Extracellular Signal-Regulated Kinase-1 and -2 Respond Differently to Mitogenic and Differentiative Signaling Pathways in Myoblasts

Dos D. Sarbassov, Linda G. Jones and Charlotte A. Peterson

Departments of Medicine and Biochemistry and Molecular Biology University of Arkansas for Medical Sciences and the Geriatric Research, Education, and Clinical Center McClellan Veterans Hospital Little Rock, Arkansas 72205


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this report we show that extracellular signal-regulated kinase-1 and -2 (ERK-1 and -2) respond differently to signals that elicit proliferation and/or differentiation of myoblasts using the C2C12 cell line and nondifferentiating mutant NFB4 cells derived from them. Induction of differentiation by withdrawal of serum rendered ERKs in C2C12 myoblasts relatively insensitive to restimulation by serum. Instead, myogenic differentiation of C2C12 cells was associated with sustained activation of ERK-2 dependent on the insulin-like growth factor II (IGF-II) autocrine loop. By contrast, mutant NFB4 cells cultured under the same conditions remained proliferative and demonstrated robust activation of ERKs in response to serum. Similarly, a Gi-dependent signaling pathway induced activation of ERKs in NFB4 cells, but not in C2C12 cells, after stimulation by lysophosphatidic acid (LPA). In NFB4 cells partially rescued by prolonged IGF-I treatment, ERK activity remained responsive to Gi-dependent LPA stimulation, whereas rescue of NFB4 cells by constitutive expression of myogenin or MyoD, associated with activation of the IGF-II autocrine loop, rendered the Gi-signaling pathway refractory to LPA stimulation. Relatively high levels of G{alpha}i2 were detected in NFB4 cells and IGF-I treated NFB4 cells, which correlated with responsive Gi signaling. Activation of the IGF-II autocrine loop in C2C12 and NFB4 myoblasts or treatment with IGF-II was associated with loss of G{alpha}i2 and inhibition of Gi-dependent signaling. Thus, IGF-I and IGF-II activate distinct signaling cascades, with IGF-II eliciting a stronger differentiation effect correlated with down-regulation of G{alpha}i2 protein. Short-term stimulation of NFB4 cells with IGF-I, a mitogenic signal for myoblasts, also induced ERK-1 and -2 activation. Transient stimulation of NFB4 cells with IGF-I while blocking activation of Gi-proteins is with pertussis toxin resulted in preferential activation of ERK-2 characteristic of differentiated C2C12 cells, suggesting that proliferation induced by IGF-I is Gi-dependent and separable from the IGF-I-signaling pathway that leads to differentiation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Many growth factor tyrosine kinase receptors transduce signals to the cytoplasm through a common pathway involving the GTP-binding protein p21ras, thereby leading to activation of sequential protein kinase reactions. This pathway diverges upon activation of extracellular signal-regulated kinases (ERKs), also referred to as mitogen-activated protein kinases (MAPKs) (1, 2). GTP-bound p21ras activates raf kinase by its recruitment to the plasma membrane. Raf phosphorylates and thereby activates MAPK/ERK kinase (MEK), which in turn phosphorylates ERKs on threonine and tyrosine residues that are separated by one amino acid. This dual phosphorylation of ERKs is required for their activation (3, 4, 5). The biological role of ERKs, a typical family of serine/threonine kinases, has been the subject of intensive study. Two members of this family, ERK-1 and -2, are widely expressed and well characterized. Activation of these ERKs is involved in cellular proliferation and differentiation, indicating that downstream cellular responses initiated by ERKs may vary and trigger mutually exclusive events (6, 7, 8). For instance, stimulation of neuronal cells by a variety of growth factors resulted in ERK activation leading to different cellular responses. Insulin-like growth factor-I (IGF-I) and epidermal growth factor appeared to be mitogenic for these cells, whereas neuronal differentiation was induced by basic fibroblast growth factor and nerve growth factor. Cellular proliferation correlated with a transient peak of ERK activity, and sustained activation of ERKs was observed during differentiation (9, 10, 11). These observations indicated that the duration of ERK activity is critical for cell-signaling decisions.

Seven membrane-spanning receptors that interact with Bordella pertussis toxin (PT)-sensitive heterotrimeric G proteins also are able to mediate p21ras-dependent activation of ERKs (12, 13). Receptor activation stimulates nucleotide exchange and dissociation of the G protein, releasing the G{alpha}i subunit in its GTP-bound state from the Gß{gamma} complex. Gß{gamma} complex mediates src-dependent phosphorylation of the epidermal growth factor receptor and thereby activation of the p21ras pathway (14). Employing this mechanism, G protein-coupled receptors can also contribute to p21ras-dependent cellular response.

The role of ERK activity in myogenic growth and differentiation is not well understood. The formation of skeletal muscle in embryogenesis proceeds through commitment of mesodermal progenitors to the myogenic lineage and subsequent differentiation of skeletal myoblasts into terminally differentiated myotubes. Growth factors, such as basic fibroblast growth factor and transforming growth factor-ß, play a central role in maintenance of myoblasts in the proliferative state that is nonpermissive for the expression of muscle-specific genes (15, 16). Basic fibroblast growth factor-stimulated proliferation is accompanied by robust and transient ERK activation (17). Exit from the cell cycle that can be forced by serum withdrawal from the medium induces terminal differentiation. Transition of myoblasts to myotubes is accompanied by activation of the IGF-II autocrine loop and appears absolutely required (Ref. 18; reviewed in Ref.19). The IGF-I receptor is the main mediator of both IGF-I and IGF-II signaling in myoblasts (20), where IGF-I promoted proliferation followed by differentiation, whereas IGF-II demonstrated less mitogenic effect (21). Activation of the IGF-I receptor by binding of IGFs induces tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), the major substrate for insulin and type I IGF tyrosine kinase receptors, and Shc proteins. The Grb-2 adaptor protein links IRS-1, and alternatively Shc proteins, to the p21ras-signaling pathway, resulting in activation of ERK-1 and -2 (22, 23). Recently it was shown that the ERK pathway mediates primarily the proliferative effects of IGF-I on myoblasts (24). In our previous study, we characterized the IGF signal transduction pathway during myogenic differentiation of C2C12 myoblasts and nondifferentiating mutant NFB4 cells that fail to activate the IGF-II autocrine loop and require exogenous IGFs to induce differentiation (25). Exogenous IGF-I partially rescued the mutant phenotype, making these cells useful tools for manipulating the IGF pathway and analyzing downstream signaling components.

In this study we analyzed ERK activity in C2C12 cells during myogenic differentiation and also in nondifferentiating mutant NFB4 cells derived from them by chemical mutagenesis. After exposure of cells to low serum, ERK-1 and -2 in NFB4 cells remained responsive to stimulation by mitogens whereas they were much less responsive in C2C12 cells. Reactivation of ERKs occurred in response to different mitogenic signals and appeared dependent on signaling through Gi proteins. Furthermore, induction of differentiation in both cell types was correlated with preferential and sustained activation of ERK-2. Activation of ERK-1 and -2 was an early response to IGF-I, which could be blocked by PT leading to sustained activation of ERK-2. Thus, the biphasic response of myoblasts to IGF-I, proliferation followed by differentiation, was correlated with specific ERK activity.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ERK Reactivation in Response to Serum Stimulation Is Characteristic of the Mutant NFB4 Phenotype (Fig. 1Go)
To determine whether differences in ERK activity are associated with the nondifferentiated phenotype of NFB4 cells, functional in-gel kinase assays were performed using myelin basic protein as a substrate. As myelin basic protein is a substrate for a variety of serine/threonine kinases, several protein bands, which possessed kinase activity that was not altered after serum stimulation in C2C12 and NFB4 cells, were visualized (Fig. 1AGo). Both C2C12 and NFB4 cell lines demonstrated low kinase activity in the mol wt range of ERK-1 and -2, indicated as p44 and p42, in differentiation medium (DM) low in serum (Fig. 1AGo, lanes 1 and 3). After exposure to DM for 24 h, stimulation of NFB4 cells by medium containing high serum (growth medium, GM) induced activation of the p44 and p42, as well as a 97-kDa kinase (Fig. 1AGo, lane 4; p42, p44, and p97) that might represent a novel kinase of this family, ERK-5 (26). By contrast, ERK activation in response to serum was significantly reduced in C2C12 cells (Fig. 1AGo, lane 2). Similar to NFB4 cells, C2C12 cells blocked from differentiation by stable expression of constitutively active Ras also demonstrated highly inducible ERK activity after stimulation by GM (data not shown).



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Figure 1. ERK Activity, Phosphorylation, and Abundance in C2C12 and NFB4 Cells after Serum Stimulation

Cell extracts from C2C12 (lanes 1 and 2) and NFB4 cells (lanes 3 and 4) incubated in DM for 48 h with (lanes 2 and 4) or without (lanes 1 and 3) stimulation by GM for 10 min were analyzed by in-gel kinase assay (panel A) and Western blot with ERK-1 and –2 (panel B) and phospho-MAPK (panel C) antibodies.

 
To confirm that p44 and p42 were, in fact, ERK-1 and -2, Western blots with an antibody recognizing phosphorylated and nonphosphorylated ERK-1 and -2 were performed. This analysis showed that in C2C12 cells, ERK-2 was approximately 4 times more abundant than ERK-1 (Fig. 1BGo, lanes 1 and 2). A small proportion of ERK-2 appeared to be phosphorylated as detected by a shift in mobility, and this was relatively unaffected by serum stimulation, consistent with in-gel kinase results. ERK-2 abundance in NFB4 cells was comparable to C2C12s (1.5-fold), whereas ERK-1 was 4 times more abundant in NFB4 cells, as abundant as ERK-2 (Fig. 1BGo, lanes 3 and 4). After stimulation by GM, both kinases became phosphorylated in NFB4 cells but to different extents. ERK-1 became completely phosphorylated whereas approximately half of the ERK-2 demonstrated altered mobility.

Phosphorylation of ERK-1 and -2 in C2C12 compared with NFB4 cells was quantitated using a phospho-MAPK antibody specifically selected to recognize only activated (phosphorylated) forms of ERK-1 and -2 (Fig. 1CGo). This antibody reacted strongly with ERK-1 and -2 only in NFB4 cells stimulated by GM (Fig. 1CGo, lane 4). Much less reactivity (~20-fold less) was detectable in unstimulated cells (Fig. 1CGo, lane 3). ERKs were weakly recognized in C2C12 cells, which increased slightly after stimulation by GM (Fig. 1CGo, lanes 1 and 2). This was most apparent for ERK-2 due to its abundance (see Fig. 1BGo). Overall, phosphorylation of ERK-1 and -2 was increased 5-fold in NFB4 compared with C2C12 cells in response to serum stimulation. These data indicated that kinase activity of ERK-1 and -2 was correlated with their phosphorylation state, and that NFB4 myoblasts that continued to proliferate in low serum had ERK activity that was strongly serum inducible.

Sustained Activation of ERKs during Myogenic Differentiation Is Dependent on Activation of the IGF-II Autocrine Loop ( Figs. 2–4GoGoGo)
It was shown above that NFB4 cells that do not differentiate upon serum withdrawal retained highly inducible ERK-1 and -2. Moreover, activated ERK-2 was detectable in differentiated C2C12 cells (Fig. 1Go, B and C, lane 1), suggesting that sustained ERK activation may be involved in myogenic differentiation. This was examined by performing a time course of ERK phosphorylation during differentiation of C2C12 cells by phospho-MAPK Western blot (Fig. 2Go). C2C12 cells have been well characterized, and IGF-II is secreted into the medium within 24 h of exposure to DM, and fully formed myotubes are present within 72 h (19, 20, 25). In GM and early during differentiation of C2C12 cells, low level ERK phosphorylation was observed (Fig. 2Go, top panel, lanes 1 and 2). Phosphorylation of ERK-2 was increased slightly (1.5-fold) by 12 h and within 36 h of incubation in DM, ERK-2 became preferentially phosphorylated (4-fold over GM levels) that was sustained (Fig. 2Go, top panel, lanes 5–7), indicating that activation of ERK-2 was associated with myogenic differentiation of C2C12 cells. ERK-1 phosphorylation also increased slightly with differentiation, although this was less apparent due to low protein abundance. Neither ERK-1 nor ERK-2 abundance changed during differentiation demonstrated with a p44/42 MAPK antibody recognizing phosphorylated and nonphosphorylated ERKs (Fig. 2Go, bottom panel). ERK-2 phosphorylation correlated with tyrosine phosphorylation of IRS-1 in C2C12 cells after 36 h of incubation in DM, dependent on the IGF-II autocrine loop (D. D. Sarbassov and C. A. Peterson, unpublished observations), implying involvement of the IGF signal transduction pathway in sustained activation of ERK-2. To test this idea, ERK activation in C2C12 cells in which the IGF-II autocrine loop was inhibited by IGF-II antisense expression was analyzed (Fig. 3Go). It has been shown that expression of IGF-II antisense oligonucleotides in C2C12 cells blocked myogenic differentiation and induced apoptosis (18). In our study, expression of IGF-II antisense oligonucleotides in C2C12 cells resulted in loss of accumulation of IGF-II precursor protein (Fig. 3AGo, lane 4) compared with control (Fig. 3AGo, lane 3) and vector transfected (Fig. 3AGo, lane 1) cells, similar to the NFB4 phenotype (Fig. 3AGo, lane 2). Furthermore, IGF-II antisense-expressing cells differentiated poorly as indicated by inhibition of myogenin and myosin heavy chain (MyHC) accumulation after exposure to DM (Fig. 3BGo, lane 4), again, similar to the NFB4 phenotype (Fig. 3BGo, lane 2). Treatment of IGF-II antisense-expressing cells with exogenous IGFs restored the differentiated phenotype (Fig. 3BGo, lane 5), as in control C2C12 cells (Fig. 3BGo, lane 3) and vector-transfected cells (Fig. 3BGo, lane 1). Moreover, C2C12 cells expressing IGF-II antisense oligonucleotides were not able to activate ERK-2 in DM (Fig. 3CGo, lanes 1 and 3). Exogenous IGF-I induced a 3-fold increase in phosphorylation of ERK-2 (Fig. 3CGo, lane 4) that required greater than 24 h incubation with the growth factor (Fig. 3CGo, lane 2). Thus, the IGF signal transduction pathway was necessary for activation of ERK-2 during myogenic differentiation of C2C12 cells.



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Figure 2. Sustained Activation of ERKs Occurs during Myogenic Differentiation

Cell extracts from C2C12 cells in GM (lane 1) and after exposure for the indicated times to DM (lanes 2–7) were analyzed by Western blot with phospho-MAPK (top panel) or ERK-1 and -2 (bottom panel) antibodies.

 


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Figure 3. Sustained Activation of ERKs Is Dependent on the IGF-II Autocrine Loop

A, Cell extracts from control C2C12 cells (C, lane 3), C2C12 cells transfected with empty vector (V, lane 1), C2C12 cells expressing IGF-II antisense oligonucleotide (AS, lane 4), and NFB4 cells (lane 2) were analyzed by Western blot with an antibody recognizing IGF-II precursor protein. B, Cell extracts from C2C12 cells (C, lane 3), C2C12 cells transfected with empty vector (V, lane 1), C2C12 cells expressing IGF-II antisense oligonucleotide (AS, lane 4), NFB4 cells (lane 2), and antisense-expressing C2C12 cells treated with IGF-I (15 ng/ml, lane 5), were analyzed by Western blot with MyHC and myogenin antibodies simultaneously. All cells in panels A and B were cultured 48 h in DM. C, Cell extracts from C2C12 cells expressing IGF-II antisense oligonucleotide incubated for the indicated times in DM ± IGF-I (15 ng/ml) were analyzed by Western blot with phospho-MAPK antibody.

 


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Figure 4. Blocking Sustained ERK Phosphorylation by Inhibition of MEK1 Activity Interferes with Myogenic Differentiation

A, Cell extracts from C2C12 cells incubated for 36 h in DM with (+) or without (-) the MEK1 inhibitor PD98059 were analyzed by Western blot with phospho-MAPK (top panel) or myogenin (bottom panel) antibodies. B, Cells treated as in panel A were analyzed immunocytochemically for MyHC accumulation.

 
To determine whether ERK phosphorylation in response to autocrine IGF-II was required for differentiation, the inhibitor PD98059 that prevents activation of MEK, thereby inhibiting phosphorylation and activation of ERKs, was used (Fig. 4Go). After 36 h exposure to DM + PD98059, ERK phosphorylation was significantly inhibited in C2C12 cells (Fig. 4AGo, top panel), whereas ERK accumulation was unaffected by the inhibitor (data not shown). PD98059 treatment appeared to be detrimental to the differentiation process. Although the expression of myogenin was unaffected (Fig. 4AGo, bottom panel) and myotubes did form, they did not survive (Fig. 4BGo). In the presence of the inhibitor, the cells rounded up and detached from the plate compared with untreated cells, suggesting that sustained ERK phosphorylation is required for cell survival during differentiation.

Differential ERK Activation in Response to Gi-Dependent Signaling Pathways ( Figs. 5–7GoGoGo)
ERK activation in NFB4 cells in response to serum was also produced by specific signaling molecules. Lysophosphatidic acid (LPA) acts through its cognate receptor that interacts with PT-sensitive heterotrimeric G proteins (13, 28, 29, 30). LPA activated ERKs in NFB4 cells but not in C2C12 cells assayed by the in-gel kinase assay (Fig. 5Go, compare lanes 1 and 2). Under these conditions activation of ERK-1 (p44) was more pronounced. Blocking the Gi-dependent pathway by PT in NFB4 cells inhibited activation of ERKs by LPA (Fig. 5Go, lane 3). These data suggested that activation of ERKs in NFB4 cells occurred through a Gi-dependent pathway. Taken together with the results described above, it appears that increased phosphorylation of ERK-1 relative to ERK-2 is associated with proliferation, whereas high levels of phosphorylated ERK-2 relative to ERK-1 is characteristic of differentiation.



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Figure 5. ERK Activity Is Responsive to Gi-Dependent Signaling in NFB4 Cells

Cell extracts from C2C12 (lane 1) and NFB4 (lanes 2 and 3) cells in DM with (lane 3) and without (lanes 1 and 2) PT preincubation were stimulated by LPA for 10 min and analyzed by in-gel kinase assay.

 


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Figure 6. Distinct Responses to Gi-Dependent Pathways in Partially Rescued NFB4 Cells

In-gel kinase assays of cell extracts from IGF-I treated NFB4 cells (lanes 1–3) and cells expressing myogenin (lanes 4–6) or MyoD (lanes 7–9) stimulated for 10 min by GM (lanes 2, 5, and 8) or LPA (lanes 3, 6, and 9).

 


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Figure 7. Abundance of G{alpha}i2 Protein and Its Correlation with Myogenic Differentiation

A, Western blot with G{alpha}i1-2 antibody of cell extracts from C2C12 cells (lane 1), untreated NFB4 (lane 2), IGF-I-treated (lane 3), or IGF-II-treated (lane 6) NFB4 cells, and NFB4 cells expressing myogenin (lane 4) or MyoD (lane 5). All cells were incubated in DM for 24 h. IGF-I was used at a concentration of 50 ng/ml and IGF-II at 100 ng/ml, because of differences in their affinity to the IGF-I receptor (21, 39). B, Cellular extracts from NFB4 (lane 1) and IGF-I-treated (lane 2) or IGF-II-treated (lane 3) NFB4 cells mentioned in panel A were analyzed by Western blot with myogenin antibody.

 
In parallel, we analyzed activation of ERKs in partially rescued NFB4 cells by IGF-I treatment or by overexpressing myogenin (NFB4/myogenin) or MyoD (NFB4/MyoD). Mutant NFB4 cells fail to accumulate significant levels of either transcription factor or activate IGF-II expression (25). Prolonged treatment of NFB4 cells with IGF-I resulted in activation of the myogenin gene in the absence of significant endogenous IGF-II expression, whereas overexpression of myogenin and MyoD in these cells resulted in activation of the IGF-II autocrine loop (25). IGF-I treated NFB4, NFB4/myogenin, and NFB4/MyoD cells incubated in DM demonstrated low ERK activity (Fig. 6Go, lanes 1, 4, and 7). Stimulation of IGF-I treated NFB4 cells by either GM or LPA strongly induced activation of ERKs (Fig. 6Go, lanes 2 and 3), whereas ERKs in NFB4/myogenin and NFB4/MyoD cells were responsive only to GM (Fig. 6Go, lanes 5 and 8) but not to LPA (Fig. 6Go, lanes 6 and 9). Thus, NFB4 cells rescued by direct IGF-I treatment and those rescued by myogenin or MyoD expression differed in their response to LPA. In IGF-I-rescued NFB4 cells, ERKs, in particular ERK-1, were still responsive to the Gi-dependent pathway, whereas in myogenin or MyoD-transfected cells this pathway was inhibited.

The mitogenic effect of LPA involving activation of ERKs is mediated through its receptor coupled to G{alpha}i2 protein. It might be that activation of ERKs through a Gi-dependent pathway was dependent on the abundance of G{alpha}i2 protein in C2C12 and NFB4 cells. Similar levels of G{alpha}i1 protein were detected in C2C12, NFB4, and rescued NFB4 cells, whereas the levels of G{alpha}i2 were different (Fig. 7AGo). A low level of G{alpha}i2 was found in C2C12 cells (Fig. 7AGo, lane 1), and a 10-fold higher level of this protein was detected in NFB4 cells relative to G{alpha}i1 (Fig. 7AGo, lane 2). Northern analysis also revealed overexpression of G{alpha}i2 mRNA in NFB4 cells compared with C2C12 cells (data not shown). IGF-I-rescued NFB4 cells (Fig. 7AGo, lane 3) continued to accumulate G{alpha}i2 whereas the protein was 3 times less abundant in NFB4/myogenin and NFB4/MyoD cells relative to G{alpha}i1 (Fig. 7AGo, lanes 4 and 5). Thus, the abundance of G{alpha}i2 correlated with the ERK response to LPA stimulation.

We and others showed that IGF-I treatment inhibits activation of the IGF-II autocrine loop in myoblasts (25, 31). Activation of the IGF-II autocrine loop is normally associated with myogenic differentiation of C2C12 cells and was also detected in NFB4/myogenin and NFB4/MyoD cells (25), implying that the IGF-II-signaling pathway may lead to down-regulation of G{alpha}i2. This appears to be the case as incubation of NFB4 cells with exogenous IGF-II induced down-regulation of G{alpha}i2 by 4-fold relative to G{alpha}i1 (Fig. 7AGo, lane 6). These results suggest that NFB4 cells respond differently to long-term exposure to IGF-I vs. IGF-II.

To determine whether the different effects of IGF-I and IGF-II on the level of G{alpha}i2 protein correlated with induction of myogenic differentiation in NFB4 cells, we analyzed expression of myogenin. Myogenin accumulated to very low levels in NFB4 cells (Fig. 7Go, lane 1). Incubation of NFB4 cells with IGF-I induced expression of myogenin (Fig. 7BGo, lane 2), whereas a higher level of myogenin was detected in NFB4 cells incubated with IGF-II (Fig. 7BGo, lane 3). Thus, IGF-II appeared to be a stronger myogenic factor, compared with IGF-I, consistent with previous reports (21, 25).

PT Alters ERK Activity in Response to IGF-I (Fig. 8Go)
Rapid phosphorylation of ERKs in response to serum stimulation and LPA treatment that was preferential for ERK-1 was characteristic of the proliferative phenotype of the mutant NFB4 cells, whereas sustained activation of ERK-2 was associated with IGF-dependent myogenic differentiation. These observations suggest that ERK activity in NFB4 cells may be an indicator of the biphasic effects of IGF-I on myoblasts: proliferation is the early response followed by differentiation (21). This idea was tested by analyzing ERK activity in NFB4 cells incubated in DM and stimulated by IGF-I for 10 min. Low level phosphorylation of ERKs was detected in DM by the phospho-MAPK antibody (Fig. 8Go, lane 1). Short-term IGF-I stimulation induced activation of both ERK-1 and -2, whereas LPA stimulation resulted in preferential activation of ERK-1 (Fig. 8Go, compare lanes 2 and 4; see also Fig. 5Go). This effect was abrogated by preincubation with PT (Fig. 8Go, lane 5). Surprisingly, in cells preincubated with PT, IGF-I induced preferential phosphorylation of ERK-2 relative to ERK-1 (Fig. 8Go, lane 3). Thus, in response to IGF-I, high levels of G{alpha}i2 protein in NFB4 cells were linked with activation of ERK-1 and -2. Blocking Gi signaling by PT resulted in preferential activation of ERK-2, similar to the pattern of ERK phosphorylation in differentiated C2C12 cells treated with IGF-I (Fig. 8Go, lane 6). These results suggest that early IGF-I signaling events associated with proliferation can be altered by blocking activation of G{alpha}i proteins, thereby mimicking signaling events occurring during myogenic differentiation dependent on the IGF-II autocrine loop.



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Figure 8. Alteration of the IGF-Signal Transduction Pathway by PT in NFB4 Cells

Cell extracts from C2C12 (lane 6) and NFB4 cells (lanes 1–5) incubated in DM for 24 h with (lanes 3 and 5) or without (lanes 1, 2, 4, and 6) PT preincubation stimulated by IGF-I (lanes 2, 3, and 6) for 10 min were analyzed by Western blot with phospho-MAPK antibody.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this study we showed an interplay of IGF-signaling pathways with Gi proteins that appeared to be relevant in the activation of ERKs and in the control of myoblast proliferation and differentiation. We characterized activation of ERKs by high serum in NFB4 cells that remained proliferative in DM, and in C2C12 cells that withdraw from the cell cycle and differentiated under similar conditions. Dramatic activation of ERKs was demonstrated for NFB4 cells after stimulation, consistent with reports that these kinases remain responsive in other cell types with a proliferative phenotype (6, 7, 11). A modest activation of ERK-2 was shown in differentiated C2C12 cells stimulated by high serum. In both C2C12 and NFB4 cells, ERK activation by serum correlated with the relative abundance of the proteins: ERK-1 and -2 accumulated to high, comparable levels in NFB4 cells, whereas C2C12 cells demonstrated a higher abundance of ERK-2 compared with ERK-1. Phosphorylation was also correlated with abundance in the sustained activation of ERKs associated with myogenic differentiation in C2C12 cells. Sustained activation of ERKs has previously been observed during differentiation of neuronal cells (9, 11). Inhibition of MEK activation during differentiation interfered with sustained ERK phosphorylation and differentiation consistent with the conclusion that ERKs play a positive role during differentiation of C2C12 cells (32). However, as myogenin gene expression was unaffected by inhibition of ERK phosphorylation, it appears that sustained activation of ERKs is necessary to maintain cells in a viable state, allowing differentiation to proceed, rather than initiating the process. In any case, these results are in apparent disagreement with those of Coolican et al. (24), who recently reported that PD98059 accelerates myogenic differentiation in L6A1 cells in response to exogenous IGF-I. The most likely explanation is that the signaling pathways activated by autocrine IGF-II in C2C12s myoblasts in the experiments described here are not identical to those activated in response to exogenous IGF-I in L6A1 myoblasts (Ref. 21; also discussed below) resulting in a different cellular response to inhibition of ERK phosphorylation during differentiation.

Activation of the IGF-II autocrine loop is required for myogenic differentiation of C2C12 cells, and IGFs are survival factors for myoblasts (18, 19). NFB4 cells fail to activate IGF-II gene expression upon serum withdrawal, are unable to differentiate, and do not demonstrate activation of ERK-2 in DM. Inhibition of IGF-II accumulation in C2C12 cells also resulted in a nondifferentiated phenotype and no ERK-2 activation. Exogenous long-term IGF-I treatment was able to induce differentiation and concomitant activation of ERK-2. By contrast, a Gi-signaling pathway was activated in NFB4 cells but not in C2C12 cells in response to LPA leading to preferential ERK-1 activation. LPA acts through its cognate receptor that is coupled with G{alpha}i2 protein, and the level of G{alpha}i2 protein correlated with PT-sensitive activation of ERK-1. A low abundance of this protein was demonstrated in C2C12 and a high abundance in NFB4 cells, suggesting that G{alpha}i2-signaling contributes to the proliferative phenotype of NFB4 cells. Thus, a high ratio of phosphorylated ERK-2/ERK-1 is associated with myogenic differentiation, whereas a high ratio of phosphorylated ERK-1/ERK-2 is associated with proliferation. These results suggest that ERK-1 and -2 may be functionally distinct. The mechanism whereby these kinases may participate in different cellular processes is unknown but may involve different substrates or different subcellular localization.

Although the IGF-I receptor is the main mediator of IGF-I and IGF-II, the effects of these factors on myogenic differentiation were not identical. It has been reported previously that IGF-I has a greater mitogenic effect and IGF-II is more myogenic (21). IGF-I-treated NFB4 cells remained responsive to LPA stimulation, whereas NFB4/myogenin and NFB4/MyoD cells, able to activate the IGF-II autocrine loop, demonstrated nonresponsiveness to stimulation by LPA. Thus, rescue of the mutant phenotype by IGF-I treatment or by indirect activation of the IGF-II autocrine loop by myogenin or MyoD expression were distinct. This is confirmed by the fact that in NFB4/myogenin, NFB4/MyoD, and NFB4 cells directly treated with IGF-II (but not IGF-I), G{alpha}i2 protein was down-regulated. Specific coupling of the IGF-II receptor with G{alpha}i2 protein has been reported (33). Long-term activation of G{alpha}i2 signaling by the IGF-II receptor may result in down-regulation of G{alpha}i2 protein. IGF-I was not able to down-regulate G{alpha}i2 protein possibly due to its low affinity to the IGF-II receptor. This effect of IGF-II may also be linked to the putative atypical IGF-I receptor associated only with myogenic differentiation of myoblasts that preferentially binds IGF-II (34). Thus, activation of the IGF-II autocrine loop associated with myogenic differentiation of C2C12 cells and NFB4 cells by expression of myogenin or MyoD demonstrated low-level G{alpha}i2 protein accumulation correlated with nonresponsiveness of these cells to PT-sensitive activation of ERKs by LPA. The stronger myogenic effect of IGF-II vs. IGF-I might be linked with the ability of IGF-II to down-regulate one of the proliferative pathways associated with G{alpha}i2-signaling.

Signaling through the IGF-I receptor was altered by PT, suggesting that Gi-proteins are involved in this process. It has been shown in other cell types that blocking Gi-dependent signaling interfered with activation of ERKs by the IGF-I signal transduction pathway (33, 35). Stimulation of NFB4 cells that overexpress G{alpha}i2 protein by IGF-I induced activation of ERK-1 and -2. Similar stimulation of NFB4 cells while blocking the activity of Gi proteins with PT altered IGF signaling, resulting in preferential activation of ERK-2 that mimicked ERK-2 activation during normal differentiation of C2C12 cells. Thus, the dual effects of IGF-I on myoblasts, induction of proliferation and differentiation, appear to be mediated by different signaling pathways that result in distinct patterns of ERK activation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture and Transfection
NFB4 is a subclone of the nondifferentiating NFB cell line originally derived from the C2C12 mouse muscle cell line (25, 36). Both cell lines were grown in serum-rich growth medium (GM): DMEM (GIBCO BRL, Gaithersburg, MD) supplemented with 10% FBS and 10% defined bovine serum (all serum from Hyclone, Logan, UT) at 37 C in a humidified 10% CO2, 90% air atmosphere. Confluent cells were washed with serum-free DMEM and maintained in DM: DMEM plus 2% horse serum. For IGF-I (provided by Elena Moerman, University of Arkansas for Medical Sciences, Little Rock, AR) or IGF-II (obtained from PeproTech, Rocky Hill, NJ) treatment, cells were maintained in DMEM containing 0.4% horse serum plus IGF-I or IGF-II at the indicated concentrations added fresh daily. For short-term IGF-I stimulation, IGF-I was added at 150 ng/ml for 10 min. LPA (Sigma Chemical Co., St. Louis, MO) was added in serum-free DMEM at a concentration of 20 µM for 10 min with crystalline BSA (Calbiochem, San Diego, CA) at 0.5 mg/ml. PT (List Biological Laboratories, Campbell, CA) was applied in the medium at a concentration of 100 ng/ml and preincubated for 2 h before each experiment. The MEK1 inhibitor PD98059 (New England Biolabs, Inc., Beverly, MA) was applied in DM at a concentration of 50 µM and was added fresh every 24 h as recommended by the manufacturer.

Introduction of a pEMSVscribe{alpha}2/IGF-II antisense expression plasmid (18) or empty vector into C2C12 cells was performed by the calcium phosphate coprecipitation method as described (36). C2C12 cells were plated at 1.5 x 105 cells/100-mm tissue culture plate and 24 h later, cells were washed and cotransfected with 10 µg of a pEMSVscribe{alpha}2/IGF-II antisense expression plasmid together with 1 µg pSV2neo. The DNA was removed 24 h later with the addition of fresh GM. After an additional 24 h, cells were split 1:4 and refed with medium to which 400 µg/ml of G418 (Geneticin, GIBCO/BRL, Gaithersburg, MD) had been added. Selection proceeded for 14 days and G418-resistant clones were picked randomly. Pooled clones of NFB4 cells constitutively expressing MyoD or myogenin have been described previously (25).

Western Blot Analysis
After incubation and stimulation of cells, 100-mm dishes were washed twice with cold PBS and lysed in 0.5 ml cold lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 10 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 3 µg/ml of each leupeptin and aprotinin, 6 µg/ml of antipain, 30 µg/ml of benzamidine, 1 mM Na3VO4, 1% Triton X 100) for 10 min by shaking. All manipulations of cell lysates were at 4 C. Lysates were scraped into microcentrifuge tubes and cleared of nuclei and detergent-insoluble material by centrifugation for 10 min at 14,000 rpm. Samples (35 µg of protein) were resolved by discontinuous electrophoresis through 7.5% SDS polyacrylamide gels and electrophoretically transferred to PVDF membrane (Immobilon P, Millipore, Bedford, MA). Blots were blocked for 1 h in 5% milk in PBS plus 0.5% Tween-20 (PBST). Phospho-MAPK and ERK-1 and -2 antibodies (New England Biolabs) were applied at a 1:1000 dilution in PBST containing 3% BSA (heat shock treated, Fisher, Pittsburgh, PA) overnight at 4 C. G{alpha}i1 and G{alpha}i2 (Calbiochem, San Diego, CA), myogenin (hybridoma F5D, Dr. Woodring Wright, University of Texas Southwestern Medical Center, Dallas, TX), MyHC (A4.1025, 25), and IGF-II (sc-1417, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) antibodies were applied at 1:1000 dilution in PBST containing 3% milk and incubated for 1 h. Blots were washed 5 times with PBST for a total of 30 min, before and after incubating with horseradish peroxidase-conjugated secondary antibody (Pierce, Rockford, IL) diluted 1:4000 in PBST containing 4% milk for 1 h. Renaissance Chemiluminescence Reagent (DuPont/NEN, Wilmington, DE) was used as the detection system. Signals were quantitated using a computing densitometer with LasarQuant software (Molecular Dynamics, Sunnyvale, CA). Blots were stripped and reprobed as described (37).

Immunocytochemistry
Immunocytochemical analyses were performed essentially as described (25). Briefly, cells were washed in PBS, fixed with 1% paraformaldehyde, and then treated with ice-cold methanol. Cells were incubated with undiluted MyHC A4.1025 hybridoma tissue culture supernatant for 1 h at room temperature followed by incubations with horseradish peroxidase-conjugated anti-mouse IgG. Peroxidase reactivity was visualized using the DAB substrate kit (Vector Laboratories, Burlingame, CA).

In-Gel Kinase Assay
The in-gel kinase assay was performed essentially as described (38). Cell lysates were boiled in Laemmli sample buffer for 2 min and resolved by discontinuous electrophoresis through 7.5% SDS polyacrylamide gel containing 0.5 mg/ml myelin basic protein (Sigma). The gel was fixed by four washes with 20% 2-propanol in buffer A (50 mM Tris-HCl buffer, pH 8.0, containing 2 mM dithiothreitol) for 2 h, and SDS was removed by washing the gel for 2 h in several volumes of buffer A, changing the solution every 15 min. Proteins in the gel were denatured in 6 M guanidine HCl for 2 h in buffer A and then renatured overnight at 4 C in buffer A containing 0.04% Tween 40 (Sigma). After preincubation of the gel for 1 h in 5 ml of 40 mM HEPES, pH 8.0, containing 2 mM dithiothreitol and 10 mM MgCl2, the kinase reaction was carried out by incubation of the gel for 1 h at 25 C in 5 ml of 40 mM HEPES, pH 8.0, containing 25 µCi of [{gamma}-32P]ATP, 40 µM ATP, 0.5 mM EGTA, 2 mM dithiothreitol, and 10 mM MgCl2. After the kinase reaction the gel was washed several times in 5% (wt/vol) trichloroacetic acid containing 1% pyrophosphate until the radioactivity reached background levels. After washes, the gel was dried and exposed to x-ray film.


    ACKNOWLEDGMENTS
 
We thank Dr. Peter Rotwein for providing pEMSVscribe{alpha}2/IGF-II antisense expression plasmid, Dr. Woodring Wright for the myogenin hybridoma, Elena Moerman for IGF-I, and Jane Taylor-Jones for assistance with artwork.


    FOOTNOTES
 
Address requests for reprints to: Charlotte A. Peterson, Veterans Affairs Hospital, Research 151, 4300 West 7th Street, Little Rock, Arkansas 72205.

This work was supported by grants from the National Institutes of Health-National Institute on Aging (to C.A.P).

Received for publication May 27, 1997. Revision received September 8, 1997. Accepted for publication September 23, 1997.


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