regulatory sequences, is degraded by the liver (Leonard and Skeel, 1996).
suggestingthatMSPgeneexpressioncanberegulated Conversion of pro-MSP to MSP is thought to occur
byanumberofcytokinesandhormones(Waltzetal., at extravascular sites where MSP target cells are
1997). Treatment of HepG2 cells with retinoic acid, located. Pro-MSP convertases and their inhibitors
thyroid hormone, dexamethasone, progesterone or (see Tables 2 and 3) are components of a pro-MSP/
estrogen caused a decrease in the expression of MSP MSPregulatorysystem,inwhichtheconcentrationof
mRNA (Muraoka et al., 1999b). The proposed componentsmayplayacriticalrole.Atpresent,three
mechanism of MSP gene suppression is due to 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
Aprotinin Serum pro-MSP convertases Wang et al., 1994b
Wound fluid pro-MSP convertase Nanney et al., 1998
Soybean trypsin inhibitor Serum pro-MSP convertases Wang et al., 1994b
Macrophage membrane-associated Wang et al., 1996
pro-MSP degrading proteases
AEBSF Macrophage membrane-associated Wang et al., 1996
pro-MSP degrading proteases
C1-inhibitor Serum kallikrein Wang et al., 1994b
Nerve growth factor (cid:13) Wang et al., 1994a
(cid:11) -Antichymotrypsin, high concentrations Macrophage membrane-associated Skeel and Leonard, 2001
1
(7mM as in human plasma) pro-MSP degrading proteases
Macrophage membrane-associated Skeel and Leonard, 2001
pro-MSP convertases
low concentrations (0.4mM as expected Macrophage membrane-associated Skeel and Leonard, 2001
in extracellular fluid) pro-MSP degrading proteases
10 Alla Danilkovitch-Miagkova and Edward J. Leonard
proteases of the coagulation cascade (Wang et al., (cid:12) chain binds to RON, it does not induce cellular
1994b), enzymes associated with the macrophage responses. Only the MSP (cid:11)(cid:12) chain heterodimer
membrane (Wang et al., 1996) and a protease from induces biological activity and presumably receptor
wound fluid (Nanney et al., 1998). These proteases dimerization.
have the potential to regulate tissue levels of MSP. It has been recently suggested that MSP induces
Murineresidentperitonealmacrophageshavetwocell RON dimerization by a mechanism comparable to
surface proteolytic activities that cleave pro-MSP to growth hormone-induced dimerization of its receptor
MSP or to inactive fragments (Wang et al., 1996). (Wells, 1996), in which a bivalent ligand engages
The degrading protease activity is inhibited by low binding sites of two receptors (Miller and Leonard,
concentrations of blood plasma, which allows the 1998). Detection of (cid:11) chain binding to RON
specificconvertasetocleavepro-MSPtoMSP(Wang (Danilkovitch et al., 1999) suggests that MSP has
et al., 1996). The blood plasma inhibitor was identi- two independent binding sites with high and low
fied as (cid:11) -antichymotrypsin (Skeel and Leonard, affinities located in (cid:12) and (cid:11) chain respectively. This
1
2001). At the expected concentration range (approxi- fact supports the hypothesis that two MSP sites
mately 0.4mM) in extracellular fluid under together mediate RON dimerization and subsequent
non-inflammatory conditions, (cid:11) -antichymotrypsin activation. Mapping of the binding site on the MSP
1
preferentially inhibits the pro-MSP-degrading (cid:12) chain showed the importance of the S1 substrate
enzyme, which allows the macrophage convertase to specificity pocket within the serine protease domain
cleavepro-MSPtoMSP.Incontrast,ininflammatory (Danilkovitch et al., 1999). Location of the low-
exudates, (cid:11) -antichymotrypsin approaches its 7mM affinity binding site within the MSP (cid:11) chain remains
1
concentration in blood plasma, and at these levels unknown.
it inhibits both macrophage enzymes (Skeel and Investigations of mechanisms of MSP/RON inter-
Leonard, 2001). Thus, the MSP detected in inflam- actions revealed that both high- and low-affinity
matoryexudatesisprobablygeneratedbyfluidphase MSP binding sites are located within the SEMA
enzymes that can cleave pro-MSP to MSP. domain of RON (corresponding the extracellular
part of RON, amino acid residues 57–508 in human
RON) (Angeloni et al., unpublished data).
RECEPTOR UTILIZATION
IN VITRO ACTIVITIES
MSP is a ligand for RON.
The first investigation of MSP domains that are
involved in RON bindingand activation showed that In vitro findings
the first two N-terminal kringle domains of the MSP
(cid:11)chainstimulateRONtyrosinephosphorylationina MSP has a number of biological activities that
dose-dependent manner (Gaudino et al., 1994). The were detected predominantly by in vitro assays
minimal portion of the MSP molecule involved in (Figure 2; see also Table 1 in the chapter on RON
RON binding is located within these first two N- receptor). MSP activities can be grouped into several
terminal kringles. Binding assays of radioiodinated broad categories including adhesion and motility,
MSP or its chains to RON-expressing cells showed mediator production, growth, differentiation and
that MSP or its (cid:12) chain specifically bound to RON. survival.
The calculated K is 0.6–0.8nM for MSP (cid:11)(cid:12)
d
heterodimer and 1.4nM for its (cid:12) chain (Wang et al.,
Regulatory molecules: Inhibitors
1997). Despite high-affinity binding of the (cid:12) chain to
RON, functionalstudies indicatedthat onlythe MSP and enhancers
(cid:11)(cid:12) chain heterodimer induces RON tyrosine phos-
phorylation and biological responses, such as macro-
The level of biologically active MSP can be regulated
phageshapechangesandepithelialcellmigrationand
by a variety of trypsin-like proteases (Tables 2 and 3,
proliferation (Waltz et al., 1997; Wang et al., 1997).
and the section Eliciting and inhibitory stimuli,
To initiate signal transduction from the cell surface
including exogenous and endogenous modulators).
to the cell interior, ligand binding has to cause
receptordimerization,followedbyreceptorautophos-
Bioassays used
phorylation and activation (Ullrich and Schlessinger,
1990). Ligand-induced receptor dimerization is
believed to be the key mechanism for initiation of MSPhasanumberofbiologicalactivitiesthatcanbe
signal transduction cascades. Although the MSP detected by a variety of in vitro tests (see Table 1 in