Fer Is a Downstream Effector of Insulin and Mediates the Activation of Signal Transducer and Activator of Transcription 3 in Myogenic Cells

Michal Taler, Sally Shpungin, Yaniv Salem, Hana Malovani, Orel Pasder and Uri Nir

Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel

Address all correspondence and requests for reprints to: Professor Uri Nir, Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel. E-mail: nir{at}mail.biu.ac.il.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Fer is an intracellular tyrosine kinase that associates with signal transducer and activator of transcription 3 (Stat3) in mammalian cells. However, the signaling pathways in which this interaction plays a functional role have not been revealed. Herein, we show that insulin up-regulates the levels of the fer mRNA and Fer protein in myoblasts that undergo insulin-induced myogenic differentiation. Moreover, insulin directs the interaction of Fer with members of the Janus family of tyrosine kinases (Jak)-Stat3 signaling pathway. Although in untreated cells Fer binds Jak1 and its tyrosine phosphorylation level is low, insulin treatment induced the phosphorylation of Fer and its dissociation from Jak1.

The up-regulation of Fer and its dissociation from Jak1 were accompanied by an augmented association of activated Fer with Stat3 and by a concomitant increase in the tyrosine phosphorylation of Stat3. Dissociation of Fer and Jak1, as well as elevation in the level of Fer and in the tyrosine phosphorylation of Stat3, depended on the activity of phosphatidylinositol 3-kinase (PI3K) and was abolished by a PI3K inhibitor. However, Fer and Stat3 were only mildly affected by low concentrations of IGF-I, another activator of the PI3K pathway that can also induce myogenic differentiation. RNA interference directed toward the fer mRNA did not affect the cellular levels of Stat3 but led to a dramatic reduction in the tyrosine phosphorylation level of this transcription factor. Thus, Fer is a downstream effector of insulin and mediates the activation of Stat3 in myogenic cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
FER IS AN intracellular tyrosine kinase, which was found to reside both in the cytoplasm and nucleus of mammalian cells (1, 2, 3). Together with c-fes, Fer constitutes a distinct subfamily of intracellular tyrosine kinases that share a unique structure. Both kinases bear an extended N-terminal tail that contains an Fps/Fes/Fer/CIP4 homology domain followed by three coiled-coil forming regions (4). Although the Fps/Fes/Fer/CIP4 homology domain could mediate the association of Fer and c-fes with microtubular structures, the coiled-coil forming regions were shown to direct the oligomerization of these kinases (4). The kinase domain of these enzymes resides at their carboxy-terminal part and is 70% identical in the two proteins (4). Fer is activated by growth factors such as epidermal growth factor and platelet-derived growth factor in fibroblastic cells (5) and by occupation of the Fc{gamma} receptor in mast cells (6). Activation of Fer in these systems could be linked to the modulation of cell-cell and cell-substratum interactions, because Fer was shown to associate with, and phosphorylate, adherence molecules (4, 7, 8, 9). However, the essential role of Fer in the proliferation of malignant cells (10, 11) and its survival factor activity in irradiated pre-T cells (12) suggest its involvement in the growth of mammalian cells.

Fer was also shown to associate with cellular regulatory proteins such as phosphatidylinositol-3 kinase (PI3K) (13), Stat3 (14), and the cytoskeletal linker protein plectin (15). In the case of PI3K, Fer binds the active form of this enzyme in insulin-treated adipocytes (13), suggesting the involvement of Fer in insulin-signaling pathways. However, Fer was not activated upon stimulation of adipocytes with insulin (13).

Stat3 is a downstream effector of both cytokines and growth factors (16). These signaling cascades engage intracellular tyrosine kinases that associate with the activated receptor and thereafter directly phosphorylate Stat3 on tyrosine 705 (17). This leads to the dimerization of Stat3 and to its subsequent translocation to the nucleus, where it induces the expression of defined sets of genes (18). Genes induced by Stat3 encode proteins that are involved in either proliferation (18), differentiation (19), or survival of cells under stress conditions (20).

Insulin and IGF-I were both shown to induce the activation of Stat3. However, whereas the activation of Stat3 in IGF-I signaling is mediated by the intracellular tyrosine kinases JAK-1 and JAK-2 (Janus family of tyrosine kinases 1 and 2) (21), intracellular mediators of the insulin-induced activation of Stat3 have not been identified (22). The engagement of Fer in insulin-signaling pathways, on the one hand (13), and the association of Fer and Stat3, on the other hand (14), prompted us to examine whether Fer could mediate the activation of Stat3 in an insulin-elicited signaling cascade. Fer phosphorylates Stat3 on tyrosine 705 and can consequently activate the binding of that transcription factor to its cognate DNA target sites (14). The association of Fer and Stat3 is regulated by interferon-{gamma}, which stabilizes the mutual association of these two proteins in their inactive states. This implies that stable association of Fer and Stat3 does not depend on the activation and autophosphorylation of that kinase (23).

Muscle cells express relatively high levels of insulin receptor (24), and numerous physiological processes are affected by this hormone and its closely related growth factor, IGF-I, in myogenic cells (25, 26). Both insulin and IGF-I can induce myogenic differentiation of myoblastic cells (27, 28), and both ligands exert this activity through the induction of the PI3K/Akt pathway (29, 30, 31). Involvement of p38-MAPK, but not of p42/p44-MAPK, had also been demonstrated in the myogenic differentiation induced by these two ligands (28, 30).

Myogenic differentiation can be induced also by growth factor withdrawal from myoblast cultures. However, the molecular mechanisms that underlie this process differ from those elicited by insulin or IGF-I in differentiating myoblasts. Although insulin directly induces the expression of myogenic genes via the activation of PI3K (31), mitogen deprivation induces myogenic differentiation by down-regulating the inhibitor of differentiation-Id in myoblastic cells (32).

Although activating common downstream signaling cascades in many cell types, including muscle cells, insulin and IGF-I also elicit specific biochemical and cellular effects (33). Characterization of novel regulatory cascades that are modulated by insulin in muscle cells could therefore have both scientific and clinical implications.

We therefore sought to examine the involvement of Fer in the activation of Stat3 in myoblasts that undergo insulin-stimulated myogenic differentiation. In the current study, we show that Fer mediates the activation of Stat3 in myogenic cells and that this pathway is up-regulated in insulin-driven, but not in growth factor withdrawal or IGF-I-induced, myogenic differentiation of C2C12 cells.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Up-Regulation of Fer in Insulin-Treated C2C12 Cells
To study the effect of insulin on the tyrosine kinase Fer in myogenic cells, C2C12 myoblasts were exposed to 1 µg/ml insulin for different periods of time (34). The insulin-driven differentiation of these cells from myoblasts to myogenic myotubes (35) enabled us to follow the effect of that treatment on Fer in nondifferentiated myoblasts and in differentiating myogenic cells. To dissect effects of insulin from effects of other inducers of myogenic differentiation, C2C12 cells were subjected in parallel to low-serum growth conditions that lead to insulin-independent myogenic differentiation (32). Progression of the myogenic differentiation process was monitored by following the cellular levels of the muscle-specific transcription factor, myogenin (32). The cellular levels of Fer were determined in untreated, actively growing cells, in cells exposed to insulin, and in cells subjected to low-serum growth conditions. Although the level of Fer decreases gradually concomitantly with the progression of myogenic differentiation in C2C12 cells propagated under low-serum growth conditions (Fig. 1AGo), insulin-induced myogenic differentiation was accompanied by a gradual increase in the level of that kinase (Fig. 1BGo). The elevation in the level of Fer began after 24 h of insulin treatment, and it became maximal after 48 h, where it was tripled compared with the basal Fer level in untreated cells (Fig. 1CGo). Appearance of myotubes in the differentiating culture occurred after 48 h of insulin treatment (data not shown). This was preceded by the appearance of myogenin (32) (Fig. 1BGo) and by the activation of the Akt kinase, as was reflected by its serine (473) phosphorylation level (36) (Fig. 1DGo). Both events occurred after 24 h of insulin treatment. Hence, up-regulation of Fer paralleled the activation of the downstream effector of insulin, Akt, and the appearance of early myogenic markers such as myogenin (32). Moreover, the activation of Akt was maximal after 48 h of insulin treatment (Fig. 1DGo) and, overall, the activation profile of Akt resembled the induction profile of the cellular levels of Fer (Fig. 1CGo). The level of Fer did not change after 48 h of insulin treatment and remained constant despite the increase in the myotubes content in cultures treated with insulin for 72 h (data not shown).



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Fig. 1. Insulin Up-Regulates the Level of Fer in C2C12 Cells

Whole-cell lysates were prepared from: A, Untreated C2C12 cells grown in the presence of 15% FCS (lane 1) or cells grown under low-serum (0.5% FCS) for the indicated periods of time (lanes 2–4). B, Untreated cells grown with 15% FCS (lane 1) or cells grown in 10% horse serum (HS) containing 1 µg/ml insulin for the indicated periods of time (lanes 2–4). Proteins were resolved in 7.5% SDS-PAGE and were then reacted with anti-Fer (upper panel) or antimyogenin (lower panel) in a Western blot analysis [immunoblotting (IB)]. C, Quantification of the Fer protein levels in the blots presented in panels A and B. The data represent one of three independent experiments that gave similar results. D, The lysates in panel B were reacted with anti-Akt (upper panel) or with anti-pS-Akt (lower panel), in a Western blot analysis. E, Lysates were prepared from cells that were either grown in the presence of 15% FCS (lane 1) or in the presence of 10% HS for the indicated period of time (lanes 2–4). Proteins were reacted with anti-Fer antibodies in a Western blot analysis.

 
The increase in the level of Fer was driven by the presence of insulin and not by other constituents of the differentiation medium, because horse serum, which substitutes fetal calf serum in the C2C12 differentiation medium, did not, by itself, lead to an elevation in the level of Fer (Fig. 1EGo).

To determine whether insulin up-regulates the expression of Fer at the protein or at the fer RNA level, RNA was extracted from nonstimulated and from C2C12 cells that were treated with insulin for 48 h. The RNA samples were subjected to a quantitative RT-PCR analysis, and the amplification products were resolved in an agarose gel. A significant increase in the level of the fer RNA RT-PCR product could be seen after 48 h of insulin treatment (Fig. 2Go). Thus, the elevation in the level of the Fer protein in insulin-treated C2C12 cells most probably reflects the increase in the accumulation level of the fer transcript. This, by itself, could result from an effect of insulin on the transcription of the FER gene or on the stability of the fer mRNA.



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Fig. 2. Insulin Up-Regulates the Level of the Fer mRNA in Myogenic Cells

A, Total RNA was extracted from C2C12 cells that were grown in 15% FCS (lane 1) or in 10% horse serum (HS) containing 1 µg/ml insulin for 48 h (lane 2). Samples were subjected to quantitative RT-PCR analysis using a pair of primers that are specific for the fer mRNA. These primers amplified the expected 299-bp product (upper panel). The same RNA samples were subjected to a quantitative RT-PCR analysis using specific 18S rRNA primers (lower panel). B, The level of the Fer protein in these experiments was detected by immunoblotting. The data represent one of three independent experiments that gave similar results.

 
An insulin-driven increase in the cellular level of Fer could be accompanied by an up-regulation of the kinase activity. The tyrosine phosphorylation level was found to reflect the activation level of Fer (4). We therefore examined the presence of Fer among tyrosine-phosphorylated proteins in insulin-treated C2C12 cells. Tyrosine-phosphorylated proteins were immunoprecipitated from insulin-treated cells and were then reacted with anti-Fer antibodies. Although the level of tyrosine-phosphorylated Fer was low in untreated cells and in cells exposed to insulin for 24 h, it increased after 48 h and was significantly elevated after 72 h of insulin treatment (Fig. 3AGo). The increase in the specific phosphorylation level of Fer could be clearly seen when the levels of tyrosine-phosphorylated Fer were normalized to the total cellular levels of Fer (Fig. 3BGo).



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Fig. 3. Tyrosine Phosphorylation of Fer in Insulin-Treated C2C12 Cells

A, Whole-cell protein extracts were prepared from untreated C2C12 cells grown in 15% FCS (lane 1) and from cells that were grown in 10% horse serum (HS) containing 1 µg/ml insulin for the indicated periods of time (lanes 2–4). Tyrosine-phosphorylated proteins were immunoprecipitated (IP) from all samples using anti-pY antibodies, and precipitated proteins were resolved in 7.5% SDS-PAGE (upper panel). The original whole-cell extracts were resolved in SDS-PAGE as well (lower panel). Fer was detected with anti-Fer antibodies in a Western blot analysis. B, The autoradiograms in panel A were scanned, and the level of tyrosine-phosphorylated Fer at a defined time point (upper panel in A) was divided by the corresponding cellular levels of that kinase (lower panel in A). The relative pY-Fer-specific activities were plotted. The data represent one of three independent experiments that gave similar results.

 
Insulin Induces the Association of Fer and Stat3 in Myogenic Cells
Fer activates Stat3 by inducing its phosphorylation on tyrosine 705 (14). Moreover, Fer associates with Stat3 in C2C12 cells (14), suggesting that Stat3 is a downstream effector of Fer. We, therefore, examined the effect of insulin on the cellular and activation levels of Stat3 and on the association profiles of Fer and Stat3. Although the level of Fer was up-regulated by insulin (Fig. 1BGo), the cellular levels of Stat3 were down-regulated in insulin-stimulated cells (Fig. 4AGo). However, the tyrosine phosphorylation levels of Stat3, which reflect the activation state of this transcription factor, were significantly elevated in the treated myogenic cells (Fig. 4AGo). To obtain a quantitative estimation of the activation level of Stat3, the relative levels of tyrosine-phosphorylated Stat3 (pY-stat3) were normalized according to the levels of the Stat3 protein. This gave specific activity values of pY-Stat3. The specific activity of pY-Stat3 increased concomitantly with insulin treatment and was highest after 72 h of insulin treatment (Fig. 4CGo). The relative specific activity of pY-Stat3 during myogenic differentiation of serum-starved cells was much lower then the values obtained in insulin-treated cells. Thus, insulin elevates the specific activity of tyrosine-phosphorylated Stat3 in myogenic cells.



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Fig. 4. Activation of Stat3 in Insulin-Treated C2C12 Cells

Whole-cell lysates were prepared from the following. A, Untreated C2C12 cells grown in 15% FCS (lane 1) and from cells grown in 10% horse serum (HS) containing 1 µg/ml insulin for the indicated periods of time (lanes 2–4). B, Untreated cells grown in 15% FCS (lane 1) and cells that were grown under low serum (0.5% FCS) for the indicated periods of time (lanes 2–4). Proteins were reacted with anti-Stat3 (upper panel) or anti-pY-Stat3 (lower panel) antibodies in a Western blot. C, Images in panels A and B were scanned, and the pY-Stat3-specific activities were calculated by dividing the values obtained for the pY-Stat3 levels (lower panel in each figure) by the levels obtained for Stat3 (upper panel in each figure). The data represent one of three independent experiments that gave similar results.

 
The insulin-induced phosphorylation of Fer and Stat3 suggests an effect of the hormone on the interaction between these two proteins. Stat3 was immunoprecipitated from untreated C2C12 myoblasts and from cells exposed to insulin for increasing periods of time. Precipitated proteins were resolved in SDS-PAGE and were then reacted with anti-Fer and anti-Stat3 antibodies in a Western blot analysis. Although the levels of the precipitated Stat3 were lower in insulin-treated cells, the level of Fer associated with Stat3 was significantly augmented after insulin treatment. Maximal association of Fer and Stat3 was seen after 48 h of insulin stimulation, and the complex dissociated to its basal level after 72 h of insulin treatment (Fig. 5AGo, upper panel). A similar profile was obtained when Fer was immunoprecipitated and the levels of coprecipitated Stat3 were detected in a Western blot analysis (Fig. 5BGo, upper panel). Hence, the increase in the tyrosine phosphorylation level of Stat3 was accompanied by an induced association of Fer and Stat3. When the presence of tyrosine-phosphorylated proteins in the Fer-Stat3 complex was analyzed, a band of 94 kDa was detected. This tyrosine-phosphorylated band, which does not comigrate with Stat3, most probably corresponds to the 94-kDa Fer. The tyrosine phosphorylation level of this protein was increased after 48 h of insulin treatment (Fig. 5AGo, lowest panel), suggesting that active Fer associates with Stat3 after the extended treatment of C2C12 cells with insulin. Similarly, when Fer immunoprecipitates were subjected to anti-pYStat3 antibodies, a prominent level of tyrosine-phosphorylated Stat3 was found to be associated with Fer after 48 h of insulin treatment.



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Fig. 5. Insulin Exacerbates the Association of Fer and Stat3 in Myogenic Cells

A, Whole-cell lysates were prepared from untreated C2C12 cells grown in 15% FCS (lane 1) and from cells grown in 10% horse serum (HS) containing 1 µg/ml insulin (lanes 2–4). Stat3 was immunoprecipitated from each sample using anti-Stat3 antibodies. Precipitates were resolved in 7.5% SDS-PAGE and were then reacted with anti-Fer (upper panel), with anti-Stat3 antibodies (second panel), or with anti-pY, in a Western blot analysis. B, The same extracts as in panel A were immunoprecipitated with anti-Fer antibodies and were then reacted with anti-Stat3 (upper panel) or with anti-pY-Stat3 (lower panel) in a Western blot analysis.

 
Dissociation from Jak1 Precedes the Enhanced Association of Fer with Stat3
The insulin-augmented association of Fer and Stat3 prompt us to look for regulated interactions of Fer with other constituents of the Jak-Stat3 signaling pathway. Jak1 and Jak2 are two activators of Stat3 that were both shown to function in muscle cells (20). To follow a possible association of Fer with Jak1, Fer was immunoprecipitated from nontreated and from insulin-treated C2C12 cells. Precipitated proteins were resolved in SDS-PAGE and were then reacted with Jak1 antibodies in a Western blot analysis. Surprisingly, Fer coimmunoprecipitated with Jak1 in nontreated proliferating myoblasts, and the two kinases associated to the same extent after 24 h of insulin treatment (Fig. 6AGo). Fer dissociated from Jak1 after 48 h of insulin treatment, and the two kinases did not coimmunoprecipitate after 48 and 72 h of insulin stimulation (Fig. 6AGo). The shift in the association profile of Fer and Jak1 did not reflect a change in the cellular level or tyrosine phosphorylation level of Jak1, both of which remained relatively constant throughout the exposure of cells to insulin (Fig. 6BGo).



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Fig. 6. Fer Associates with Jak1 in C2C12 Cells

A, Whole-cell protein lysates were prepared from untreated cells grown in 15% FCS (lane 1) and from cells grown in 10% horse serum (HS) containing 1 µg/ml insulin for 24, 48, and 72 h (lanes 2–4). Fer was immunoprecipitated from each sample using anti-Fer antibodies. Precipitates were resolved in SDS-PAGE and were then reacted with anti-Jak1 (upper panel) or anti-Fer antibodies (lower panel), in a Western blot analysis. B, Cellular (upper panel) and tyrosine phosphorylation levels (lower panel) of Jak1 in the original samples were determined using a Western blot analysis.

 
Jak2 was previously found to be involved in the activation of Stat3 in C2C12 cells (21), and we could detect Jak2 before and after insulin treatment (data not shown). However, we did not detect association of Fer with Jak2 in C2C12 cells before and after 24 h of insulin treatment (data not shown).

Thus, Fer associates with Jak1 in nontreated myoblastic C2C12 cells, and the dissociation of these two kinases parallels the association of Fer with Stat3.

PI3K Mediates the Up-Regulation of Fer in Insulin-Treated Cells
PI3K is a key downstream effector of insulin signaling (37). We therefore examined whether activation of PI3K is a mediating step in the insulin-dependent up-regulation of Fer. C2C12 cells were exposed to insulin in the absence or presence of the PI3K inhibitor LY294002 (38). Addition of LY294002 to the cells’ growth medium abolished the above described effects of insulin on C2C12 cells. Insulin stimulation under this condition did not induce the accumulation of myogenin and failed to increase the cellular level of Fer (Fig. 7AGo). Moreover, in contrast to cells treated with insulin alone, the level of Stat3 did not change in the presence of LY294002, and the activation level of Stat3 was decreased during the exposure of cells to insulin (Fig. 7AGo). Thus, PI3K mediates the insulin-induced myogenic differentiation of C2C12 cells, and it is also involved in the activation of both Fer and Stat3, which concomitantly occurs with this differentiation process. The involvement of PI3K in the activation of Stat3, during the myogenic differentiation of C2C12, implicated a role for this kinase in the interaction of Fer with Stat3 and with Jak1. Exposure of C2C12 cells to insulin in the presence of LY294002 antagonized the increase in the association level of Fer and Stat3 after 48 h of insulin treatment (Fig. 7BGo, upper panel). The role of PI3K in mediating the effect of insulin on the interaction between Fer and Stat3 suggested the involvement of that kinase in modulating the interaction between Fer and Jak1, as well. Inclusion of the LY294002 inhibitor in the insulin differentiation medium prevented the dissociation of Fer and Jak1 after 48 h of insulin treatment, and their association levels before and after insulin treatment were similar (Fig. 7BGo, middle panel). The key role of PI3K in the up-regulation of the Fer-Stat3 pathway by insulin could be corroborated by the effect of LY2940032 on the increase in the level of the fer mRNA. Addition of the PI3K inhibitor abolished the induction of the fer mRNA level by insulin (Fig. 7CGo). Hence, activation of PI3K is a key step in the insulin-elicited signaling cascade that led to the up-regulation of the Fer-Stat3 regulatory pathway.



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Fig. 7. Inhibition of PI3K Activity Abolishes the Effect of Insulin on the Fer-Stat3 Pathway

A, Whole-cell lysates were prepared from untreated cells grown in 15% FCS (lanes 1 and 5) and from cells grown in 10% horse serum (HS) containing 1 µg/ml insulin which were either untreated (lanes 2–4 and 9) or treated with the PI3K inhibitor LY294002 (lanes 5–8) for the indicated periods of time. Proteins were resolved in 7.5% SDS-PAGE and were then exposed to anti-Fer (upper panel), anti-Stat3 (middle panel), anti-pY-Stat3 (third panel), or antimyogenin (lowest panel on the right) antibodies in a Western blot analysis. B, Whole-cell lysates were prepared from C2C12 grown in 15% FCS (lane 1) or from cells grown in 10% HS containing 1 µg/ml insulin exposed to the PI3K inhibitor LY294002 for 48 h (lane 2). Proteins were immunoprecipitated with anti-Fer antibodies, resolved in SDS-PAGE, and were then reacted with anti-Stat3 (upper panel), anti-Jak1 (middle panel), or anti-Fer (lowest panel) antibodies in a Western blot analysis. C, Whole-cell RNA was prepared from untreated C2C12 cells grown in 15% FCS (lane 1), and from cells grown in 10% HS containing 1 µg/ml insulin for 48 h, in the presence (lane 2) or in the absence of LY294002 (lane 3). RNA samples were subjected to a quantitative RT-PCR analysis, using Fer-specific primers (upper panel) and 18S rRNA-specific primers (lower panel).

 
IGF-I shares some common signaling cascades with insulin, and PI3K is a common downstream effector of these two factors (37). To test whether IGF-I can also lead to the up-regulation of Fer, C2C12 cells were treated with 15 nM IGF-I for up to 72 h and the level of Fer was determined in the different time points. Unlike 180 nM insulin, 15 nM IGF-I only slightly induced the level of Fer after 72 h; likewise, the level of pY-Stat3 was not significantly increased (Fig. 8Go). The profiles of IGF-I-induced accumulation of myogenin and activation of PI3K differed also from the profiles induced by insulin in the C2C12-treated cells. Although insulin induced the accumulation of myogenin and activation of PI3K after 24 h of treatment (Fig. 1DGo), IGF-I led to the accumulation of myogenin after 48 h (Fig. 8Go, second panel) and to the activation of PI3K only after 72 h of exposure to this growth factor (Fig. 8Go, lowest panel). Thus, the myogenic differentiation processes elicited by insulin and IGF-I in C2C12 cells differ in their kinetics, and the up-regulation of the Fer-Stat3 pathway is specific to treatment with high levels of insulin.



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Fig. 8. IGF-I Does Not Up-Regulate the Level of Fer in C2C12 Cells

C2C12 cells were either grown in 15% FCS (lane 1) or were grown in 10% horse serum (HS) containing 15 nM IGF-I for the indicated periods of time (lanes 2–4). Whole-cell lysates were prepared and resolved in 7.5% SDS-PAGE. Proteins were reacted with anti-Fer (upper panel), antimyogenin (second panel), anti-Stat3 (third panel), anti-pY-Stat3 (fourth panel), anti-Akt (fifth panel), and anti-pS-Akt (lowest panel) antibodies, in a Western blot analysis.

 
Down-Regulation of Fer Decreases the Activation Level of Stat3 in C2C12 Cells
The concomitant activation of Stat3 and its association with Fer suggests that Fer plays a role in the activation of Stat3 in myogenic cells. To directly verify this notion, we sought to specifically down-regulate Fer in C2C12 cells. A synthetic double-stranded small interfering RNA (siRNA) molecule, which should lead to a specific degradation of the fer mRNA, was designed. C2C12 cells were exposed to the fer-siRNA for 48 h, and the effect of that treatment on the level of the Fer protein was followed. Whole-cell protein extracts were prepared from cells treated with siRNA and from untreated cells, and the level of Fer was determined using immunoblot analysis. Exposure of the cells to a fer siRNA reduced the level of the Fer tyrosine kinase (Fig. 9AGo, upper panel). This reduction was specific because the cellular level of Stat3 in the same samples prepared from treated and from untreated cells was similar (Fig. 9AGo, middle panel). However, the activation state of Stat3, as reflected by its tyrosine phosphorylation level, was dramatically compromised in cells subjected to the Fer siRNA interference process (Fig. 9CGo). Thus, down-regulation of Fer leads to a reduced activation of Stat3 in C2C12 cells, a fact that directly proves the involvement of Fer in the activation of Stat3 in these cells.



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Fig. 9. Down-Regulation of Fer Decreases the Activation Levels of Stat3

A, Proteins were extracted from untreated C2C12 cells grown in 15% FCS. Cells were untreated (lane 1), treated with the transfection vehicle metafecten alone (lane 2), or treated with metafecten and siRNA duplexes targeting the fer mRNA for 72 h (lane 3). Proteins were resolved in SDS-PAGE and were then reacted with anti-Fer (upper panel), anti-Stat3 (middle panel), and anti-pY-Stat3 (lower panel) antibodies in Western blot analysis. B, Autoradiograms in the upper and middle panels were scanned and the relative levels of Fer and Stat3 in the different samples were plotted. C, pY-Stat3 levels were determined by scanning, and the pY-Stat3-specific activity was determined by dividing the pY-Stat3 values by the Stat3 cellular level values that were obtained in panel B. The data represent one of three independent experiments that gave similar results.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Fer is a ubiquitously expressed tyrosine kinase that accumulates in most mammalian cells (1). Although present in a wide variety of tissues and cells, the role of Fer has been recently elucidated only in cells that take part in an innate immune response (39). In this study, we showed that insulin treatment leads to an elevation in both the Fer mRNA and protein levels in myogenic cells (Figs. 1Go and 2Go). The up-regulation of Fer by insulin in myogenic cells defines this tyrosine kinase as a downstream effector of insulin and attributes new physiological roles to this kinase.

Insulin induces the myogenic differentiation of C2C12 cells (35), and the level of Fer increased concomitantly with the appearance of myogenic markers, such as myogenin, in the cells. Hence, the up-regulation of Fer could be linked to the myogenic differentiation process that is induced by insulin in the C2C12 myoblasts. However, the up-regulation of Fer is not essential for the differentiation process, because differentiation elicited by other conditions, such as growth-factor withdrawal, did not lead to an increase in the expression of Fer (Fig. 1Go). Insulin was found to be the key inducing component of Fer, and other constituents in the insulin differentiation medium, such as horse serum, did not cause an increase in the level of Fer (Fig. 1Go). Thus, up-regulation of Fer in myogenic cells is specific to the differentiation process elicited by insulin. The insulin-driven increase in the level of Fer could be seen after 24 h, when myogenic markers such as myogenin started to accumulate in the cells. However, the level of Fer became maximal only after 48 h, when myotubes began to be formed in the culture (data not shown). Hence, extended insulin treatment affects the expression of Fer in myogenic, rather then in nondifferentiated, myoblastic C2C12 cells. Insulin affected also the activation state of the Fer tyrosine kinase after extended stimulations of myogenic C2C12 cells. However, the kinase became maximally activated in terminally differentiated myotubes, after 72 h of insulin treatment (Fig. 3Go). These results imply that insulin up-regulates Fer in differentiating myogenic cells by affecting both the expression of the FER gene and the activity of the Fer kinase.

Insulin also directed the association of Fer with constituents of the Jak-Stat3 signaling pathway. Although in proliferating myoblasts Fer was mainly associated with Jak1 (Fig. 6AGo), after 48 h of insulin treatment Fer dissociated from Jak1, became activated, and associated with its putative substrate Stat3 (Fig. 5Go). The coordinated activation of Fer, which occurs concomitantly with its dissociation from Jak1 and its consequent association with Stat3, suggests a regulatory link between these processes. Insulin induces the activation of Fer, a process that could lead to its dissociation from Jak1 and its subsequent interaction with Stat3. Insulin-induced activation of Stat3 in C2C12 cells may, therefore, result from unique regulatory steps that lead to the activation of Fer, which then phosphorylates and activates Stat3.

The enhanced interaction between Fer and Stat3 occurred 48 h after the onset of insulin treatment, in parallel to the appearance of myotubes in the treated cultures (Fig. 5Go). This suggests that insulin modulates the association of Fer and Stat3 and coordinates it with the onset of the myogenic differentiation state. In similarity to the C2C12 myogenic differentiation system, insulin drove the association of Fer with insulin receptor substrate 1 and with activated PI3K in differentiated 3T3-L1 adipocytes (13).

The increased association of Fer and Stat3 after 48 h did not result from an elevation in the cellular level of Stat3, because extended insulin treatment led to a decline in the level of this transcription factor (Fig. 4Go). However, the elevated association of Fer and Stat3 was accompanied by an increase in the level of Fer and its dissociation from Jak1 (Fig. 6AGo).

Although the cellular level of Stat3 declined in insulin-treated cells, the activation state of that downstream effector of Fer was significantly increased after 48 h of insulin treatment. Moreover, Stat3 stayed highly phosphorylated and activated in terminally differentiated C2C12 cells after 72 h of insulin treatment. The induced activation of Stat3 after 48 h of insulin stimulation paralleled its augmented association with an activated Fer (Fig. 5Go) and could result from the interaction between these two proteins. However, whereas Stat3 and Fer remained highly activated after 72 h of insulin treatment (Fig. 3AGo and Fig. 4AGo), their mutual association was markedly reduced at that stage (Fig. 5AGo). The relatively high activation level of Stat3 after 72 h of insulin treatment could therefore reflect high stability of the tyrosine phosphorylation state of Stat3, in terminally differentiated myotubes. It should be noted that unlike interferon-{gamma}, which stabilizes the association of inactive Fer and Stat3 (23), both activated Fer and Stat3 could be found in the complex induced by insulin in myogenic cells (Fig. 5Go).

In C2C12 cells that underwent myogenic differentiation under low-serum growth conditions, the tyrosine phosphorylation level of Stat3 marginally increased after 48 h. It then dropped back, after 72 h of insulin treatment, to the basal level seen in proliferating C2C12 myoblasts (Fig. 4BGo). Thus, the maintenance of a relatively high activation state of Stat3 in myogenic cells is insulin specific.

The significant decline in the tyrosine phosphorylation level of Stat3 in C2C12 cells that were subjected to a fer RNA interference demonstrates the involvement of Fer in the insulin-driven activation of Stat3 in these cells (Fig. 9Go). This experiment strongly suggests that up-regulation of Fer in insulin-treated cells probably leads to the relatively high activation level of Stat3 in differentiated myogenic cells (Fig. 4AGo).

Two major regulatory functions have been attributed to insulin in muscle cells. These are modulation of glucose uptake (25) and induction of cellular hypertrophy (26). The up-regulation of the Fer and Stat3 proteins that can act as survival factors to salvage cells from necrotic and apoptotic stress in thymocytes (12), and in muscle cells (20, 40), endows insulin with a new role in the protection of muscle cells from these death-causing conditions.

Insulin activates a variety of signaling cascades in stimulated cells (33). One of the key downstream effectors of insulin is PI3K, which also mediates the insulin-induced myogenic differentiation in C2C12 cells (Fig. 7AGo). The up-regulation of Fer and the modulation of its interaction with Jak1 and Stat3 were also found to depend on the activation of PI3K (Fig. 7Go, B and C). This suggests that the insulin-induced myogenic differentiation and the activation of the Fer-Stat3 cascade are linked in a regulatory manner.

Insulin-dependent activation of Stat3 was also reported in fibroblastic cells (22). However, no intracellular mediators of this process have been described. Moreover, in contrast to our findings, no involvement of PI3K in the activation of Stat3 was found in those cells (22). These results suggest that the repertoire of intracellular factors that mediate the activation of Stat3 after stimulation by insulin varies from one cell type to another.

IGF-I, which is another activator of PI3K pathways (37), induces myogenic differentiation in C2C12 cells (Fig. 8Go). Moreover, IGF-I was also shown to activate Stat3 in C2C12 cells (21). However, in our comparative study, the activation level of Stat3 that was elicited by 15 nM IGF-I was much lower than the one observed in 180 nM insulin-treated cells (Figs. 4Go and 8Go). The activation of Stat3 by IGF-I was previously shown to be mediated by JAK1 and JAK2 (21). In addition, no significant increase in the level or activation state of Fer could be detected in IGF-I-treated C2C12 cells (Fig. 8Go and data not shown). Hence, activation of Stat3 through up-regulation of Fer in C2C12 cells is specific to high levels of insulin and could involve intracellular signaling pathways that are primarily activated by high levels of insulin and not by low levels of IGF-I, which were used in the current work (33). The specific activation of Fer by insulin and not by IGF-I did not result from the differential activation of IRS1 or IRS2 by these ligands (33), because, in contrast to insulin-stimulated adipocytes (13), Fer did not associate with IRS1 in insulin-stimulated C2C12 cells (data not shown). The activation of the Fer-Stat3 cascade by insulin endows this important hormone with a new physiological role in muscle cells.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Cultures and Treatments
Murine myogenic C2C12 cells were maintained in growth medium DMEM supplemented with 15% fetal calf serum (FCS) (Biological Industries, Beth Haemek, Israel) in 5.5% CO2 at 37 C. The cultures were divided every 3–4 d by removing the cells with a mixture of 0.25% trypsin and 0.05 M EDTA in PBS. For experimental purposes, cells were seeded at 1 x 106 cells per 10-cm tissue culture dish. After 1 d, cultures were shifted to DMEM supplemented with 10% horse serum and 1 µg/ml insulin (Eli Lilly & Co., Indianapolis, IN) or to a low-serum medium: 0.5% FCS in DMEM. For IGF-I treatment, the cultures were shifted to DMEM supplemented with 10% horse serum and 15 nM IGF-I (Roche, Indianapolis, IN). For inhibition of PI3K activity, cells were treated with the PI3K inhibitor, LY 294002 (Calbiochem, La Jolla, CA) at 20 µM in DMEM supplemented with 10% horse serum and 1 µg/ml insulin.

Western Blot Analysis
Whole-cell proteins were extracted in lysis buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 2 mM Na2VO4] and were rotated for 1 h at 4 C. The proteins were cleared by centrifugation at 14,000 x g for 40 min at 4 C. Subcellular fractionation was carried out essentially as described (41). Protein concentration was determined by Bradford analysis. For Western blot analysis, 30 µg protein from each sample were resolved by 7.5% SDS-PAGE. Electroblotted proteins were detected using specific {alpha}FER C2 antibodies, {alpha}Stat3 monoclonal antibodies (Transduction Laboratories, Inc., Lexington, KY), {alpha}pY-Stat3 monoclonal antibodies (directed toward Stat3 phosphorylated on tyrosine705, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), {alpha}-myogenin monoclonal antibodies (BD PharMingen, San Diego, CA), {alpha}Akt, {alpha}pS-Akt ({alpha}-phosphoserine473-Akt), {alpha}Jak1, and {alpha}-phosphotyrosines1022/1023-Jak1 antibodies (Santa Cruz Biotechnology, Inc.). Bound antibodies were visualized using chemiluminescence reaction (Pierce Chemical Co., Rockford, IL).

The blots were scanned using an optical scanner, UMAX Astra 3400, and the optical density of each band was compiled using the TINA-2 software application.

Immunoprecipitation
Extracted proteins (500 µg) were incubated overnight at 4 C with 1:100 diluted {alpha}Stat3 polyclonal antibody (Santa Cruz Biotechnology, Inc.), with 4G-10 {alpha}pY monoclonal antibody (Upstate Biotechnology, Lake Placid, NY), or with polyclonal {alpha}Jak1 antibodies (Santa Cruz Biotechnology, Inc.). Antigen-antibody complexes were precipitated with protein A-Sepharose for 2 h at 4 C and were washed four times with TGET buffer [20 mM Tris HCl, 10% glycerol (pH 7.5), 1 mM EDTA, 0.1% Triton X-100]. The first two washes were carried out with TGET buffer supplemented with 150 mM NaCl, and the last two washes were done with TGET containing 75 mM NaCl. Precipitated proteins were resolved by SDS-PAGE, blotted onto nitrocellulose membranes, and were then reacted with {alpha} FER C2 or with monoclonal {alpha}Stat3 antibodies.

Quantitative RT-PCR Analysis
Total RNA was extracted from C2C12 cells using TRI Reagent (Molecular Research Center, ICN Biochemicals, Inc., Costa Mesa, CA) following the manufacturer’s instructions. Total RNA (25 ng) was reverse transcribed using AccessQuick RT-PCR System (Promega, Madison, WI) with specific oligonucleotides for the mouse fer mRNA: forward (5'-GCT GTG TTA AAG TTG CAA GAC TGG-3') and reverse (5'-CTT CTT CAC TTG CTG CTT GCT CTT-3'). The selected primers are derived from separated exons (1 and 2), which flank an intron of the murine FER gene. PCR was run for 27 cycles which was found to be optimal for quantitative comparison of the fer mRNA levels. The expected 299-bp PCR product is specific to the mature fer mRNA and could be differentiated from genomic DNA contamination PCR products. 18S rRNA primer pairs (Quantum RNA, Ambion, Inc., Austin, TX) were used to yield a 489-bp fragment of 18S rRNA as an internal control. PCR products were separated in 1.4% agarose gel and were visualized with ethidium bromide.

RNA Interference
Synthetic siRNA was designed for targeting sequence in the open reading frame of the murine fer mRNA. siRNA corresponding to nucleotides 63–74 of the murine fer mRNA open reading frame (5'-AAC TAC GGT TGC TGG AGA CAG-3') was used for targeting the murine fer mRNA. The siRNA duplex was synthesized and purified by Pharmacon, Inc. (Warszawa, Poland). Selected siRNA target sequences were also submitted to a BLAST search against other murine genome sequences to ensure target specificity.

C2C12 cells (7 x 104) were seeded in 10-cm tissue culture dishes in growth medium without antibiotics. Transfections were performed with Metafectene (Biontex Laboratories GmbH, Munich, Germany). Metafectene (15 µl) was mixed with Opti-MEM (Life Technologies, Inc., Gaithersburg, MD) for 7 min. This mixture was gently added to a solution containing 22.5 µl of the siRNA duplex in 225 µl Opti-MEM. After 20 min at room temperature, 892.5 µl Opti-MEM and 225 µl FCS were added, and the whole solution containing 300 nM siRNA was laid over the cells. Twelve hours after transfection, cells were supplemented with additional 1.5 ml Opti-MEM containing 15% FCS. Proteins were extracted 72 h after transfection.


    ACKNOWLEDGMENTS
 
We thank Dr. R. Wides for his critical comments on the manuscript, and Mrs. Sharon Victor for typing the manuscript.


    FOOTNOTES
 
This work was supported by grants from CaPCURE—Association for the Cure of Cancer of the Prostate—Israel and from the Weinkselbaum Family Research Fund.

Abbreviations: FCS, Fetal calf serum; Jak, Janus family of tyrosine kinases; PI3K, phosphatidylinositol-3 kinase; {alpha}pS-Akt, {alpha}-phosphoserine473-Akt; pY-Stat3, tyrosine-phosphorylated Stat3; siRNA, small interfering RNA; Stat3, signal transducer and activator of transcription 3.

Received for publication September 17, 2002. Accepted for publication May 2, 2003.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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