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Centro de Estudios Moleculares de la Célula and Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Casilla 70005, Santiago 6530499, Chile
Address all correspondence and requests for reprints to: Dr. Enrique Jaimovich, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Independencia 1027, Casilla 70005, Santiago 6530499, Chile. E-mail: ejaimovi{at}machi.med.uchile.cl.
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Introduction |
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Materials and Methods |
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Cell cultures
Rat myotubes were cultured as reported previously (27). Briefly, myoblasts were obtained from rat neonatal hind limbs, and the tissue was mechanically dispersed and then treated with 10% (wt/vol) collagenase for 15 min at 37 C under mild agitation. The suspension was filtered through a Nytex (Sartorius, Goettingen, Germany) membrane and spun down at low speed, preplating was used to partially eliminate fibroblasts, and finally, cells were plated onto dishes (60 mm) at a density of 1.2 x 106/dish. The culture medium was DMEM/F-12 without phenol red, 10% bovine serum, 2.5% fetal calf serum, 100 mg/liter penicillin, 50 mg/liter streptomycin, and 2.5 mg/liter amphotericin B. To eliminate remaining fibroblasts, 10 µM cytosine arabinoside was added on the third day of culture for 24 h. The medium was then replaced with serum-free medium. Myotubes with an estimated purity of more than 90% were visible after the fifth day of culture. Unless indicated, we used 6- to 8-d-old cultures exhibiting a fairly homogeneous population of myotubes with central nuclei and measuring 200–300 µm long and 20–40 µm wide, corresponding to young, not fully differentiated cells.
Intracellular calcium measurement
Cytosolic calcium images were obtained from single noncontracting myotubes preloaded with Fluo-3-AM using an inverted confocal microscope (Axiovert 135M, LSM Microsystems, Carl Zeiss, New York, NY) or a fluorescence microscope (T041, Olympus Corp., New Hyde Park, NY) equipped with a cooled CCD camera and image acquisition system (MCD 600, Spectra Source Instruments, Westlake Village, CA). Myotubes were washed three times with Krebs buffer [145 mM NaCl, 5 mM KCl, 2.6 mM CaCl2, 1 mM MgCl2, 10 mM HEPES-sodium, and 5.6 mM glucose (pH 7.4)] to remove serum and loaded with 5.4 µM Fluo-3-AM (coming from a stock in 20% pluronic acid-dimethylsulfoxide) for 30 min at room temperature. After loading, myotubes were washed with the same buffer and used within 2 h. The cell-containing coverslips were mounted in a 1-ml capacity plastic chamber and placed in the microscope for fluorescence measurements after excitation with a 488-nm wavelength argon laser beam or filter system. Hormones were added directly to the medium, or the solution was fast changed in the chamber (1 sec). The fluorescent images were collected every 0.4–2.0 sec for fast signals and were analyzed frame by frame with the image data acquisition program (MCD 600, Spectra-Source) of the equipment. An objective lens PlanApo x40 (numerical aperture, 1.4) was generally used. In most of the acquisitions, the image dimension was 512 x 120 pixels. The inhibitors were added during the dye incubation; times and concentrations are indicated in Results. To assess the role of G proteins, myotubes were incubated either with 1 µg/ml PTX for 5 h or in a permeabilization solution [100 mM KCl, 20 mM NaCl, 5 mM MgSO4, 1 mM NaH2PO4, 25 mM NaHCO3, 3 mM EGTA, 1 mM CaCl2, 20 mM Tris-HCl (pH 7.4), 0.1% BSA, 1 mM ATP, 0.1% glucose, and 40 mg/ml saponin] for 5 min in the presence or absence of 100 nM GDPßS, a nonhydrolyzable analog of GDP, before hormone stimulation. Intracellular calcium was expressed as a percentage of fluorescence intensity relative to basal fluorescence, which was stable for at least 5 min during resting conditions. The increase in fluorescence intensity is proportional to the rise in intracellular calcium level (28).
Digital image processing
Elimination of out of focus fluorescence was performed by software. Both "no-neighbors" deconvolution algorithm and Castleman’s point spread function theoretical model were used. Complementary to restoration methods, a procedure was created to segment the images. To section an image, an initial contour can be entered manually, and a recursive algorithm that adapts automatically to the region of interest (adaptable contour) can be applied (6). To quantify fluorescence, the summed pixel intensity was calculated for the section delimited by a contour. As a way of increasing the efficiency of these data manipulations, action sequences were generated. To avoid possible interference in the fluorescence by changes in volume after exposure to steroids, the area of fluorescent cell was determined by image analysis using adaptive contour and then creating a binary mask, which was compared with a brightfield image.
Determination of IP3 levels
IP3 levels were determined as described previously (27). Briefly, a crude rat cerebellum membrane preparation was obtained after homogenization in 50 mM Tris-HCl (pH 7.7), 1 mM EDTA, 2 mM ß-mercaptoethanol, and centrifugation at 20,000 x g for 15 min. This procedure was repeated three times, resuspending the final pellet in the same solution plus 0.3 M sucrose and freezing it at -80 C until use. The membrane preparation was calibrated for IP3 binding with 1.6 nM [3H]IP3 and 1–120 nM cold IP3, carrying out the sample analysis in a similar way, but adding an aliquot of the neutralized supernatant instead of cold IP3. The remaining membrane-bound [3H]IP3 radioactivity was measured by liquid scintillation.
Immunocytochemistry
Intracellular androgen receptor was localized using indirect immunofluorescence. Myotubes (control and testosterone stimulated) were washed and then fixed with 100% methanol at -20 C for 20 min and treated with a blocking solution of 1% BSA in PBS for 30 min. Cells were incubated with the primary polyclonal antiintracellular androgen receptor antibody (1:100) overnight at 4 C. Later, myotubes were washed in PBS and incubated with fluorescein isothiocyanate-conjugated goat antirabbit IgG diluted 1:200 for 2 h at room temperature. Cells were washed, and Vectashield (Vector Laboratories, Inc., Burlingame, CA) was added to prevent bleaching. Myotubes were examined with a confocal microscope (135-M LSM Microsystems, Carl Zeiss). Controls were performed both without the primary antibody and by displacement with the antigenic peptide as a test for specificity.
ERK1/2 phosphorylation by steroids and Western blot
Myotubes were grown on 60-mm plates and serum-starved for 24 h before being exposed to steroids for various times, as indicated in Results and the figure legends. Cells were washed with ice-cold PBS and scraped off plates in 70 µl harvesting lysis buffer [150 mM NaCl, 1 mM EGTA, 1% Nonidet P-40, 50 mM Tris-HCl (pH 7.4), 5 mM sodium orthovanadate, 20 mM NaF, 1 µg/ml aprotinin, 1 µM pepstatin, 20 µM leupeptin, 1 mM benzamidine, and 0.2 mM 4-(2-aminoethyl)benzene sulfonyl fluoride]. They were then sonicated and subjected to centrifugation at 15,000 x g for 20 min. Supernatants were removed and divided into portions for ERK1/2 determination and protein assay. Equal amounts of proteins (20 µg) were denatured at 100 C in 30% glycerol, 8% sodium dodecyl sulfate, 10% 2-mercaptoethanol, 25 mM Tris-HCl (pH 6.8), and 0.1% bromophenol blue; resolved by 10% SDS-PAGE; and transferred to 0.22-µm pore nitrocellulose filters. Nonspecific staining was blocked with Tris-buffered saline containing 0.1% Tween 20 (TBST) and 5% nonfat dry milk at room temperature for 1 h. Nitrocellulose membranes were incubated overnight at 4 C with primary antibody raised against phosphorylated ERK1/2 (1:1000). Next, the membranes were washed three times in TBST, incubated with peroxidase-conjugated secondary antibody (1:2000) for 2 h at room temperature, and washed three additional times in TBST. Enhanced chemiluminescence techniques were used to visualize the immunoblots according to the manufacturer’s protocol. The membranes were stripped according to conventional methods and immunoblotted with pan-ERK (phosphorylated and nonphosphorylated ERK1/2) for total ERK1/2 protein. Digitized images of the immunoblots were used for densitometric measurements with ScionImage software (NIH, Bethesda, MD). Relative enzyme activation was determined by normalization of densities image to that of the total ERK1/2 from the same membrane.
Transient transfection of primary myotubes
Cells were grown in 60-mm dishes to 60% confluence. Before transfection, the cells were washed to remove serum and were transiently transfected with either empty vectors or dominant negative Ras, MEK, or pON249 (2–4 µg) using Lipofectamine (Invitrogen Life Technologies, Inc., Gaithersburg, MD) following the manufacturer’s instructions. A single plate of transfected cells was then used to set-up the experimental cultures required for each assay, ensuring equal transfection efficiencies between different treatments, and cells were cultured for an additional 24 h before treatment with androgens. For reporter gene assays, myotubes were cotransfected with pCRE-Luc (2 µg/ml) and pON249 (2 µg/ml) plasmids, and then luciferase and ß-galactosidase activities were measured. The ratio between luciferase values and ß-galactosidase values was used to correct for differences in transfection efficiency.
Statistics
Data are expressed as the mean ± SEM. Differences between basal and poststimulated points were determined using a paired t test. P < 0.05 was considered statistically significant.
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, which represents a sequence of fluorescence images acquired at the times indicated. The response was characterized by fast and transient increases (seconds to minutes) in the fluorescence of myotubes preloaded with fluo-3. Frequently, calcium oscillations and waves were observed (n = 72 from 101 cells responding to testosterone). In this particular experiment, an increase in basal fluorescence after hormone exposition can be seen, and it is possible to note that fluorescence oscillates in certain regions of the myotube, producing a propagated calcium wave from one end to the center of the myotube. Rapid testosterone effects (within the first minute) involving second messengers have been reported in other cell types, and nongenomic mechanisms of signal transduction have been proposed (5, 29, 30). To evaluate whether the effect of the hormone is mediated by extracellular membrane receptors, we tested the effect of testosterone covalently bound to albumin (T-BSA). This compound does not cross the plasma membrane, nor has it been reported to act on intracellular androgen receptors. T-BSA produces intracellular calcium increases in myotubes (Fig. 1, B and D) and this response is similar to that obtained with the free hormone. Albumin (0.1%) does not produce any change in the intracellular Ca2+ (Fig. 1D). When calcium signals produced by testosterone and T-BSA were compared, no differences were found in the onset of the signal (time to peak, 34 ± 8 and 38 ± 12 sec, respectively) or the maximal intracellular Ca2+ response (84 ± 22% and 88 ± 20% with respect to basal fluorescence, respectively). For both hormones, the intracellular Ca2+ oscillations diminish in fluorescence intensity over time and have a mean frequency of 0.083 ± 0.029 Hz. To investigate whether other steroids produce similar responses, we used several known hormones. Nandrolone, a synthetic androgen, mimics the effects of testosterone (Fig. 2A), suggesting that the intracellular calcium increase is a common pathway for androgen steroid action in skeletal muscle cells. The response was specific to androgens, because the use of 17ß-estradiol, progesterone, or dexamethasone (Fig. 2A) produced no detectable calcium rises.
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). Furthermore, the intracellular androgen receptor translocation from the cytoplasm to nucleus upon hormone binding is necessary for its activation and subsequent action on transcriptional machinery (32). To determine whether this critical step occurs within the time frame in which testosterone-induced Ca2+ increases are produced, immunocytochemistry was used to locate the intracellular androgen receptor in control myotubes and after testosterone stimulation at different times. Figure 3A shows the cytoplasmic location of androgen receptors in a control myotube (no stimulus). After incubation for 5 min with 100 nM testosterone, the intracellular distribution was similar to that under control conditions (Fig. 3B), and only after 60 min of testosterone addition was receptor translocation to the nuclear compartment observed (Fig. 3C). This time is far larger than that required to produce effects on intracellular Ca2+. To confirm that the dose of cyproterone used was able to inhibit androgen receptor translocation, myotubes were incubated with 1 µM of the antagonist for 30 min before testosterone stimulation. Figure 3D shows that cyproterone alone did not affect the cytosolic localization of androgen receptors and largely inhibited the testosterone-induced nuclear translocation normally seen after 1 and 2 h (Fig. 3, E and F, respectively), indicating that this concentration of cyproterone was effective in inhibiting the genomic effects of the hormone. Collectively, these results indicate that intracellular androgen receptors are not involved in the testosterone-induced intracellular Ca2+ increases in myotubes.
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). Interestingly, under these conditions, the intracellular Ca2+ oscillations were completely lost in most experiments. Furthermore, nifedipine (10 µM), an inhibitor of L-type voltage-gated calcium channels, did not modify the Ca2+ oscillations induced by testosterone (data not shown). Thus, androgen-induced calcium increases as well as the Ca2+ oscillations must originate from intracellular calcium stores.
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). Pretreatment of myotubes with ryanodine (20 µM) did not modify the calcium response (data not shown), suggesting that IP3 is involved in the androgen-induced intracellular calcium release in myotubes. To further probe this point, we measured IP3 mass in response to androgen. Figure 4B shows the time course of both testosterone and nandrolone effects on IP3 mass in cultured myotubes. The basal value of IP3 mass increased significantly after 30 sec of hormone exposure and reached a maximum 45 sec after hormone addition for both steroids. The IP3 mass then gradually returned to its basal value. The increase in IP3 mass preceded the fast oscillatory calcium transient (see Fig. 1A). Furthermore, neither the basal value of IP3 mass nor the increased mass in response to testosterone were significantly affected when myotubes were incubated for 30 min with 1 µM cyproterone (basal, 47.3 ± 10.2 vs. 58.2 ± 8.5 pg/mg protein; post stimuli, 140.6 ± 5.2 vs. 138.1 ± 10.1 pg/mg protein).
To determine the early events involved in the Ca2+ signal produced by testosterone, myotubes were incubated for 20 min with 50 µM genistein, a tyrosine kinase inhibitor, previous to hormone stimulation. This inhibitor did not modify the testosterone-induced intracellular Ca2+ increases (Fig. 5A). On the other hand, a role for a G protein in this effect was evaluated. Myotubes were permeabilized for 5 min with saponin in the presence of 100 nM GDPßS, a nonhydrolyzable analog of GTP. Permeabilization did not modify testosterone-induced responses in the myotubes, whereas GDPßS suppressed the Ca2+ increases induced by the hormone (Fig. 5B). Furthermore, myotubes were incubated with PTX (1 µg/ml) before testosterone stimulation. Figure 5C shows that PTX inhibited the Ca2+ signals produced by the hormone. A similar effect of these inhibitors was observed when the IP3 mass was analyzed; genistein did not affect the testosterone-induced IP3 increase, whereas both GDPßS and PTX almost totally blocked this increase (Fig. 5D). These results suggest that testosterone action requires a PTX-sensitive G protein to produce both the intracellular IP3 and Ca2+ increases in myotubes.
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). Total-ERK1/2 staining (nonphosphorylated and phosphorylated ERK1 and -2 proteins) confirmed that within a particular experiment the individual lanes were loaded with equal amounts of proteins (Fig. 6B). To evaluate the time course of ERK1/2 phosphorylation during androgen stimulation, myotubes were incubated with 100 nM testosterone or nandrolone for 1, 5, 15, 30, and 60 min (Fig. 6C). Incubation of skeletal muscle cells with testosterone or nandrolone resulted in the dual phosphorylation of ERK1/2, as detected by Western blot, that can be detected after 1 min and significantly increased after 5 min for both hormones (Fig. 6A, lanes 2 and 3 for testosterone, lanes 5 and 6 for nandrolone; 1 and 5 min, respectively). Phosphorylation was fast and transient, and after 60 min of ERK1/2 phosphorylation returned to basal levels (Fig. 6A, line 4, and Fig. 6C). The effect of nandrolone-induced ERK1/2 phosphorylation was similar to that induced by testosterone. Densitometric analysis showed that the androgen-induced phosphorylation at 5 min was 2.0- to 2.5-fold basal values (Fig. 6C).
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, the Western blot shows that the testosterone-induced ERK1/2 phosphorylation in myotubes was dose dependent. Significant increases in phosphorylation were observed at a concentration of 10 nM testosterone. At 1 µM testosterone, the response continued to increase (Fig. 7C). We did not study concentrations above this range because they may not have physiological relevance and may be due to less specific effects. Furthermore, cyproterone was used in these experiments to test whether a classical genomic transcription-mediated pathway mediates the effect of androgen steroids on ERK1/2 phosphorylation. When cells were incubated with cyproterone acetate (1 µM) for 30 min before the addition of testosterone or nandrolone, no effects on the basal phosphorylation of ERK1/2 were seen (Fig. 7D, first two columns). Both testosterone and nandrolone stimulation for 5 min produced a rapid increase in ERK1/2 phosphorylation in cultured myotubes (Fig. 7D, third and fourth columns), indicating that this response was not dependent on an intracellular androgen receptor. Furthermore, T-BSA (100 nM) increased ERK1/2 phosphorylation, in a time scale similar to that of testosterone or nandrolone (Fig. 7D, last column). These results indicate that ERK1/2 phosphorylation does not involve the intracellular androgen receptor and is probably mediated by interactions at the plasma membrane level.
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, A and B, ERK1/2 phosphorylation was about 26% lower in magnitude, but did not disappear. Second, we incubated the myotubes with BAPTA-AM, a compound that crosses the cell membrane and chelates intracellular calcium ions. As observed in Fig. 8, A and B, the testosterone-induced ERK1/2-phosphorylation in myotubes preincubated with BAPTA-AM was blocked. These observations suggest that an elevation of intracellular calcium is needed for ERK1/2 phosphorylation. To confirm the role of PLC as a mediator of intracellular calcium release and subsequent ERK1/2 phosphorylation, we used inhibitors of the IP3 pathway. In myotubes preincubated with 10 µM U73122 or 40 µM xestospongin B, no effect on basal phosphorylation of ERK1/2 was evident; however, complete inhibition of testosterone-induced ERK1/2 phosphorylation was observed (Fig. 8C). These results indicate that the calcium involved in ERK1/2 phosphorylation is released from intracellular stores after activation of PLC, IP3 generation, and binding to IP3 receptors.
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). Similarly, the incubation of the myotubes with 1 µg/ml PTX for 5 h before testosterone stimulation blocked the response to the hormone (Fig. 9). These results suggest that testosterone-induced ERK1/2 phosphorylation requires the participation of a PTX-sensitive G protein.
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, results from myotubes incubated for 20 min with 10 µM PD 98059 are shown. This compound almost completely blocked testosterone-induced ERK1/2 phosphorylation without an effect on the basal phosphorylation of this protein. The role of Ras in the signal transduction cascade mediated by androgens was also evaluated using dominant negatives for MEK and Ras proteins. Figure 10B shows a selective blockade of testosterone-induced ERK1/2 phosphorylation by dominant negatives for both MEK and Ras. These results are consistent with ERK1/2 phosphorylation induced by testosterone through the Ras/MEK/ERK pathway in cultured myotubes.
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), and the effect was completely blocked by cyproterone, indicating a genomic effect mediated by intracellular androgen receptors. Furthermore, testosterone linked to BSA was not able to mimic this effect (Fig. 11).
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Discussion |
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The skeletal muscle cell is a target for androgen action (1, 20). Binding of steroid hormones to intracellular receptors mediates a variety of genomic responses in this cell type (20, 21, 32). However, several observations demonstrate that androgens also produce nonclassical effects, particularly, the activation of intracellular mechanisms of signal transduction (4, 5). In myotubes we found that the time course analysis of the changes in Ca2+ signals show that the calcium increase is fast and transient, occurring within seconds after the addition of either testosterone or its structural cognate nandrolone. Calcium increased at both the cytosolic and the nuclear level. As we are not using a ratiometric dye and as the dye may be compartmentalized, higher fluorescence in the nucleus does not necessarily mean a higher calcium concentration in this compartment; however, the nuclear component of the calcium signal was evident. Interestingly, we observed intracellular Ca2+ oscillations, sometimes propagating as Ca2+ waves along the myotubes. The generation of Ca2+ waves may represent an important early step for the coordination of cell functions in skeletal muscle cells (10). The increases in intracellular calcium have different spatial and temporal patterns, and release from different subcellular compartments influences the transcriptional response (10, 12). Thus, a different oscillatory pattern may mean a different mechanism of calcium release and recapture as well as a different function for calcium as an intracellular signal.
Our data show that the Ca2+ increase elicited by androgens is due to Ca2+ released from intracellular stores. There is evidence that calcium signals evoked by stimulation of myotubes with androgens involve the formation of IP3 (6). This is probably due to activation of phospholipase C, because the responses are produced in Ca2+-free medium, and inhibitors of the IP3-mediated pathway almost completely blocked the calcium increases in response to testosterone. Moreover, increases in IP3 mass precede the oscillatory calcium transients, suggesting a sequential relationship between IP3 increases and Ca2+ increases. Interestingly, these oscillations are not observed in calcium-free medium. In many cell types, depletion of calcium from intracellular stores is followed by an influx of calcium from the extracellular space through activation of store-operated channels (33); thus, androgen-evoked Ca2+ oscillations in myotubes may involve interactions between events in the plasma membrane and internal stores, with calcium release from sarcoplasmic reticulum being a key event for this mechanism. It has been proposed that the generation and propagation of intracellular Ca2+ oscillations may reduce the thresholds for action of this second messenger, and different Ca2+ oscillatory patterns can preferentially activate or inactivate calcium-dependent processes (34).
Testosterone exerts its effects through binding to and activation of cytosolic androgen receptor, which translocates to the nucleus and functions as a transcription factor (3, 30). In our system cyproterone, an antagonist of the intracellular androgen receptor was able to block both the receptor translocation and CRE-mediated transcription, events probably mediated by the intracellular androgen receptor. Nevertheless, androgen-induced second messenger generation was not blunted by cyproterone, and the membrane-impermeant testosterone conjugate (T-BSA) induced similar effects on calcium compared with the free hormone, suggesting that these rapid effects are mediated by the actions of androgens on membrane components. The IP3-mediated calcium release from internal stores can be due to activation of tyrosine kinase as well as G protein-coupled membrane receptors. The treatment of myotubes with genistein, a tyrosine kinase inhibitor, did not modify the intracellular Ca2+ or IP3 increases induced by testosterone. On the other hand, several lines of evidence demonstrate that androgens can activate PTX-sensitive G proteins (5, 35); to determine whether testosterone may interact with a G proteins in myotubes, the Ca2+ increase was evaluated in myotubes treated with GDPßS and PTX. The fact that G protein inhibitors blocked fast effects of testosterone reinforces the idea of a membrane receptor for this hormone. The presence of membrane binding sites for androgens has been previously suggested (4, 5, 29). According to our results in myotubes, testosterone-induced intracellular calcium increases have also been described in other cells, including rat osteoblasts (4), mice splenic T cells (29), and macrophages (5). Interestingly, macrophages, lacking classical intracellular androgen receptors, respond to testosterone with intracellular Ca2+ increases. Thus, a transient increase in calcium should affect certain cellular processes directly (nongenomic pathway), or alternatively, they could act through Ca2+-binding proteins to regulate slower processes (genomic pathway) via protein synthesis.
Steroid hormones have been associated with the growth and differentiation of muscle cells, but to date little evidence has accumulated on the role of intracellular signaling pathways in these processes. Upon activation by phosphorylation, ERK1/2 translocate from the cytoplasm into the nucleus (14), and this results in the phosphorylation or induction of transcription factors, leading to the expression of genes involved in the control of cellular growth. The results reported here show that testosterone, nandrolone, and T-BSA can induce dual phosphorylation of ERK1/2 within a few minutes. In skeletal muscle an increase in the ERK1/2 phosphorylation correlates with its activity (15, 19). The rapid phosphorylation of ERK1/2 by androgens was transient and dose dependent, demonstrating that myotubes respond to testosterone in the physiological range of concentrations. The relationship among intracellular calcium increases, the IP3 pathway, and ERK1/2 phosphorylation was illustrated by pharmacologically manipulating the calcium sources. Thus, testosterone-induced ERK1/2 phosphorylation was slightly inhibited in a nominally Ca2+-free medium, a possible explanation being that the effect of intracellular Ca2+ increases depends on the time course, i.e. ERK1/2 phosphorylation will be different depending on whether the calcium increase occurs with an oscillatory pattern or not. A similar effect has been suggested for the effects of ouabain, a steroid derivative, on the transcription factor nuclear factor-
B, which reaches maximal activation during calcium oscillations, but not during maintained Ca2+ increase (36). On the other hand, steroid-induced ERK1/2 phosphorylation was completely blocked by chelation of intracellular calcium with BAPTA-AM or the use of either U-73122, a PLC inhibitor, or xestospongin B, an inhibitor of IP3 receptors. Moreover, G protein-coupled receptors can induce ERK1/2 activation (13, 14). Testosterone-induced ERK1/2 phosphorylation in the myotubes was inhibited by treatment with GDPßS and PTX, suggesting the participation of a PTX-sensitive G protein in this response. These results are in accordance with the inhibition of intracellular Ca2+ and IP3 increases by the G protein inhibitors and the Ca2+ requirement for the ERK1/2 phosphorylation induced by testosterone in myotubes.
Some researchers have shown that antagonists for intracellular receptors can block steroid-induced ERK1/2 phosphorylation (37). However, after incubation of myotubes with 1 µM cyproterone in the present study, neither basal ERK1/2 phosphorylation nor testosterone-dependent phosphorylation was modified. Even though these effects were not blocked by cyproterone in skeletal muscle cells, which suggests that activation of the transcription machinery was not involved, nongenomic effects may play a physiological role in events previous to the androgen receptor activation. A likely possibility is phosphorylation of coactivators that amplify the androgen action, as has been proposed by Zhu et al. (38), or a biphasic system with both high affinity nuclear as well as low affinity membrane androgen receptors, as described in rat Sertoli and human prostatic cell lines (39). Several mitogens can produce trans-activation of steroid receptors through phosphorylation (40, 41). An interesting hypothesis to be explored is that intracellular calcium increases and ERK1/2 activation are events that elicit androgen receptor activation in skeletal muscle cells. Moreover, the MAPK cascade triggered by the binding of hormones or growth factors to surface receptors may produce the phosphorylation events that have been implicated in cell growth and differentiation (18). Gredinger et al. (16) have shown that the MAPK pathway plays a positive role in myogenesis and that ERK activity is substantially increased during terminal muscle differentiation in muscle cells and cooperates with MyoD to activate muscle-specific transcription. In accordance with the rapid effects of steroid hormones, Morelli et al. (23) have recently shown that 1,25-dihydroxyvitamin D3 activates ERK1/2 in skeletal muscle cells and has implicated the MAPK cascade in hormone control of myoblast proliferation. ERK1/2 are activated by phosphorylation of both threonine and tyrosine residues; these reactions are catalyzed by MEK. MEK itself is activated by at least two types of kinases, Raf-1 and MEK kinase (13, 14, 18). Raf-1, which is also activated by phosphorylation, appears to be a central point in receiving signals from upstream activated kinases, located at the plasma membrane. Most of the signaling pathways that have been proposed to be involved in the activation of ERK1/2 after increasing intracellular Ca2+ converge on Ras, thus indicating a key role for this protein in this process (14, 18). The observations that overexpression of dominant negative mutants of Ras and MEK inhibits testosterone-mediated ERK1/2 phosphorylation reflects that this hormone activates the Ras/MEK/ERK pathway in myotubes. The mechanism of Ca2+-mediated Ras activation remains elusive; however, in other cell types, a Ras-dependent activation of ERK1/2 by either PKC (18) or Ca2+-regulated protein kinase such as calmodulin (12) has been described. Buitrago et al. (42) recently demonstrated in avian myotubes that 1
,25-dihydroxyvitamin D3-induced MAPK activation occurs through both a Raf-1 via Ras pathway as well as a PKC-dependent pathway. The intracellular targets for the steroid-stimulated ERK1/2 cascade have not been established. It has been reported that androgens can activate the transcription factor CREB (43). However, in myotubes the transcriptional CREB activity induced by testosterone, but not by T-BSA, indicates that a hormone-bound intracellular androgen receptor is necessary for this effect. There is evidence that activation of several signaling pathways involving protein kinase A and C leads to stimulation of androgen-regulated trans-activation through interaction of components of these pathways with the androgen receptor (41). It is possible, then, that the interaction of activated androgen receptor with some of the signaling pathways will eventually lead to activation of the element-binding protein by phosphorylation (43). A possible role for the ERK pathway would be to phosphorylate hormone-bound androgen receptors (44) and modulate transcriptional activity (22, 30). Another possibility could be that ERK-activated pathways could influence the genomic response through a synergistic mechanism by phosphorylation of coactivators of the intracellular receptor at the nuclear level, as reported for both thyroid receptor and estrogen receptor (45, 46).
A scheme of the proposed G protein-dependent action for androgens in skeletal muscle cells is depicted (Fig. 12). It includes a putative membrane androgen receptor coupled to PTX-sensitive G protein, PLC activation, and calcium release; ERK1/2 phosphorylation appears to be triggered by calcium. Taken together, our results indicate that increases in intracellular calcium as well as activation of the Ras/MEK/ERK1/2 pathway represent an intermediate step in the intracellular signaling toward as yet undefined downstream effects of androgen steroids on skeletal muscle cells.
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Footnotes |
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Abbreviations: BAPTA-AM, 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM; CRE, cAMP response element; CREB, CRE-binding protein; GDPßS, guanosine 5'-O-(2-thiodiphosphate); IP3, inositol, 1,4,5-trisphosphate; MEK, MAPK kinase; PLC, phospholipase C; PTX, pertussis toxin; T-BSA, testosterone bound to albumin; TBST, Tris-buffered saline containing 0.1% Tween 20.
Received December 18, 2002.
Accepted for publication May 1, 2003.
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,25(OH)2-vitamin D3 signal transduction in skeletal muscle cells. J Biol Chem 275:36021–36028i2, 3. J Biol Chem 277:21379–21388. mediates 1,25(OH)2-vitamin D3 regulation of the mitogen-activated protein kinase pathway in muscle cells. J Biol Chem 278:2199–2205This article has been cited by other articles:
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 G. Bagchi, J. Wu, J. French, J. Kim, N. H. Moniri, and Y. Daaka Androgens Transduce the G{alpha}s-Mediated Activation of Protein Kinase A in Prostate Cells Cancer Res., May 1, 2008; 68(9): 3225 - 3231. [Abstract] [Full Text] [PDF]
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 L. A. Burnes, S. J. Kolker, J. F. Danielson, R. Y. Walder, and K. A. Sluka Enhanced muscle fatigue occurs in male but not female ASIC3-/- mice Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2008; 294(4): R1347 - R1355. [Abstract] [Full Text] [PDF]
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 B. D. Phillis, M. Y. Abeywardena, M. J. Adams, J. A. Kennedy, and R. J. Irvine Nandrolone Potentiates Arrhythmogenic Effects of Cardiac Ischemia in the Rat Toxicol. Sci., October 1, 2007; 99(2): 605 - 611. [Abstract] [Full Text] [PDF]
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P. Kumar, Q. Wu, K. L. Chambliss, I. S. Yuhanna, S. M. Mumby, C. Mineo, G. G. Tall, and P. W. Shaul Direct Interactions with G{alpha}i and G{beta}{gamma} Mediate Nongenomic Signaling by Estrogen Receptor {alpha} Mol. Endocrinol., June 1, 2007; 21(6): 1370 - 1380. [Abstract] [Full Text] [PDF]
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 T.-V. V. Nguyen, M. Yao, and C. J. Pike Flutamide and Cyproterone Acetate Exert Agonist Effects: Induction of Androgen Receptor-Dependent Neuroprotection Endocrinology, June 1, 2007; 148(6): 2936 - 2943. [Abstract] [Full Text] [PDF]
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M. C. Gonzalez-Montelongo, R. Marin, T. Gomez, and M. Diaz Androgens Differentially Potentiate Mouse Intestinal Smooth Muscle by Nongenomic Activation of Polyamine Synthesis and Rho Kinase Activation Endocrinology, December 1, 2006; 147(12): 5715 - 5729. [Abstract] [Full Text] [PDF]
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 M. Estrada, A. Varshney, and B. E. Ehrlich Elevated Testosterone Induces Apoptosis in Neuronal Cells J. Biol. Chem., September 1, 2006; 281(35): 25492 - 25501. [Abstract] [Full Text] [PDF]
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 M Yoshioka, A Boivin, P Ye, F Labrie, and J St-Amand Effects of dihydrotestosterone on skeletal muscle transcriptome in mice measured by serial analysis of gene expression. J. Mol. Endocrinol., April 1, 2006; 36(2): 247 - 259. [Abstract] [Full Text] [PDF]
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J. W. Gatson, P. Kaur, and M. Singh Dihydrotestosterone Differentially Modulates the Mitogen-Activated Protein Kinase and the Phosphoinositide 3-Kinase/Akt Pathways through the Nuclear and Novel Membrane Androgen Receptor in C6 Cells Endocrinology, April 1, 2006; 147(4): 2028 - 2034. [Abstract] [Full Text] [PDF]
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 A. Corbould, H. Zhao, S. Mirzoeva, F. Aird, and A. Dunaif Enhanced Mitogenic Signaling in Skeletal Muscle of Women With Polycystic Ovary Syndrome Diabetes, March 1, 2006; 55(3): 751 - 759. [Abstract] [Full Text] [PDF]
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J. M. Vicencio, C. Ibarra, M. Estrada, M. Chiong, D. Soto, V. Parra, G. Diaz-Araya, E. Jaimovich, and S. Lavandero Testosterone Induces an Intracellular Calcium Increase by a Nongenomic Mechanism in Cultured Rat Cardiac Myocytes Endocrinology, March 1, 2006; 147(3): 1386 - 1395. [Abstract] [Full Text] [PDF]
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 P. Balagopal, R. Olney, D. Darmaun, E. Mougey, M. Dokler, G. Sieck, and D. Hammond Oxandrolone enhances skeletal muscle myosin synthesis and alters global gene expression profile in Duchenne muscular dystrophy Am J Physiol Endocrinol Metab, March 1, 2006; 290(3): E530 - E539. [Abstract] [Full Text] [PDF]
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 M. Estrada, P. Uhlen, and B. E. Ehrlich Ca2+ oscillations induced by testosterone enhance neurite outgrowth J. Cell Sci., February 15, 2006; 119(4): 733 - 743. [Abstract] [Full Text] [PDF]
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C. Cardenas, J. L. Liberona, J. Molgo, C. Colasante, G. A. Mignery, and E. Jaimovich Nuclear inositol 1,4,5-trisphosphate receptors regulate local Ca2+ transients and modulate cAMP response element binding protein phosphorylation J. Cell Sci., July 15, 2005; 118(14): 3131 - 3140. [Abstract] [Full Text] [PDF]
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Y. Chen, J. D Zajac, and H. E MacLean Androgen regulation of satellite cell function J. Endocrinol., July 1, 2005; 186(1): 21 - 31. [Abstract] [Full Text] [PDF]
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 D.J. Haisenleder, L.L. Burger, K.W. Aylor, A.C. Dalkin, H.E. Walsh, M.A. Shupnik, and J.C. Marshall Testosterone Stimulates Follicle-Stimulating Hormone {beta} Transcription via Activation of Extracellular Signal-Regulated Kinase: Evidence in Rat Pituitary Cells Biol Reprod, March 1, 2005; 72(3): 523 - 529. [Abstract] [Full Text] [PDF]
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M Estrada, A Espinosa, C J Gibson, P Uhlen, and E Jaimovich Capacitative calcium entry in testosterone-induced intracellular calcium oscillations in myotubes J. Endocrinol., February 1, 2005; 184(2): 371 - 379. [Abstract] [Full Text] [PDF]
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J. A. Huhtakangas, C. J. Olivera, J. E. Bishop, L. P. Zanello, and A. W. Norman The Vitamin D Receptor Is Present in Caveolae-Enriched Plasma Membranes and Binds 1{alpha},25(OH)2-Vitamin D3 in Vivo and in Vitro Mol. Endocrinol., November 1, 2004; 18(11): 2660 - 2671. [Abstract] [Full Text] [PDF]
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 I. Sinha-Hikim, W. E. Taylor, N. F. Gonzalez-Cadavid, W. Zheng, and S. Bhasin Androgen Receptor in Human Skeletal Muscle and Cultured Muscle Satellite Cells: Up-Regulation by Androgen Treatment J. Clin. Endocrinol. Metab., October 1, 2004; 89(10): 5245 - 5255. [Abstract] [Full Text] [PDF]
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Y. Zagar, G. Chaumaz, and M. Lieberherr Signaling Cross-talk from G{beta}4 Subunit to Elk-1 in the Rapid Action of Androgens J. Biol. Chem., January 23, 2004; 279(4): 2403 - 2413. [Abstract] [Full Text] [PDF]
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; T) or vehicle (
; <0.01% ethanol). D, T-BSA (
; 100 nM). The calcium increase reached 50-fold the basal value, and some oscillations were present (n = 32 cells in 16 independent experiments). Other steroid hormones, 17ß-estradiol (
; 1 µM), progesterone (
; 1 µM), and dexamethasone (
; 1 µg/ml) for 5 h and then stimulated with testosterone. PTX blocked the Ca2+ increases induced by the hormone. D, Cells were incubated with PTX (1 µg/ml for 5 h), GDPßS (100 nM for 5 min) or genistein (gen; 50 µM for 20 min) and then stimulated with testosterone (100 nM) for 45 sec. Both PTX and GDPßS inhibited the IP3 increases induced by testosterone. The IP3 mass correspond to the mean ± SEM of three experiments performed in triplicate. *, P < 0.05 compared with the control.
