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
; 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.
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-β, 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.
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
), could play a role in the differentiation of the myogenic mouse C2 cell line.
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
; 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.
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
). 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
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
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.
We have shown that the expression of HGF
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
; Florini et al., 1991
; Olson, 1992
). For example, the endogenous expression of FGF and TGF-β, 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
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
). 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
The observed HGF
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
; Sonnenberg et al., 1993
). Hence, HGF/SF could generate a signal interfering with the differentiating program, just like FGF and TGF-β. Research is in progress to determine the possible role of myogenic factors in the downregulation of both met
and HGF/SF during C2 differentiation.
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-β (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
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
). 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.
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.