* Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Biotecnologie Cellulari ed Ematologia, Sezione di Genetica
Molecolare, Università di Roma La Sapienza, 00161 Roma; and Institute for Cancer Research, Università di Torino, Facoltà di
Medicina, Torino, Italy
As a rule, hepatocyte growth factor/scatter factor (HGF/SF) is produced by mesenchymal cells, while its receptor, the tyrosine kinase encoded by the met proto-oncogene, is expressed by the neighboring epithelial cells in a canonical paracrine fashion. In the present work we show that both HGF/SF and met are coexpressed by undifferentiated C2 mouse myoblasts. In growing cells, the autocrine loop is active as the receptor exhibits a constitutive phosphorylation on tyrosine that can be abrogated by exogenously added anti-HGF/SF neutralizing antibodies. The transcription of HGF/SF and met genes is downregulated when myoblasts stop proliferating and differentiate. The coexpression of HGF/SF and met genes is not exclusive to C2 cells since it has been assessed also in other myogenic cell lines and in mouse primary satellite cells, suggesting that HGF/SF could play a role in muscle development through an autocrine way.
To analyze the biological effects of HGF/SF receptor activation, we stably expressed the constitutively activated receptor catalytic domain (p65tpr-met) in C2 cells. This active kinase determined profound changes in cell shape and inhibited myogenesis at both morphological and biochemical levels. Notably, a complete absence of muscle regulatory markers such as MyoD and myogenin was observed in p65tpr-met highly expressing C2 clones. We also studied the effects of the ectopic expression of human isoforms of met receptor (h-met) and of HGF/SF (h-HGF/SF) in stable transfected C2 cells. Single constitutive expression of h-met or h-HGF/SF does not alter substantially the growth and differentiation properties of the myoblast cells, probably because of a species-specific ligand-receptor interaction. A C2 clone expressing simultaneously both h-met and h-HGF/SF is able to grow in soft agar and shows a decrease in myogenic potential comparable to that promoted by p65tpr-met kinase. These data indicate that a met kinase signal released from differentiation-dependent control provides a negative stimulus for the onset of myogenic differentiation.
Considerable evidence has been accumulated indicating the importance of hepatocyte growth factor
(HGF)1 in liver physiology (for review see Michalopoulos and Zarnegar, 1992 Moreover, HGF has been shown to be identical to scatter factor (SF), a fibroblast-derived soluble polypeptide
that disperses cohesive epithelial colonies, increasing cell
motility and invasiveness (Stoker et al., 1987 In virtue of its mitogenic, motogenic, and morphogenic
properties, it is believed that HGF/SF could be involved in
many processes where both cell growth and migration are
required, such as embryonal development, tissue repair,
and organ regeneration. This notion is further strengthened by the observation that HGF/SF mRNA is detected
in several organs (Tashiro et al., 1990 HGF/SF shares structural homologies with plasminogen
(38% amino acid sequence identity) and other blood coagulation serine proteases, although it has no protease activity
(Miyazawa et al., 1989 The pleiotropic action of HGF/SF is transduced by a single transmembrane receptor encoded by the met protooncogene (Bottaro et al., 1991 While its ligand is produced prevalently by stromal cells,
met receptor exhibits a complementary pattern of distribution, with the highest expression levels observed primarily in epithelial cells (Chan et al., 1988 HGF/SF binding triggers met kinase activation and its
autophosphorylation on specific tyrosine residues that will
represent docking sites for multiple transductional proteins (Ponzetto et al., 1994 In this study we have examined the expression of HGF/
SF and its receptor in in vitro mouse muscle cells so as to
determine whether HGF/SF might be implicated in myogenesis. In C2 myoblasts, we have detected the presence of
transcripts specific for both the receptor and its ligand.
The synthesis of the corresponding protein products has
also been demonstrated. The hypothesis that HGF/SF
could be an autocrine factor for C2 myoblasts has been confirmed by the observation that its cognate receptor is
highly tyrosine phosphorylated and that this phosphorylation is inhibited by an anti-HGF/SF neutralizing antibody.
Moreover, the finding that other myogenic cell lines and
primary satellite cell cultures coexpress both HGF/SF and
met receptor supports a broader physiological relevance of
this growth factor in muscle development.
The expression of both murine met and HGF/SF genes
was found to be downregulated in concomitance with C2
myogenic differentiation. In fact, we observed a coordinate
transcriptional repression during the transition from proliferating myoblasts to differentiated myotubes. Such interesting behavior of endogenous genes prompted us to investigate the consequences of a constitutive met activation.
Here we show that the expression of p65tpr-met kinase
(Cooper et al., 1984 Cell Cultures
Cells of the mouse myogenic C2 cell line (Yaffe and Saxel, 1977 C3H-10T1/2 mouse embryonic fibroblasts and HepG2 human hepatocarcinoma cells (used as positive control for the expression of met receptor in Northern and Western blot experiments) were cultured in 10%
FCS-containing DME. C3H-10T1/2-MyoD is a C3H-10T1/2 subclone stably expressing the myogenic determinant MyoD (Davis et al., 1987 Agar selection was carried out by seeding 103 or 104 cells per 60-mmdiam dish in DME containing 5% FCS and 0.35% Bacto-Agar (Difco
Laboratories, Detroit, MI) and layering this suspension in quadruplicate
onto a base of 0.7% agar.
Indirect Immunofluorescence Staining
Cells grown on glass coverslips were fixed by immersion in methanolacetone (3:7 vol/vol) for 15 min at To detect the embryonic myosin heavy chain (MHCe), we used the
mouse mAb MF20 (Bader et al., 1982 Plasmids and Probes
The expression vectors used in the transfection assay were: (a) pRK5-met,
in which the entire coding sequence of human met receptor is cloned under the control of the cytomegalovirus promoter; (b) pPEB-HGF/SF, containing the full-length human HGF/SF cDNA cloned downstream of the
murine sarcoma virus LTR; and (c) pMT2-tpr-met, in which the human
tpr-met cDNA is cloned downstream of the adenovirus late promoter
(Ponzetto et al., 1994 For Northern blot hybridization experiments, probes were labeled with
Cell Transfections
Cells were transfected by the calcium phosphate precipitation method
(Wigler et al., 1977 2 d after transfection, cells were split 1:7 in selective medium. As required, 400 µg/ml G418, 200 µg/ml hygromycin, or 1 µg/ml puromycin was
used. The medium was changed every 2-3 d and, after 10-15 d, the surviving colonies were isolated and separately amplified.
RNA Extraction and Northern Analysis
Total RNA was prepared according to the acid guanidinium thiocyanatephenol-chloroform single step method as described by Chomczynski and
Sacchi (1987) For Northern blot analysis, 10-15 µg of each RNA sample was fractionated by electrophoresis through 0.8% agarose gel containing 0.66 M formaldehyde and 1× MOPS buffer (20 mM MOPS, 5 mM NaAc, 1 mM EDTA,
pH 7.0). After running, gel was washed twice in 10× SSC (1.5 M NaCl,
0.15 M sodium citrate, pH 7.2) for 30 min at room temperature to remove
formaldehyde, and RNAs were transferred to nylon filters (Hybond N;
Amersham Intl., Little Chalfont, UK) by a capillarity fluid of the same
buffer (Sambrook et al., 1989 Comparative quantitation of RNAs was estimated by staining the gel
with ethidium bromide and rehybridizing the filters with a probe for a
constitutively expressed gene (GAPDH) (glyceraldehyde-3-phosphatedehydrogenase) (Piechaczyk et al., 1984 Immunoprecipitation and Western Blotting
Subconfluent cell cultures were washed three times in cold PBS and lysed
for 30 min on ice in a buffer containing 50 mM Tris-Cl pH 7.5, 150 mM
NaCl, 10% glycerol, 5 mM EGTA, pH 7.5, 50 mM NaF, pH 8, 1.5 mM
MgCl2, 1% (vol/vol) Triton X-100, supplemented with various protease
and phosphatase inhibitors (leupeptin, aprotinin, PMSF, sodium-orthovanadate). After clearing by centrifugation at 15,000 rpm for 20 min at
4°C, the protein content of samples was quantified by the Biorad method.
Equal amounts of cellular extracts were pretreated with 50 µl protein
A-Sepharose (Pharmacia, Uppsala, Sweden) (50% vol/vol in PBS + 3%
BSA) to remove nonspecific binding, and then incubated for 1 h at 4°C
with anti-met antibodies. The following antibodies were used: (a) rabbit
anti-h-met antiserum (C-12 antibody) and (b) rabbit anti-m-met antiserum (SP260 antibody), both purchased from Santa Cruz Biotechnology,
Inc. (Santa Cruz, CA).
The immunocomplexes were collected by adding 50 µl protein A-Sepharose (incubation for 1 h at 4°C) and washed four times with NET-gel
buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% NP-40, 1 mM EDTA,
pH 8.0, 0.25% gelatin, 0.02% sodium azide). Finally, the immunocomplexes were solubilized in boiling Laemmli buffer containing 5% Western blot analysis was carried out by using the anti-met polyclonal
antibodies described above or an anti-phosphotyrosine mAb (Upstate Biotechnology Inc., Lake Placid, NY). The staining of the blots was performed by the enhanced chemiluminescence system (ECL; Amersham
Intl.).
The autocrine mode of action of HGF/SF in C2 cells was investigated
by measuring the degree of tyrosine phosphorylation of met receptor in
different culture conditions. Briefly, C2 cells were treated for 5 min on ice
with an acid solution (20 mM acetic acid, 150 mM NaCl, 0.25% BSA), and
subsequently incubated with 0.5 M NaCl-containing DME for 20 min at
37°C to dissociate HGF/SF molecules from cell surface (Sturani et al., 1988 Scatter Assay
The production of HGF/SF by C2 cells and transfected clones was investigated by measuring the ability of the corresponding conditioned medium
to dissociate epithelial MDCK cell colonies (Stoker et al., 1987 To verify the extension of HGF/SF processing, the scatter assay was
performed using mouse liver progenitor cells (Medico et al., 1996 Expression of Endogenous met and HGF/SF in
Myogenic Cells
To determine whether C2 myoblasts could be a potential
target for HGF/SF action, we analyzed the expression of
HGF/SF receptor (met) in proliferating cells by Northern
blotting of total RNA. Using the full-length human met
cDNA as a probe, we have detected a transcript of ~8.5 kb,
which represents the major met mRNA expressed in murine tissues and in human hepatocarcinoma (Hep G2) cells (Fig. 1 A).
Met protein was visualized from C2 cell lysates by immunoprecipitation and Western blotting with an anti-
murine met polyclonal antibody (Fig. 1 B). In this experiment both the 170-kD single chain precursor (p170 To determine if met activation was due to an autocrine
stimulation, the production of HGF/SF by C2 cells was investigated, since muscle cells belong to the mesenchymal
compartment that is known to be a preferential expression
site of met ligand (Sonnenberg et al., 1993
The existence of a natural HGF/SF autocrine loop in C2
cells prompted us to assess if this autocrinia could reflect a
common situation in cells belonging to the myogenic lineage and not represent a peculiarity of C2 myoblasts only.
Therefore, we analyzed the expression of met and HGF/SF
in mouse primary satellite cells and in the PCD2 teratocarcinoma-derived mouse cell line, which undergoes myogenic differentiation (Boon et al., 1974
Met and HGF/SF Genes Are Coordinately
Downregulated during Myogenic Differentiation
Previous works have shown that the expression of different genes encoding for growth factors and their cognate
receptors is modulated during myogenesis (Ewton et al.,
1988
The observation that mRNA decline is differentiation
dependent enables us to infer that the mechanisms responsible for cell growth-arrest and muscle-specific gene activation can also be involved in the control of both met and
HGF/SF gene expression. To test this hypothesis, we have
analyzed the expression of met and HGF/SF genes in C3H10T1/2 mouse fibroblasts, which can be converted to myogenic lineage by stable ectopic expression of MyoD (Davis
et al., 1987 Effect of tpr-met Expression in C2 cells
The observation that endogenous met and HGF/SF genes
are coordinately downregulated during myogenic differentiation of C2 cells raises the question if HGF/SF-mediated
signals could exert a negative constraint on muscle differentiation. To test this hypothesis, we have studied the consequences of the expression of p65tpr-met, a constitutively
activated version of the met kinase (Cooper et al., 1984 C2 cells were transfected with pMT2-tpr-met construct
in which the human tpr-met cDNA is cloned downstream
of the adenovirus late promoter. The construct pBABEPuro encoding for the puromycin resistance was used as
a selection marker. 20 puromycin-resistant clones were
isolated, individually amplified, and tested for the presence of p65tpr-met protein by Western blot analysis. All together, eight clones resulted to express variable levels of
the heterologous protein and three of them were found to
express high levels of p65tpr-met (clones 2, 5, and 11; Fig.
6 A). In all the positive clones, p65tpr-met was strongly evidenced by an anti-phosphotyrosine antibody in a Western
blot performed on the immunoprecipitated protein (Fig. 6 B); this argues for a full activation of p65tpr-met kinase in
transfected myoblast cells. Moreover, the degree of tyrosine phosphorylation parallels the protein content. As
expected, kinase activity of p65tpr-met is retained also in
cells grown at a low serum condition (data not shown).
The p65tpr-met highly expressing clones exhibit a singular
cell morphology that is shown in Fig. 7. In detail, cells are
very spindle-shaped and little adherent to the substrate,
implying a profound reorganization of the cytoskeletal
apparatus. This scattered appearance is identical to the motogenic response observed when epithelial cells are stimulated by HGF/SF (see Fig. 2 B). The increase of cell motility in p65tpr-met highly expressing clones was confirmed by
the Boyden chamber assay (data not shown). The morphology of the clones that express low quantities of p65tpr-met
is comparable to that of C2 parental myoblasts (not
shown).
It has been shown that p65tpr-met promotes cell proliferation and tumorigenesis (Cooper et al., 1984 The motogenic properties of p65tpr-met highly expressing
clones are accompanied by a marked inability to differentiate when confluent cultures are deprived of mitogens.
Immunofluorescence staining of MHC protein synthesized
after 48 h of incubation in differentiating medium shows
few MHC-positive cells, which appear as single isolated and fusiform cells and very sporadically as multinucleated
cells (Fig. 7).
To characterize at a biochemical level the decrease of
myogenic potential of p65tpr-met expressing clones, we investigated the expression of the muscle-specific regulatory
genes MyoD and myogenin (Weintraub, 1993
In the p65tpr-met moderate expressing clones, the transcripts for MyoD and myogenin are detectable, but the relative hybridization signals have an intensity lower than
those of C2 parental cells, suggesting that the degree of
myogenic inhibition correlates with the levels of p65tpr-met
expressed (Fig. 8). Control C2 cells stably transfected with the empty pMT2 construct did not show any growth and
differentiating alterations (data not shown).
Effect of Heterologous h-met and h-HGF/SF Double
Expression in C2 Cells
To determine whether the expression of an autocrine loop
for HGF/SF released from differentiation control could
result in a phenotype similar to that of p65tpr-met-expressing
C2 cells, we analyzed the properties of C2 cells double transfected with the heterologous h-met and h-HGF/SF
coding sequences. The choice to use the human isoforms
allowed us to distinguish them from the endogenous ones
both at mRNA and protein levels. Stable transfection of
C2 cells was performed using pRK5-met, pPEB-HGF/SF,
and pRSV-neo constructs at a 5:5:1 ratio. From 40 clones
selected for G418 resistance, only two of them (clones 9 and 33) resulted positive for the synthesis of both h-met
and h-HGF/SF mRNAs. The remaining 38 G418-resistant
clones expressed h-met (eight clones), h-HGF/SF (nine
clones), or neither of them (data not shown). Subsequent
studies have revealed that, after three to five passages,
clone 9 did not express the h-met mRNA any more; the
loss of h-met transcript is probably the result of an unstable genomic integration of human DNA sequence. For this
reason, only clone 33 should be considered as a real double transfectant (Fig. 9 A). As determined by Western blot
analysis, clone 33 expresses a highly tyrosine-phosphorylated form of h-met (Fig. 9 B). This is consistent with the
productive binding and activation by h-HGF/SF.
Cells from clone 33 show several alterations in the
growth properties, particularly in the ability to grow in soft
agar, whereas C2 clones expressing only h-met or h-HGF/
SF, as well as C2 parental cells, are not able to grow independently from anchorage (data not shown).
The clone 33 was also tested for its differentiating potential. Confluent cells were transferred in DME containing 10% HS. After a 48-h incubation in this medium, cells
were analyzed for the expression of MHC by indirect immunofluorescence staining. As shown in Fig. 9 C, clone 33 presents a significant inhibition of the differentiating process, since few cells express myosin and most of them are
mononucleated and fusiform, similar to those observed in
p65tpr-met-expressing clones. In differentiation studies performed by shifting to very low percentages of serum (0.5%
or 1% FCS), we have observed a partial restoration of the
differentiating capacity in clone 33 (data not shown). The
reacquiring of the differentiated phenotype can be explained by the prevention of HGF/SF processing in these culture conditions, clearly indicating a lack of HGF/SF biological activity (Naka et al., 1992 In view of these data, although they refer to only one
h-met/h-HGF/SF-expressing clone, we can infer that a constitutive autocrine loop for h-HGF/SF causes growth alterations and myogenic inhibition.
The single stable expression of h-met or h-HGF/SF in
C2 cells was not found to induce appreciable phenotypic
changes in proliferative conditions, indicating that a high
species-specificity of ligand-receptor interaction is required
to activate met signaling. Indeed, C2 clones selected for
expression of the human isoform of the receptor showed
consistent protein levels of h-met, which, in a different way
from the endogenous m-met, is weakly or not at all phosphorylated on tyrosine residues (Fig. 9 D). This is in accordance with previous data obtained using mouse fibroblast
cells (Rong et al., 1992 Although we have had no particular problems in selecting C2 clones expressing p65tpr-met kinase, we presume that
the simultaneous expression of h-met and h-HGF/SF results in severe cell growth disturbances and is therefore
more restrictive for C2 myoblast survival. Two lines of evidence support this hypothesis, with the first being the low
ratio of positive double transfectants as compared with
that of C2/h-met and C2/h-HGF/SF single transfectants.
Here it should be noted that the h-met/h-HGF/SF transfection was repeated a second time yet without success
(from 30 clones selected, none were double positive). Secondly, cells of clone 33 are subject to cell death when cultured in growth-restrictive conditions. Preliminary experiments indicate that the observed cell death is to be
classified as apoptosis (data not shown).
It is well known that muscle development is strongly influenced by serum components, first of all by peptide growth
factors (for review see Florini and Magri, 1989 In this context, the local concentration of growth factors
(of own production or added exogenously to the culture
medium) results in being critical for maintaining cells as
proliferating myoblasts or inducing them to acquire the differentiated phenotype. Until now, much of the work done
was centered on distinct growth factors such as FGF, TGF- In the present work we have addressed the question of
whether HGF/SF, a pleiotropic protein able to elicit multiple biological responses (for review see Goldberg and
Rosen, 1993 We have shown that C2 myoblasts express both HGF/
SF and its receptor, met tyrosine kinase. Despite the fact
that HGF/SF has been considered essentially as a paracrine factor secreted by mesenchymal cells and effective on
epithelial cells, some examples of natural autocrine cells
for HGF/SF have been described (Adams et al., 1991 Different studies indicate that HGF/SF can exert an important function in muscle development. It has been shown
that met and HGF/SF transcripts are present in muscle formation sites during mouse embryogenesis (Sonnenberg
et al., 1993 We have shown that the expression of HGF/SF and met
genes is subordinated to the proliferative state of myoblast
cells since a transcriptional decline for both of them was
consistently observed after induction of differentiation.
The modulation of genes encoding for growth factors and
growth factor receptors seems to be quite a common strategy adopted by muscle cells when they enter the differentiating pathway (Florini and Magri, 1989 The disappearance of growth factor receptors with the
onset of differentiation is important in ensuring an irreversible withdrawal from cell cycle and, consequently, a
stable expression of muscle-specific phenotype. In fact, fusion-defective muscle cells differentiate with neither terminal commitment nor receptor decline in such a way that
they can reverse muscle phenotype after growth factor exposure (Hu and Olson, 1990 The observed HGF/SF and met gene downregulation
suggests a functional shut-off in the autocrine stimulation
by HGF/SF during C2 myogenic differentiation. Our data
are in agreement with the previous observation that in rodents HGF/SF and met receptor are expressed in skeletal
muscle tissue during embryonic development and in the
first days after birth, while the levels of the relative transcripts are weak or not detectable in adult skeletal muscle (Jennische et al., 1993 We have found a similar differentiation-dependent downregulation of HGF/SF-met system in other met-HGF/SF
coexpressing cells, C3H-10T1/2 mouse fibroblasts converted to myoblasts by stably expressed MyoD. This finding further supports the incompatibility between met kinase signaling and the differentiating program.
The transcriptional downregulation of HGF/SF and met
genes during muscle differentiation allows us to think that
a premature met kinase signaling interruption could result
in a major propension of cells to differentiate. However,
our attempts to force C2 myoblasts to enter the differentiating pathway by cultivating them in growth medium supplemented with anti-HGF/SF neutralizing antibodies were not successful; in fact, no sign of precocious myogenesis
was detected (data not shown). It is possible that the high
levels of HGF/SF produced by C2 cells would make the interruption of the autocrine stimulation by the neutralizing
antibodies very difficult, at least in the conditions we used.
Furthermore, met kinase inactivation could be necessary,
but not by itself sufficient, to permit muscle differentiation
since additional inhibitory constraints would continue to
function in these conditions.
Nevertheless, strong evidence for a negative effect of met
kinase signaling on myogenic differentiation comes from
the characterization of C2 clones expressing the constitutively activated p65tpr-met fusion protein. As determined by
immunofluorescence staining, these clones are defective
for the synthesis of muscle-specific structural proteins
(MHC) and myotube formation. The extent of this myogenesis inhibition corresponds with the expression levels of p65tpr-met.
A reduced expression of different myogenic helix-loophelix transcription factors, such as MyoD and myogenin,
was also detected in these clones. Once again, the p65tpr-met
highly expressing clones exhibit the most extreme phenotype with a complete failure in myogenin gene activation
and with the silencing of MyoD gene expression. A negative regulation of both gene expression and functional activity of MyoD has been already reported for FGF and
TGF- In addition, we have shown that the activity of p65tpr-met
kinase induces remarkable changes in cell morphology.
Particularly in p65tpr-met highly expressing clones, cells appear spindle-shaped and refractile, denoting a poor adherence to substrate, witnessed also by a major sensitivity to
trypsinization. The presence of a network of threadlike cytoplasmic extensions is reminiscent of the scattered appearance of epithelial cells upon HGF/SF stimulation, indicating that p65tpr-met signaling triggers an increased motogenic
response.
Attempting to obtain further insight into the role of
HGF/SF in C2 differentiation and to overcome the differentiation-dependent decrease of HGF/SF and met endogenous genes, we have also studied the effects of an ectopic
constitutive expression of the human isoforms of HGF/SF
and of its cognate receptor in C2 myoblasts.
Clones selected for the single expression of h-met or
h-HGF/SF do not exhibit significant phenotypic alterations
compared with those of the parental cell line, although
they produce detectable quantities of each product. From
the results it may be inferred that a species-specific correspondence between the ligand and its related receptor is
required to create an effective autocrine loop for h-HGF/
SF in C2 myoblasts. This is noteworthy if we consider the
high degree of homology between murine and human
HGF/SF proteins, which is >90% (Liu et al., 1993 On the other hand, the establishment of an autocrine
loop for h-HGF/SF through its coexpression with h-met in
C2 cells results in soft agar growth ability and in a simultaneous myogenic inhibition, just as for p65tpr-met-expressing
clones. In this case, h-met exhibits a quantitative tyrosine
phosphorylation, indicating a full activation by h-HGF/SF.
In light of these observations, we believe that a met receptor stably activated through an autocrine HGF/SF
stimulation could be equivalent to the expression of tprmet kinase, despite the fact that p65tpr-met has a cytoplasmic
localization and a constitutive kinase activity independent
from HGF/SF. Yet some differences must be present, since
we were able to select only one C2 clone expressing simultaneously h-met and h-HGF/SF from two distinct transfection assays. This difficulty was further confirmed by the
loss of one human product in a second clone and by the cell
death observed when cells of clone 33 were transferred to
differentiation medium. It appears therefore that the establishment of a species-specific autocrine loop for h-HGF/SF,
released from differentiation-dependent control, has a major negative action on C2 myoblasts.
In sum, our results support the notion that HGF/SF plays a
role in the motogenic and growth properties of myogenic
cells through an autocrine loop that needs to be downregulated during differentiation, since the constitutive activation
of met kinase is incompatible with myogenesis.
). This protein is a potent mitogen for mature hepatocytes in primary culture (Miyazawa et al., 1989
; Nakamura et al., 1989
) and functions in
vivo as a hepatotrophic factor during the regenerative events
in the liver injured by a partial hepatectomy or by hepatotoxin treatment (Ishiki et al., 1992
). In addition, mice lacking HGF exhibit severe impairment in liver development
and die in utero (Schmidt et al., 1995
). Although its role in
maintaining liver homeostasis is well accepted, other studies have shown that HGF is a multifunctional cytokine
possessing a wide spectrum of biological activities besides
the hepato-specific ones by which it was first identified
(for review see Goldberg and Rosen, 1993
). HGF has been
reported to stimulate proliferation of endothelial cells and
various epithelial cells, including melanocytes and keratinocytes (Igawa et al., 1991
; Kan et al., 1991
; Matsumoto
et al., 1991
; Rubin et al., 1991
; Bussolino et al., 1992
). The
mitogenicity exerted on renal tubular and on pulmonar
cells reflects the active role of HGF in promoting regeneration in kidney (Nagaike et al., 1991
; Kawaida et al., 1994
)
and lung (Yanagita et al., 1993
) upon tissue damage.
; Gherardi
et al., 1989
; Weidner et al., 1990
, 1991; Naldini et al.,
1991b
). HGF/SF has been also qualified as a morphogen
for its ability to induce the organization of cells into ordered tubule-like structures (Montesano et al., 1991
; Tsarfaty et al., 1992
); on endothelial cells, the morphogenic
stimulus assumes the character of angiogenesis with formation of blood vessels in vivo (Bussolino et al., 1992
;
Grant et al., 1993
).
; Zarnegar et al., 1990
;
Sonnenberg et al., 1993
), and that HGF/SF protein is still
broadly localized by immunohistochemical means (Wolf
et al., 1991
; DeFrances et al., 1992
). Finally, there is evidence of the involvement of HGF/SF in tumorigenesis and
in tumor progression toward a more aggressive phenotype
(Weidner et al., 1990
; Rong et al., 1992
, 1994; Bellusci et
al., 1994
).
; Nakamura et al., 1989
). Similarly
to the biosynthetic pathway of the serine proteases, HGF/
SF is secreted by cells of mesodermal origin as a singlechain precursor that is then processed to yield a disulfidelinked complex composed of a heavy and a light chain (Miyazawa et al., 1989
; Nakamura et al., 1989
; Weidner et al., 1990
). The proteolytic maturation is serum dependent and
is necessary for the acquisition of biological activity (Hartmann et al., 1992
; Lokker et al., 1992
; Naka et al., 1992
;
Naldini et al., 1992
, 1995).
; Naldini et al., 1991a
,b;
Weidner et al., 1993
). The met protein has a heterodimeric
structure consisting of an extracellular
subunit (50 kD)
and a membrane-spanning
subunit (145 kD) endowed
with a tyrosine kinase domain in its cytoplasmic region.
Both subunits originate from glycosylation and from processing of a common precursor of 170 kD (Giordano et al.,
1989a
,b).
; Iyer et al., 1990
;
Di Renzo et al., 1991; Prat et al., 1991
). Since HGF/SFproducing and -responding (met-positive) cells belong to
adjacent tissue compartments, HGF/SF is actually considered a major paracrine mediator of mesenchyme-epithelium interactions (Sonnenberg et al., 1993
; Comoglio and
Boccaccio, 1996
).
). It is thought that the particular cellular response (cell growth vs cell locomotion or morphogenesis) may be ensured by the integration of distinct
signaling pathways into different cell types. Recent studies
have shown an active role of HGF/SF in the control of muscle development (Bladt et al., 1995
; Maina et al., 1996
; Takayama et al., 1996
; Yang et al., 1996
).
; Park et al., 1986
; Gonzatti-Haces et al.,
1988
; Rodriguez and Park, 1993
) results in substantial alterations of cell growth and inhibition of myogenesis, as
does the constitutive activation of the human isoform of
met receptor (h-met) through a species-specific autocrine
stimulation by human HGF/SF (h-HGF/SF).
Materials and Methods
) clone 7 and of the mouse teratocarcinoma-derived myogenic PCD2 cell line
(Boon et al., 1974
) were maintained as undifferentiated myoblasts in
DME supplemented with 20% FCS in a 10% CO2 atmosphere. To induce
cell differentiation, myoblasts were grown to confluence, and then shifted
to DME supplemented with 0.5-2% FCS or 10% horse serum (HS). Extensive morphological and biochemical differentiation was obtained after
24-48 h. Mouse primary satellite cells (kindly provided by Dr. Giulio
Cossu, Institute of Histology, University of Rome "La Sapienza," Roma,
Italy) were derived from limbs of 14-d-old mice, purified according to Salvatori et al. (1993)
and amplified once.
). The
epithelial cell lines (MDCK and mouse liver progenitor) used in the scatter assay are described below.
20°C and then air dried. Coverslips
were then incubated at 37°C in a humidified atmosphere with the primary
antibody, in some cases diluted in PBS containing 1% BSA. After three
washes with PBS, the coverslips were incubated with the secondary fluorochrome-conjugated antibody diluted in PBS plus 1% BSA, washed repeatedly with PBS, and mounted with 70% glycerol in PBS.
) as undiluted hybridoma supernatant. As secondary antibody, we used a goat anti-mouse IgG rhodamineconjugated IgG fraction (Cappel Immunochemical Products, Malvern,
PA) diluted 1:100.
). As selectable markers for stably transfected cells,
we used plasmids: (a) pRSV-neo, containing the neomycin resistance gene
under the control of the Rous Sarcoma virus long terminal repeat (LTR)
(Mulligan and Berg, 1980
); (b) pSV2-hygro, containing the hygromycin resistance gene under the control of the SV-40 promoter-enhancer region
(Maione et al., 1992
); and (c) pBABE-puro, containing the puromycin resistance gene under the control of Moloney murine leukemia virus LTR
(Morgenstern and Land, 1990
).
[32P]dATP by the random priming method. To detect specific transcripts, the following probes were used: (a) the human full-length met cDNA
(Ponzetto et al., 1991
); (b) the human full-length HGF/SF cDNA (Naldini
et al., 1991); (c) the plasmid pMHC 2.2, specific for MHCe transcript
(Weydert et al., 1985
); (d) the EcoRI fragment from plasmid pEMC11s
specific for MyoD transcript (Davis et al., 1987
); and (e) the human cDNA
myf4 (Braun et al., 1989
) highly homologous to the mouse myogenin transcript, provided by Dr. Hans Henning Arnold (University of Hamburg
Medical School, Hamburg, Germany).
), with ~105 cells per 60-mm-diam dish. Single stable
transfectants were obtained by cotransfecting each dish with the following
amounts of DNA: 5 µg of the expression vector and 0.5 µg of the selection
plasmid. For h-met/h-HGF/SF double transfections, 2.5 µg of pRK5-met
plasmid, 2.5 µg of pPEB-HGF/SF plasmid, and 0.5 µg of pRSV-neo plasmid were used.
.
). Filters were fixed by Stratalinker (1,200 µJ × 100). Prehybridization and hybridization were carried out at 42°C using
the commercial Hybrisol solution (Oncor, Gaithersburg, MD).
-[32P]dATP-labeled probes were used at a 2 × 106 counts per ml concentration.
Hybridized filters were washed once at 42°C in a 2× SSPE (a 20× SSPE
solution was 3 M NaCl, 200 mM NaH2PO4·H2O, 20 mM EDTA, pH 7.4),
0.1% SDS solution, and twice at 60°C in a 1× SSPE, 0.1% SDS solution.
Finally, filters were exposed to an autoradiographic film (Fuji Photo Film
Co., Tokyo, Japan) at
70°C with intensifying screens for variable periods.
).
-mercaptoethanol, electrophoresed on 8-12% SDS polyacrylamide gels, and
transferred onto nitrocellulose filters (Hybond C; Amersham Intl.) by the
semidry blot method.
;
Naldini et al., 1992
). Then cells were lysed immediately or after an additional incubation (30 min at 37°C) with fresh 10% FCS-containing medium, C2-conditioned medium, or C2-conditioned medium previously incubated with a neutralizing goat antibody directed against mouse HGF/SF (kindly supplied by Dr. Ermanno Gherardi, Imperial Cancer Research Fund, Cambridge, UK).
). The assay was performed in 24-well Costar plates (Cambridge, MA). 4,000-6,000
cells per well were plated and exposed to serial dilutions of conditioned
medium in 10% FCS-containing DME for 15-20 h at 37°C. After being
fixed with 11% glutaraldehyde and colored with crystal violet, cell scattering was examined by a phase-contrast light microscope. Scatter activity
was generally detected in the range of 1:30-1:300 dilutions.
), which
can be cultivated in low serum; in this case, the testing conditioned medium was diluted in 1% FCS-containing DME.
Results
Fig. 1.
Synthesis of met receptor by C2 myoblast cells. (A)
Northern blot of total RNA extracted from human hepatocarcinoma HepG2 cells and C2 cells probed with human full-length
met cDNA (Ponzetto et al., 1991). The position of 8.5-kb metrelated transcript and the 28S ribosomal RNA is indicated. (B)
Activation state of met receptor expressed by C2 myoblasts. Protein extracts from proliferating C2 myoblasts were immunoprecipitated with anti-met rabbit SP260 antiserum (directed against
the COOH-terminal portion of the murine protein), electrophoresed onto 8% reducing SDS-PAGE, and immunoblotted with SP260 antiserum (
-m-met). In these conditions, both the 145-kD
subunit of mature met receptor (p145
) and the 170-kD
single-chain precursor (p170
) were detected. The same filter
was stripped and reprobed with monoclonal anti-phosphotyrosine antibodies (
-PTyr) to provide evidence for the activation
degree of p145
.
[View Larger Version of this Image (35K GIF file)]
) and
the 145-kD
subunit of met receptor (p145
) were clearly
detected. Furthermore, to establish if met receptor was activated in C2 cells, a Western blot with an anti-phosphotyrosine mAb was performed on the immunoprecipitated
protein. As shown in Fig. 1 B, p145
exhibits a high degree
of phosphorylation on tyrosine residues, indicating that
met receptor is activated in C2 myoblasts.
). Northern blot
analysis has brought evidence of the presence of a 6-kb transcript (Fig. 2 A) equivalent in size to the principal HGF/SF
mRNA species described by other authors (Nakamura et al.,
1989
; Tashiro et al., 1990
). The presence of HGF/SF protein in the supernatant of C2 cells was tested by assaying its scatter activity on MDCK epithelial cells (Fig. 2 B), in
comparison with the standard medium (Fig. 2 C). Titration
by serial dilutions indicated the production of amounts
comparable to that of HGF/SF-producing fibroblast cells
(Stoker et al., 1987
). The existence of a natural autocrine
loop for HGF/SF in C2 myoblasts was confirmed by exposing C2 cell cultures to an acid treatment followed by an
incubation with DME supplemented with 0.5 M NaCl.
These conditions have been shown to effectively dissociate
the ligand-receptor complexes and remove the HGF/SF
molecules linked to extracellular matrix (Sturani et al., 1988
;
Naldini et al., 1992
). Duplicate plates of C2 cells were
treated in the same way and then further incubated for 30 min with C2-conditioned or fresh 10% FCS-containing medium, respectively. Results shown in Fig. 3 demonstrate
that the treatment causes a net decrease of met tyrosine
kinase phosphorylation and that only C2-conditioned medium restores phosphorylation. Moreover, C2-conditioned
medium loses its capacity to stimulate met kinase if previously incubated with a neutralizing anti-HGF/SF antibody. These results demonstrate that met receptor expressed by
C2 myoblasts is activated by the endogenously produced
HGF/SF.
Fig. 2.
Synthesis of HGF/
SF by C2 myoblast cells. (A)
Northern blot analysis of total RNA extracted from human hepatocarcinoma cells (HepG2) and C2 cells to illustrate the production of a
6-kb HGF/SF mRNA. As
previously reported in literature (Shiota et al., 1992), HepG2 cells are negative for
the expression of HGF/SF
gene. (B and C) Phase-contrast micrographs of a scatter assay carried out with
MDCK epithelial cells incubated with C2 myoblast-conditioned medium (B) or standard medium (C).
[View Larger Version of this Image (48K GIF file)]
Fig. 3.
HGF/SF is an autocrine factor for C2 myoblasts. C2
proliferating cells exposed to culture conditions that remove
HGF/SF molecules from cell surface (see Materials and Methods) were lysed, immunoprecipitated with anti-murine met antibodies, and analyzed for the tyrosine phosphorylation degree of
HGF/SF receptor by Western blotting with anti-phosphotyrosine
antibodies (-PTyr). Control cells (lane 1); C2 cells acid treated
and incubated with 0.5 M NaCl-containing DME (lane 2); cells
treated as in lane 2 and further incubated with C2-conditioned
medium (lane 3) or C2-conditioned medium pretreated with an antiHGF/SF neutralizing antibody (lane 4); cells as in lane 2 and further incubated with fresh 10% FCS-containing medium (lane 5).
[View Larger Version of this Image (76K GIF file)]
). By Northern blot
analysis, we have detected specific signals for both the receptor and its ligand (Fig. 4). Here it should be noted that primary cultures were double precleared from fibroblasts
to reduce as much as possible any contribution of these
cells to the HGF/SF mRNA signal detected, since HGF/SF
is a main fibroblast-secreted cytokine (Stoker et al., 1987
;
Gherardi et al., 1989
; Weidner et al., 1990
). The persistence of high expression levels of the muscle-specific determinant MyoD confirmed the identity of these cells.
Note that PCD2 cells undergo differentiation in the absence of MyoD expression, whose function could be replaced by other members of the family of myogenic determinants (Weintraub, 1993
).
Fig. 4.
Coexpression of met
and HGF/SF in other myogenic
cells. Total RNA from C2 myoblasts, PCD2 mouse myoblasts,
and mouse primary satellite
(Sat) cell cultures was hybridized with the indicated probes.
[View Larger Version of this Image (32K GIF file)]
; Olwin and Hauschka, 1988
; Tollefsen et al., 1989a
,b;
Hu and Olson, 1990
; Lafyatis et al., 1991
; Moore et al., 1991
).
To learn whether the expression of met and HGF/SF genes
was subjected to quantitative variations during muscle differentiation, we have performed a Northern blot analysis
of total RNA prepared from undifferentiated and differentiating C2 cells. Results reported in Fig. 5 show that the
highest level of met and HGF/SF mRNA was found in actively proliferating myoblasts, cultured in 20% FCS-containing medium. When the myoblasts were induced to differentiate by a shift to low mitogen-containing medium (2%
FCS or 10% HS), an evident decrease for both transcripts was observed. The transcriptional repression is strictly
coupled with the myogenic process since the hybridization
signals are almost undetectable at day 3 postserum deprivation, when a consistent number of mature multinucleated myotubes are present. Muscle differentiation was also
assessed by measuring the expression of myosin heavy chain (MHC) mRNA, whose gene is transcriptionally activated early in differentiation.
Fig. 5.
The expression of met and HGF/SF genes is downregulated concomitantly with the onset of myogenic differentiation. C2,
C3H-10T1/2, and C3H-10T1/2-MyoD cells were cultured in DME
containing 20% FCS up to confluency, and then transferred to
low serum-containing medium (2% FCS). Total RNA extracted
before serum deprivation (lanes 1) and 1 (lanes 2), 2 (lanes 3),
and 3 (lanes 4) d after serum deprivation was assayed for the expression of met, HGF/SF, and a muscle-specific marker, MHC,
transcripts. Quantitative decrease of met and HGF/SF mRNA synthesis was observed only in the presence of muscle differentiation.
[View Larger Version of this Image (63K GIF file)]
). As reported in Fig. 5, C3H-10T1/2 cells synthesize both met and HGF/SF mRNAs that are downregulated in low serum only upon myogenic conversion.
; Park
et al., 1986
; Gonzatti-Haces et al., 1988
; Rodriguez and Park,
1993
).
Fig. 6.
Extensive p65tpr-met kinase activation in p65tpr-met-
expressing C2 clones. Total cell lysates were immunoprecipitated
with rabbit anti-met C12 antiserum (directed against the COOHterminal portion of the human protein), electrophoresed onto
12% reducing SDS-PAGE, and immunoblotted with the same
antibody (A) or anti-phosphotyrosine antibodies (B). A good
correlation was found between the p65tpr-met protein levels and its
degree of tyrosine phosphorylation. Clone 15 was negative for
the expression of p65tpr-met. Size in kD was estimated using a molecular mass standard (M).
[View Larger Version of this Image (63K GIF file)]
Fig. 7.
Phenotypic appearance and differentiating properties
of p65tpr-met-expressing C2 clones. Micrographs of p65tpr-met highly
expressing cells (B) and C2 parental cells (A) cultured either in
growth or differentiation medium. The myogenic potential was
assessed by detecting myotube formation and by immunofluorescence staining for MHC in 48-h differentiation medium-exposed
cell cultures.
[View Larger Version of this Image (106K GIF file)]
; Park et al., 1986
);
nevertheless, the growth rate of p65tpr-met-expressing clones,
independent from the protein expression levels, is not significantly modified in comparison with the C2 parental cell
line (data not shown).
). In C2 parental cells, the gene encoding for myogenin is transcriptionally activated in concomitance with the beginning of
muscle differentiation; on the contrary, MyoD gene is expressed also at the myoblast stage although the protein
product becomes functional only in differentiating conditions. By Northern blot analysis, we have found a complete absence of MyoD and myogenin transcripts in p65tpr-met
highly expressing cells (Fig. 8).
Fig. 8.
Decreased expression of muscle-regulatory genes in
p65tpr-met-expressing C2 clones. Total RNA from proliferating
(lanes 1) and differentiating (lanes 2, 3, and 4; 24-, 48-, and 72-h
postserum deprivation, respectively) cultures was hybridized with
the indicated probes. To estimate if equal amounts of RNA were
loaded, the same filter was probed with a constitutively expressed
gene (GAPDH).
[View Larger Version of this Image (51K GIF file)]
Fig. 9.
Double ectopic expression of h-met and h-HGF/
SF is required for the constitutive activation of h-met
kinase. (A) Northern blot
analysis of RNA expression
levels of h-met and h-HGF/SF
in clone 33 selected from C2
h-met/h-HGF/SF double transfection. Exogenous transcripts are larger than endogenous ones. (B) The h-met
receptor expressed by clone
33 is phosphorylated on tyrosine residues. Cells were
lysed at two different culture
passages and immunoprecipitated with anti-human met
antibodies. Proteins were resolved by 8% reducing SDSPAGE and immunoblotted
with anti-human met (-hmet) or anti-phosphotyrosine (
-PTyr) antibodies. (C) Phase-contrast and anti-MHC
immunofluorescence micrographs of clone 33 cells exposed for 48 h in differentiation medium containing 10%
HS. (D) Cell lysates from C2
parental cells and three single h-met-expressing C2 (C2/
h-met) clones were immunoprecipitated with anti-murine
met antiserum (C2 cells) or
with anti-human met antiserum (C2/h-met clones), respectively. The immunocomplexes were resolved by 8%
reducing SDS-PAGE and
immunoblotted with anti-human (
-h-met) or anti-phosphotyrosine mAbs (
-PTyr). The almost undetectable tyrosine phosphorylation
exhibited by h-met in comparison with the endogenous receptor suggests that it is not efficiently activated by murine HGF/SF.
[View Larger Versions of these Images (23 + 33K GIF file)]
; Naldini et al., 1992
, 1995).
). Likewise, no substantial alterations of muscle differentiation were observed in C2/h-met
and C2/h-HGF/SF clones in comparison with C2 parental
cells (data not shown).
Discussion
; Florini et al.,
1991
; Olson, 1992
; Maione and Amati, 1996
). The functional relationships between growth factors and muscle
differentiation have been extensively investigated by using
in vitro cultured muscle cells and taking the advantage of
the myogenic process study in monitored conditions. Myogenesis in tissue culture is accompanied by a terminal and irreversible withdrawal from cell cycle so that the postmitotic cells become committed to fusion and form multinucleated myotubes (Nadal-Ginard, 1978
). The morphological events in muscle differentiation are associated with the
expression of an array of muscle-specific gene products,
especially structural proteins of the contractile apparatus.
,
and the somatomedins (insulin, insulin-like growth factor-1
[IGF-1], and insulin-like growth factor-2 [IGF-2]) (Florini
and Magri, 1989
; Florini et al., 1991
; Olson, 1992
; Maione and Amati, 1996
). All of them have been recognized to exert a key function in the control of in vitro, and presumably also in vivo, muscle differentiation. Nevertheless, it
cannot be excluded that additional factors exist that contribute to the regulatory pathways governing the myogenic
process.
), could play a role in the differentiation of the
myogenic mouse C2 cell line.
; Rong
et al., 1992
, 1993; Tsao et al., 1993
; Ferracini et al., 1995
;
Woolf et al., 1995
; Maier et al., 1996
). We show that an autocrine loop for HGF/SF is present and active in C2 cells
since the high levels of tyrosine phosphorylation exhibited by met receptor are dependent from the endogenously
produced ligand. Further experiments have produced evidence of the coexpression of HGF/SF and its related receptor also in another myogenic cell line and in mouse primary satellite cells.
). Bladt et al. (1995)
reported that mice homozygous for a null mutation of the met locus fail to form
muscles in the limb anlage, in the diaphragm, and at the tip
of the tongue, because of the inability of myogenic precursor cells to migrate from the somites to these sites. A detailed
work by Yang et al. (1996)
confirms that, in Pax 3-deficient mice, the loss of met gene expression in somitic myogenic
precursors correlates with the lack of limb bud colonization. Similar results were obtained by Maina et al. (1996)
by using mice carrying met receptor variants that are defective in the transduction of HGF/SF signal. In addition,
they reveal a novel role of met kinase also in promoting
the proliferation of fetal myoblasts just before the formation of the secondary fibers during the late stages of muscle development. However, no clear hint about a putative
autocrine condition for HGF/SF in myogenic cell lineage
was given in these works. Rather, a general paracrine control by this growth factor was postulated. Our results appear in disagreement with this interpretation since we
have observed an autocrine loop for HGF/SF both in C2 myoblasts and in mouse primary satellite cells. Since C2
myoblasts were also derived from satellite cells (Yaffe and
Saxel, 1977
), it can be hypothesized that the autocrine
loop for HGF/SF could represent an intrinsic characteristic of satellite cells. According to this assumption, it is expected that the HGF/SF autocrine loop would become established only when satellite cells are induced to replicate
and migrate to where damaged muscle fibers must be replaced. In this regard, a study by Jennische et al. (1993)
points out an induction of HGF/SF gene expression in rat
skeletal-regenerating muscle after ischemic injury. A second element of concordance is that HGF/SF stimulates the
growth of satellite cells otherwise quiescent (Allen et al.,
1995
). It should not be completely excluded that the coexpression of met and HGF/SF genes could occur in the somitic precursors and in other cells belonging to the myogenic lineage without preventing them from responding
also to an external HGF/SF gradient. The finding that an
inappropriate expression of HGF/SF in transgenic mice
causes ectopic muscle formation in the central nervous
system (Takayama et al., 1996
) strengthens the view that a
spatially and temporally regulated HGF/SF signaling would
be required for a proper myogenic process, during both
embryogenesis and muscle regeneration. Work in this field
will be extremely informative for better understanding the
complexity of HGF/SF action in muscle development.
; Florini et al., 1991
; Olson, 1992
). For example, the endogenous expression of FGF and TGF-
, as well as that of their cognate receptors, is downregulated during myogenesis (Ewton et al.,
1988
; Olwin and Hauschka, 1988
; Hu and Olson, 1990
:
Lafyatis et al., 1991
; Moore et al., 1991
).
). However, existing data suggest that the loss of responsiveness to growth factors occurs also at a postreceptor level (Maione and Amati, 1996
).
An opposite kind of regulation has been evidenced for the
myogenic stimulators IGF-1, IGF-2, and their related receptors, whose expression increases coordinately in differentiating cells (Tollefsen et al., 1989a
,b).
; Sonnenberg et al., 1993
). Hence,
HGF/SF could generate a signal interfering with the differentiating program, just like FGF and TGF-
. Research
is in progress to determine the possible role of myogenic
factors in the downregulation of both met and HGF/SF
during C2 differentiation.
(Vaidya et al., 1989
). The mechanisms by which
the activation of the growth factor pathways interfere with
the myogenic program are under extensive investigation
(for reviews see Olson, 1992
; Maione and Amati, 1996
).
). However, a low binding affinity of HGF/SF to heterospecific
receptor molecules has been originally reported by Rong
et al. (1992)
to explain the tumorigenicity induced by met
proto-oncogene in a murine fibroblast cell line. The finding that in C2/h-met clones the human isoform of the receptor is weakly or not at all tyrosine-phosphorylated is in
line with this high species-specificity, since murine HGF/SF
is not efficient in activating h-met.
Received for publication 5 August 1996 and in revised form 10 March 1997.
1. Abbreviations used in this paper: HGF, hepatocyte growth factor; h-HGF/SF, human HGF/SF; h-met, human isoform of met receptor; HS, horse serum; IGF, insulin-like growth factor; LTR, long terminal repeat; MHC, myosin heavy chain; MHCe, embryonic MHC; SF, scatter factor.We are grateful to Dr. Carola Ponzetto for making available the tpr-met oncogene and for valuable suggestions, to Dr. Ermanno Gherardi for the generous gift of anti-mouse HGF/SF serum, and to Dr. Giulio Cossu for providing the mouse primary satellite cells. The help of Angelo Peschiaroli in some of the experiments is also acknowledged.
This work has been supported by grants of the Associazione Italiana Ricerche sul Cancro, Progetto Finalizzato Applicazioni Cliniche della Ricerca Oncologica-Consiglio Nazionale delle Ricerche and Ministero dell'Università e della Ricerca Scientifica e Tecnologica Roma, to P. Comoglio and P. Amati.