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J. Biol. Chem., Vol. 275, Issue 46, 36021-36028, November 17, 2000

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Involvement of Tyrosine Kinase Activity in 1,25(OH)₂-vitamin D₃ Signal Transduction in Skeletal Muscle Cells^*

Susana Morelli, Claudia Buitrago, Guillermo Vazquez, Ana R. De Boland, and Ricardo Boland

From the Departamento de Biologia, Bioquimica y Farmacia, Universidad Nacional del Sur, (8000) Bahia Blanca, Argentina

Received for publication, March 10, 2000, and in revised form, August 3, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In cultured chick skeletal muscle cells loaded with Fura-2, the tyrosine kinase inhibitors herbimycin A and genistein abolished both the fast inositol 1,4,5-trisphosphatedependent Ca²⁺ release from internal stores and extracellular Ca²⁺ influx induced by 1,25(OH)₂-vitamin D₃ (1,25(OH)₂D₃). Daidzein, an inactive analog of genistein, was without effects. Tyrosine phosphatase inhibition by orthovanadate increased cytosolic Ca²⁺. Anti-phosphotyrosine immunoblot analysis revealed that 1,25(OH)₂D₃ rapidly (0.5-10 min) stimulates in a concentrationdependent fashion (0.1-10 nM) tyrosine phosphorylation of several myoblast proteins, among which the major targets of the hormone could be immunochemically identified as phospholipase C (127 kDa), which mediates intracellular store Ca²⁺ mobilization and external Ca²⁺ influx, and the growth-related proteins mitogen-activated protein (MAP) kinase (42/44 kDa) and c-myc (65 kDa). Genistein suppressed the increase in phosphorylation and concomitant elevation of MAPK activity elicited by the sterol. Both genistein and the MAPK kinase (MEK) inhibitor PD98059 abolished stimulation of DNA synthesis by 1,25(OH)₂D₃. The sterol-induced increase in tyrosine phosphorylation of c-myc, a finding not reported before for cell growth regulators, was totally suppressed by the specific Src inhibitor PP1. These results demonstrate that tyrosine phosphorylation is a previously unrecognized mechanism involved in 1,25(OH)₂D₃ regulation of Ca²⁺ homeostasis in hormone target cells. In addition, the data involve tyrosine kinase cascades in the mitogenic effects of 1,25(OH)₂D₃ on skeletal muscle cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

1,25-Dihydroxy-vitamin D₃ (1,25(OH)₂D₃)¹ in addition to its classical role in the regulation of extracellular calcium homeostasis, modulates cell proliferation and differentiation and the immune system (1-5). The hormone also regulates skeletal muscle functions. Muscle weakness and atrophy are observed in vitamin D deficiency states and impaired metabolism. This myopathy is reversed by administration of physiological amounts of 1,25(OH)₂D₃ (Refs. 6-8; for a review on this topic, see Ref. 9). Studies with animal models and cultured muscle cells have shown that the hormone exerts direct effects on skeletal muscle Ca²⁺ metabolism, contractility, and growth (9-11). As in other target cells (12-15), 1,25(OH)₂D₃ elicits responses in muscle both through nuclear receptor-mediated gene transcription and a fast mechanism independent of new RNA and protein synthesis (11, 16). The non-genomic actions of 1,25(OH)₂D₃ in muscle cells involve G protein-coupled stimulation of adenylyl cyclase and phospholipases C, D, and A₂ and activation of protein kinases A and C which, in turn regulate the activity of voltage-dependent Ca²⁺ channels (VDCC) (17-22). The hormone also promotes Ca²⁺ mobilization from intracellular stores and modulates store-operated Ca²⁺ (SOC) channels as part of the 1,25(OH)₂D₃-induced Ca²⁺ entry across the plasma membrane of skeletal muscle cells (23, 24). The rapid nature and specificity by which 1,25(OH)₂D₃ activates these second messenger pathways suggest that interaction with a plasma membrane receptor is responsible for the initiation of its effects. The presence of membrane binding sites for 1,25(OH)₂D₃ in skeletal muscle (25) as well as for this and other steroid hormones in various cell types (reviewed in Refs. 26 and 27) has been described. In connection to the muscle growth-promoting activity of 1,25(OH)₂D₃, various lines of evidence have shown that the hormone stimulates both the proliferation and differentiation of myoblasts into myotubes (28-30).

Tyrosine phosphorylation is a crucial event in signal transduction mechanisms linked to the mitogen-activated protein kinase (MAPK) cascade underlying the regulation of cell proliferation and differentiation by agonists of receptor tyrosine kinases or heterotrimeric G protein-coupled receptors. Translocation of activated MAPK to the nucleus results in the phosphorylation or induction of transcription factors leading to the expression of genes involved in control of cellular growth (31, 32). There is also evidence indicating that tyrosine kinases may modulate Ca²⁺ entry both through the VDCC (33, 34) and SOC channel (35-37) pathways. Variations in cytosolic Ca²⁺ levels are also of importance in the control of the cell cycle (38). In line with the participation of this mechanism, we recently obtained preliminary evidence indicating that in skeletal muscle cells tyrosine kinase phosphorylation of cellular proteins seems to play a role in 1,25(OH)₂D₃-dependent modulation of non-genomic responses, such as fast increases in cytosolic Ca²⁺ and MAPK stimulation (39). Accordingly, it has been recently reported that 1,25(OH)₂D₃ rapidly stimulates MAP kinase phosphorylation in both promyelocytic NB4 leukemia cells (40) and enterocytes (41). On these bases, in the present study we examined the participation of tyrosine kinase(s) in the mechanism by which 1,25(OH)₂D₃ regulates cytoplasmic Ca²⁺ and exerts mitogenic effects in skeletal muscle cells and investigated hormone-dependent related changes in protein tyrosine phosphorylation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chemicals-- 1,25(OH)₂D₃ was kindly provided by Dr. Heinrich Bachmann (Hoffman-La Roche Ltd., Basel, Switzerland). Fura-2/pentaacetoxymethyl ester (Fura-2/AM), pluronic acid F-127, genistein, herbimycin, daidzein, sodium orthovanadate, Dulbecco's modified Eagle's medium, and fetal bovine serum were from Sigma. Compounds PD98059 and PP1 were supplied by Calbiochem-Novabiochem and Pfizer, respectively. Rabbit polyclonal anti-phosphotyrosine antibody was obtained from Upstate Biotechnology Inc. (Lake Placid, NY). Anti-MAP kinase antibody and anti-phospho-MAP kinase, an antibody to the active phosphorylated form of MAP kinase (reactive against p42 and p44 isoforms), were from Promega (Madison, WI). Anti-c-myc antibody was purchased from Oncogene Research Products (Cambridge, MA). Anti-phospholipase C was provided by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Secondary antibody goat anti-rabbit horseradish peroxidase-conjugated IgG and the Super Signal CL-HRP substrate system for enhanced chemiluminescence (ECL) were obtained from Amersham Pharmacia Biotech. [³²-P]ATP (3,000 Ci/mmol) was from PerkinElmer Life Sciences. Protein A-Sepharose was purchased from Pierce. All other reagents were of analytical grade.

Cell Culture-- Chick skeletal muscle cells were obtained from 13-day-old chick embryo breast muscles by stirring in Earle's balanced salt solution containing 0.1% trypsin for 30 min essentially as described previously (42). The freed cells were collected by centrifugation, and the pellet was resuspended in DMEM supplemented with 10% fetal bovine serum and antibiotic-antimycotic solution. The suspension was dispersed by pipette, filtered through nylon mesh, and "preplated" on gelatin-coated Petri dishes to remove contaminating fibroblasts. The unadsorbed cells were seeded at an appropriate density (120,000 cells/cm²) in Petri dishes (80 mm in diameter) for tyrosine phosphorylation and MAPK assays or onto glass coverslips (24 × 6 mm) for intracellular calcium measurements and cultured at 37 °C under a humidified atmosphere (air, 5% CO₂). Cells were allowed to grow until confluence (4-6 days after plating) before use. Under these conditions, myoblasts proliferate within the first 48 h and at day 4 become differentiated into myotubes expressing both biochemical and morphological characteristics of adult skeletal muscle fibers (43).

Thymidine Incorporation-- The rate of thymidine incorporation into DNA was determined by adding 2 µCi of [³H]thymidine (20 Ci/mmol)/ml Dulbecco's modified Eagle's medium to muscle cell monolayers cultured for 6-24 h, incubating for 1 h at 37 °C in Krebs-Henseleit 0.2% glucose solution, and washing three times with incubation solution. DNA and proteins were precipitated with ice-cold 12% trichloroacetic acid and dissolved in 1 N NaOH, and the radioactivity was counted in a liquid scintillation counter.

Intracellular Calcium Measurements-- Intracellular Ca²⁺ changes were monitored using the Ca²⁺-sensitive fluorescent dye Fura-2/AM (44). Cell dye loading was achieved by incubating the cells in buffer A containing 138 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 5 mM glucose, 10 mM Hepes, pH 7.4, 1.5 mM CaCl₂ plus 0.1% bovine serum albumin, 4 µM penta-acetoxymethylester derivative (membrane-permeable) Fura-2/AM, and 0.012% pluronic acid in the dark for 40 min at room temperature in order to minimize dye compartmentalization. Unloaded dye was washed out, and cells were maintained in buffer B (buffer A without bovine serum albumin, Fura-2/AM, and pluronic acid) in the dark (room temperature) for at least 40 min before use to allow for complete intracellular dye deesterification. Coverslips containing confluent cells were placed into quartz cuvettes of a thermostatically controlled (37 °C) SLM Aminco 8100 spectrofluorimeter (Spectronics Inc.) sample compartment under constant, controlled stirring. Fura-2 intracellular fluorescence intensity was monitored at an emission wavelength of 510 nm (8-nm bandpass) by alternating (300 Hz) the excitation wavelength between 340 and 380 nm (4-nm bandpass) with a dual excitation monochromator.

Signals from short and long wavelengths were compared in a ratio (r = 340/380), thus making the measurement independent of variations in cellular dye content, dye leakage, or photobleaching. Calibration of Fura-2 fluorescence signal to calculate [Ca²⁺]_i values was performed for each coverslip essentially as described by us (23, 24). Maximal (R_max) and minimal (R_min) intracellular dye fluorescence signals were determined by adding 5 µM ionomycin plus 3 mM Ca²⁺ and 10 mM EGTA, pH 9.0, respectively. Under these conditions of measurement, the dissociation constant (K_d) for the Ca²⁺-Fura-2 complex was assumed to be 224 nM, and [Ca²⁺]_i according to the algorithm of Grynkiewicz et al. (44) derives from [Ca²⁺]_i = K_d (R  R_min)/(R_max  R) × , where R is the ratio of Fura-2 fluorescence at the selected wavelengths, R_max and R_min represent ratios from Ca²⁺-saturated and Ca²⁺-free intracellular dye, respectively, and  is the ratio between the specific fluorescence of the Ca²⁺-free and Ca²⁺-bound forms of the dye at the longer wavelength (Sf₂/ Sb₂).

In some experiments, a Ca²⁺-free extracellular medium was used. In such situations, the absence of Ca²⁺ in the medium means free Ca²⁺ concentration near 1 nM, which is accomplished by preparing a nominally Ca²⁺-free buffer B (see composition above) plus 1 mM EGTA. Free Ca²⁺ levels were calculated by using the WinMaxc program, version 1.7 (45). All buffers and saline solutions used were prepared with deionized water.

Immunoprecipitation-- After treatment, muscle cells were lysed (30 min at 4 °C) in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EGTA, 25 mM NaF, 0.2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 2 µg/ml pepstatin, 2 µg/ml aprotinin, 0.25% sodium deoxycholate, and 1% Nonidet P-40. Insoluble material was pelleted in a microcentrifuge at 12,000 × g for 10 min. The protein content of the clear lysates was determined according to Lowry et al. (46). Aliquots (500-700 µg of protein) were incubated overnight at 4 °C with anti-phosphotyrosine, anti-phospho-MAP kinase (p42 and p44 isoforms), anti-PLC, or anti-c-myc antibodies followed by precipitation of the complexes with protein A conjugated with Sepharose. The immune complexes were washed five times with cold immunoprecipitation buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 0.2 mM sodium orthovanadate, 1% Triton X-100, and 1% Nonidet P-40).

SDS-PAGE and Immunoblotting-- Immunoprecipitated proteins (or lysate proteins) dissolved in Laemmli sample buffer were separated on SDS-polyacrylamide (7%) gels (47) and electrotransferred to polyvinylidene difluoride membranes. The membranes were blocked for 1 h at room temperature in TBST (50 mM Tris-HCl, pH 7.4, 200 mM NaCl, 1% Tween 20) containing 1% dry milk. For the detection of tyrosine-phosphorylated proteins, membranes were subjected to immunoblotting using a rabbit anti-phosphotyrosine antibody. Next, the membranes were washed three times in TBST, incubated with a 1:10,000 dilution of peroxidase-conjugated anti-rabbit secondary antibody for 1 h at room temperature, and washed three additional times with TBST. The membranes were then visualized using an enhanced chemiluminescent technique (ECL), according to the manufacturer's instructions. Images were obtained with a model GS-700 imaging densitometer from Bio-Rad by scanning at 600 dots per inch and printing at the same resolution. Bands were quantified using the Molecular Analyst program (Bio-Rad).

To strip the membrane for reprobing with anti-phospho-MAP kinase, the membrane was washed 10 min in TBST and then incubated in stripping buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 50 mM mercapthoethanol) for 30 min at 50 °C. The membrane was again blocked and blotted as described above, except that the primary antibody used was a 1: 1000 dilution of anti-phospho-MAP kinase.

Measurement of MAP Kinase Activity-- Muscle cells were exposed either to 0.1-10 nM 1,25(OH)₂D₃ for 1 min, 1 nM 1,25(OH)₂D₃ for 30 s¹⁰ min, or vehicle (ethanol) at 37 °C. In some experiments the cells were pretreated with genistein (100 µM, 10 min). Lysates were prepared followed by immunoprecipitation of MAP kinase (p42 and p44) as described above. After three washes with immunoprecipitation buffer and two washes with kinase buffer (10 mM Tris-HCl, pH 7.2, 5 mM MgCl₂, 1 mM MnCl₂, 1 mM dithiothreitol, 0.1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 20 µg/ml aprotinin, and 20 µg/ml pepstatin), the immune complexes were incubated at 37 °C for 10 min in kinase buffer (50 µl/sample) containing myelin basic protein as an exogenous substrate for MAP kinase (20 µg/assay), 25 µM ATP, and [³²-P]ATP (2.5 µCi/assay). To terminate the reaction, the phosphorylated product was separated from free isotope on ion-exchange phosphocellulose filters (Whatman P-81). Papers were immersed immediately into ice-cold 75 mM H₃PO₄, washed (1 × 5 min, 3 × 20 min)m and counted in a scintillation counter.

Statistical Analysis-- Statistical significance of the data was evaluated using Student's t test (48), and probability values below 0.05 (p < 0.05) were considered significant. Results are expressed as means ± S.D. from the indicated set of experiments.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Stimulation of chick embryonic skeletal muscle cells with 1,25(OH)₂D₃ triggers a rapid (30 s) and sustained increment in intracellular calcium concentration ([Ca²⁺]_i) that persists elevated as long as the cells are exposed to the hormone (Fig. 1). We have previously shown that the rapid initial [Ca²⁺]_i response to the sterol mainly results from inositol 1,4,5-trisphosphate-mediated mobilization of Ca²⁺ from a thapsigargin-sensitive store, whereas the plateau phase is entirely due to Ca²⁺ influx through VDCC and SOC channels (23, 24). In the present study, to evaluate the participation of tyrosine kinase (TK) activity in the mechanism of muscle [Ca²⁺]_i regulation by 1,25(OH)₂D₃, we first examined the effect of TK inhibition on the hormone-generated variations in intracellular Ca²⁺. Pretreatment of myoblasts with the tyrosine kinase inhibitors genistein (50-100 µM) and herbimycin (10-50 µM) completely prevented any subsequent response to 1,25(OH)₂D₃ (Fig. 2A). The effects of both inhibitors on hormone-Ca²⁺ responses are likely to be due to suppression of TK activity. At the concentrations employed or higher (up to 370 µM), genistein has been previously shown not to alter cAMP-dependent kinase, protein kinase C (PKC), and phosphorylase kinase in other cell types (49-51). Moreover, daidzein, an inactive analog of genistein, at concentrations as high as 100 µM did not block the increase in muscle [Ca²⁺]_i caused by 1,25(OH)₂D₃ (153 ± 10 and 162 ± 7 nM, for 1,25(OH)₂D₃-treated cells in the absence and presence of daidzein, respectively, at the peak of the [Ca²⁺]_i response; basal values were 98 ± 11 nM). Herbimycin inhibits protein-tyrosine kinases with higher selectivity than genistein (52, 53). The addition of either inhibitor to the medium after the plateau phase of sterol-dependent changes in [Ca²⁺]_i had been reached did not affect further intracellular Ca²⁺ levels (Fig. 2B).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Changes in intracellular Ca2+ levels ([Ca2+]i) induced by 1,25(OH)2D3 in skeletal muscle cells. Cultured chick embryo skeletal muscle cells loaded with Fura-2 were exposed to 109 M 1,25(OH)2D3 (arrow) in Ca2+ (1.5 mM)-containing medium, and [Ca2+]i was determined, as described under "Experimental Procedures." A typical response to the hormone is seen with a fast rise in [Ca2+]i due to Ca2+ mobilization from intracellular stores and a sustained, long-lasting phase corresponding to the extracellular Ca2+ entry pathway. A representative time trace from five independent [Ca2+]i recordings is shown.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Effect of tyrosine kinase inhibitors on the 1,25(OH)2D3-evoked [Ca2+]i response in skeletal muscle cells. A, cultured chick embryo skeletal muscle cells loaded with Fura-2 were incubated for 2 min (left arrow) with 100 µM genistein (continuous trace) or 10 µM herbimycin (dotted trace) and then stimulated with 109 M 1,25(OH)2D3 (right arrow). Shown is a representative time-trace curve from five independent [Ca2+]i recordings. B, the cells were stimulated with 109 M 1,25(OH)2D3, and the hormone-dependent [Ca2+]i response was monitored until the plateau (Ca2+ entry) phase was reached. Then either the tyrosine kinase inhibitors genistein (Gen, 50-100 µM) or herbimycin (Herb, 10-50 µM) or vehicle (ethanol < 0.1%, Control bar) were added, and the measurement proceeded for an additional period of 5 min. The amplitude (%) of Ca2+ entry changes after the addition of either the tyrosine kinase inhibitors or ethanol (control) was compared with the initial condition (hormone-induced plateau, 100%). Bars represent averages ± S.D. of the number of experiments indicated (in parentheses) for each experimental condition. ns, statistically not significant.

The effect of tyrosine phosphatase inhibition on the muscle cell [Ca²⁺]_i response to 1,25(OH)₂D₃ was assayed using sodium orthovanadate (vanadate). In the presence of Ca²⁺ in the extracellular medium, 1 mM vanadate alone caused a more gradual increase in [Ca²⁺]_i than the hormone, which reached a plateau level (1.5-2-fold above basal values) 3 min after its addition, whereas no changes were detected when a Ca²⁺-free medium was used (Fig. 3A). These observations suggest that in skeletal muscle cells, inhibition of tyrosine phosphatase activity promotes influx of Ca²⁺ from the extracellular millieu but not mobilization from endogenous stores. More important, the addition of vanadate to the sustained phase of the 1,25(OH)₂D₃ [Ca²⁺]_i response produced no modification in the level of Ca²⁺ influx (Fig. 3B). Conversely, adding the sterol to the medium after the vanadate response reached the steady state had no effect on such a response (data not shown). Besides its effects on tyrosine phosphatases, vanadate has been shown to inhibit the Ca²⁺-ATPase of plasma membrane (54). However, the possibility that the vanadate-induced increase in muscle cytosolic Ca²⁺ may be due to Ca²⁺-ATPase inhibition is unlikely under our experimental conditions as genistein pretreatment of cells markedly reduced the vanadate-dependent increase in [Ca²⁺]_i (data not given).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Effect of the tyrosine phosphatase inhibitor vanadate on [Ca2+]i in skeletal muscle cells under basal conditions (A) and after 1,25(OH)2D3 treatment (B). A, cultured chick embryo skeletal muscle cells loaded with Fura-2 were exposed to 1 mM vanadate (arrow) in Ca2+-containing (1.5 mM, continuous trace) or Ca2+-free (<1 nM, dotted trace) medium, and [Ca2+]i was measured as described under "Experimental Procedures." Shown are representative time-trace curves from 4 (+Ca2+) and 3 (Ca2+) independent [Ca2+]i recordings. B, the cells were stimulated with 109 M 1,25(OH)2D3, and the hormone-dependent [Ca2+]i response was monitored until the plateau (Ca2+ entry) phase was reached. Then 1 mM vanadate was added, and the measurement proceeded for an additional period of 5 min. The amplitude (%) of Ca2+ entry changes after the addition of the tyrosine phosphatase inhibitor was compared with the initial condition (hormone-induced plateau, 100%). The number of experiments for each group is given in the graph bars. ns, statistically not significant.

To determine whether tyrosine phosphorylation of skeletal muscle cell proteins is modulated by the steroid hormone, cultured chick muscle cells were briefly (1 min) stimulated with 1,25(OH)₂D₃ (0.1-10 nM). As shown in Fig. 4, immunoprecipitation and Western blot analysis of cell lysates with a polyclonal antiserum reactive with phosphotyrosine residues revealed that the hormone causes a rapid increase in tyrosine phosphorylation of various cellular proteins. The effects of 1,25(OH)₂D₃ were concentration-dependent, with maximal stimulation achieved at 1 nM. Significant changes in phosphotyrosine-containing proteins of relative molecular masses of 42-44, 65, and 127 kDa were observed in response to the hormone. Proteins of 140 and 20 kDa were also tyrosine-phosphorylated but to a lesser extent. The 1,25(OH)₂D₃-induced increment of protein phosphorylation could be suppressed by the tyrosine kinase inhibitor genistein (50-100 µM).

View larger version (31K): [in this window] [in a new window]   Fig. 4.   1,25(OH)2D3 stimulates protein tyrosine phosphorylation in skeletal muscle cells. Cultured chick embryo skeletal muscle cells were exposed for 1 min to 0.1-10 mM 1,25(OH)2D3 in the absence or presence of genistein (50-100 µM). The cells were then lysed and immunoprecipitated with anti-phosphotyrosine (anti P-Tyr) antibody and protein A-Sepharose. The immunoprecipitates were analyzed by SDS-PAGE followed by anti-phosphotyrosine immunoblotting as described under "Experimental Procedures." A mixture of brain tyrosine-phosphorylated proteins was run in parallel as the positive control (Control +). A representative immunoblot from three independent experiments is shown.

MAPK or extracellular signal-regulated kinase consists of 42- and 44-kDa isoforms and requires both tyrosine and threonine phosphorylation for activation (55). To explore the possibility that 1,25(OH)₂D₃ phosphorylates MAP kinase in muscle cells, the membranes from the experiments of Fig. 4 were stripped and reprobed with anti-phospho-MAP kinase antibody, which recognizes both the 42- and 44-kDa species of active phosphorylated MAP kinase. As shown in Fig. 5A, MAPK co-migrated with the tyrosine-phosphorylated bands at an estimated molecular mass of 42/44 kDa. Marked increases in phosphorylation could be detected after treatment with 1 and 10 nM 1,25(OH)₂D₃; unlike the experiments of Fig. 4, in which the films were sobreexposed to visualize minor changes in protein tyrosine phosphorylation, basal MAPK levels were not detected when the anti-phospho-MAP kinase antibody was used. Genistein (100 µM) blocked to a great extent the maximal response observed at a hormone concentration of 1 nM (Fig. 5B).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   1,25(OH)2D3 phosphorylates MAP kinase in skeletal muscle cells. Cultured chick embryo skeletal muscle cells were exposed for 1 min to 0.1-10 nM 1,25(OH)2D3 in the absence or presence of genistein (50-100 µM). The cells were then lysed and immunoprecipitated with anti-phosphotyrosine (anti P-Tyr) antibody and protein A-Sepharose. After immunoblot analysis of the precipitate using anti-phosphotyrosine antibody (see Fig. 4), the membranes were stripped and reprobed with anti-phospho-MAP kinase antibody as described under "Experimental Procedures." A, representative immunoblot. B, quantification by scanning volumetric densitometry of blots from three independent experiments; averages ± S.D. are given. *, p < 0.001

To evaluate the time dependence of the hormone effects on MAP kinase tyrosine phosphorylation, proteins in lysates from muscle cells treated with 1 nM 1,25(OH)₂D₃ for 0.5-10 min were separated by SDS-PAGE and directly immunoblotted with anti-phospho-MAP kinase antibody. Fig. 6 shows that the hormone significantly increased MAPK tyrosine phosphorylation within 30 s, with highest stimulation reached at 1-2 min; at 5 and 10 min, the action of 1,25(OH)₂D₃ decayed. Immunoblotting with anti-MAPK antibody confirmed that equivalent amounts of MAPK were present in samples from control and 1,25(OH)₂D₃-treated cells (Fig. 6C).

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Time course of 1,25(OH)2D3-induced MAP kinase phosphorylation. Cultured chick embryo skeletal muscle cells were treated with 1 nM 1,25(OH)2D3 or vehicle ethanol (<0.01%) for 30 s10 min. After muscle cell lysis, comparable aliquots of lysate proteins were separated by SDS-PAGE followed by Western blotting with anti-phospho-MAP kinase as described under "Experimental Procedures." A, representative immunoblot. B, quantification by scanning volumetric densitometry of blots from three independent experiments; averages ± S.D. are given. *, p < 0.001; **, p < 0.005. C, the blotted membrane shown in panel A was stripped and re-probed with anti-extracellular signal-regulated kinase 1/2 (ERK 1/2) antibody to evaluate equivalence of MAPK content among the different experimental conditions.

The extracellular signal-regulated kinase family of MAP kinases is capable of phosphorylating myelin basic protein (56, 57). To further investigate whether 1,25(OH)₂D₃ stimulates MAP kinase activity, cells were exposed to the hormone followed by immunoprecipitation of the MAPK 42/44 kDa species with anti-phospho-MAPK antibody and assay of immunocomplexes for kinase activity in the presence of myelin basic protein as substrate. 1,25(OH)₂D₃ rapidly increased MAPK activity with kinetics roughly comparable with that of phosphorylation. As shown in Fig. 7A, maximal stimulation was achieved after 1 min of exposure to 1,25(OH)₂D₃; the hormone effects on enzyme activity fell between 2 and 10 min to control levels. Similarly to MAP kinase phosphorylation, the 1,25(OH)₂D₃-dependent increase in enzyme activity was completely abolished by the tyrosine kinase inhibitor genistein (Fig. 7B).

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Stimulation of chick skeletal muscle cell MAPK activity by 1,25(OH)2D3. A, time course. Muscle cells were treated with 1 nM 1,25(OH)2D3 for 0.5 s10 min. B, myoblasts were treated with 1 nM 1,25(OH)2D3 for 1 min in the presence or absence of 100 µM genistein. Cell lysates were immunoprecipitated with anti-phospho-MAPK antibody, and MAPK activity of the immunoprecipitate was measured using myelin basic protein as a substrate as described under "Experimental Procedures." Results are the average of three independent experiments performed in duplicate ± S.D. *, p < 0.001; **, p < 0.005.

As revealed by the experiments of Fig. 4, one of the proteins that underwent a significant 1,25(OH)₂D₃-dependent increase in tyrosine phosphorylation had a relative molecular mass of 127 kDa, which matches that of PLC. This isoform of polyphosphoinositide PLC is activated and associates to membranes by tyrosine phosphorylation (58, 59). To identify this macromolecule as PLC, lysates from muscle cells incubated with 1 nM 1,25(OH)₂D₃ for 1-10 min were immunoprecipitated with anti-PLC antibody followed by anti-phosphotyrosine immunoblotting. A marked stimulation (1.5-2-fold) in the band of 127 kDa by hormone treatment was observed (Fig. 8).

View larger version (33K): [in this window] [in a new window]   Fig. 8.   Stimulation of PLC tyrosine phosphorylation by 1,25(OH)2D3 in skeletal muscle cells. Cultured chick embryo skeletal muscle cells were incubated with 1 nM 1,25(OH)2D3 for the indicated times. The cells were then lysed and immunoprecipitated with anti-PLC antibody. The immunoprecipitate was analyzed by SDS-PAGE followed by anti-phosphotyrosine (anti P-Tyr) immunoblotting as described under "Experimental Procedures." A representative immunoblot from three independent experiments is shown.

In view of the role of MAPK in the regulation of cellular growth, studies were carried out to test whether the observed activation of MAPK by 1,25(OH)₂D₃ was involved in the mitogenic effects of the hormone in proliferating skeletal muscle cells. Fig. 9 shows that both genistein (100 µM) and compound PD98059 (10 µM), which inhibits MAPK activation by the dual MAPK kinase MEK, effectively blocked the increase in myoblast DNA synthesis caused by 1 nM 1,25(OH)₂D₃ during a 6-24-h treatment interval.

View larger version (15K): [in this window] [in a new window]   Fig. 9.   1,25(OH)2D3-induced DNA synthesis in skeletal muscle cells is suppressed by genistein and MAPK kinase (MEK) inhibitor PD98059. Chick embryo skeletal muscle cells cultured for 24 h (myoblasts) were incubated for 6 to 24 h with or without 1 nM 1,25(OH)2D3 in the presence or absence of PD9805 (10 µM) or genistein (100 µM). DNA synthesis was measured by [3H]thymidine incorporation as described under "Materials and Methods." Results are the average of four independent experiments ± S.D. **,p < 0.001; *,p < 0.05.

It has been previously shown that the proliferative effects of 1,25(OH)₂D₃ in muscle cells are accompanied by enhanced mRNA levels of the nuclear proto-oncoprotein c-myc (29), known to induce the expression of genes involved in cell growth stimulation. There is evidence indicating that c-myc function may be regulated by phosphorylation (60, 61). We attempted to determine whether 1,25(OH)₂D₃ stimulates tyrosine phosphorylation of c-myc, considering that another protein whose phosphotyrosine content was rapidly increased by the hormone had a relative molecular mass of ~65 kDa, similar to that of the oncoprotein. Lysates from muscle cells incubated with 1,25(OH)₂D₃ for 1-10 min were immunoprecipitated with a highly specific anti-c-myc monoclonal antibody followed by Western blotting with anti-phosphotyrosine antibody. In agreement with the results of Fig. 4, c-myc appeared as a band ranging between 64 and 67 kDa in several independent experiments (average ± S.D. = 65.1 ± 1.3), its phosphorylation being markedly increased by 1,25(OH)₂D₃ with respect to basal levels, e.g. 10-fold at 1 min and 50-fold after 5 and 10 min of sterol exposure, respectively (Fig. 10). In separate experiments, it was observed that pretreatment of muscle cells with the Src inhibitor PP1, both at 10 and 50 µM, completely suppressed hormone-dependent tyrosine phosphorylation of c-myc, whereas compound PD98059 was without effects (data not shown).

View larger version (25K): [in this window] [in a new window]   Fig. 10.   1,25(OH)2D3 phosphorylates the transcription factor c-myc in skeletal muscle cells. Chick embryo skeletal muscle cells cultured for 24 h (myoblasts) were exposed for 1-10 min to 1 nM 1,25(OH)2D3 or vehicle ethanol. The cells were then lysed, immunoprecipitated with anti-c-myc antibody followed by Western blotting with anti-phosphotyrosine (anti P-Tyr) antibody. A, representative immunoblot. B, quantification by scanning volumetric densitometry of blots from four independent experiments; averages ± S.D. are given. *, p < 0.001

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The results of the present investigation provide the first direct evidence involving TK activity in the regulation of intracellular Ca²⁺ homeostasis by 1,25(OH)₂-vitamin D₃. In colonocytes, this has been only indirectly suggested by the finding that tyrosine phosphorylation mediates sterol activation of PLC (62), known to increase [Ca²⁺]_i via inositol 1,4,5-trisphosphate generation. In our study, pretreatment of chick skeletal muscle cells with the TK inhibitors genistein and herbimycin abolished both the transient and sustained phases of the 1,25(OH)₂D₃ [Ca²⁺]_i response, which reflect mainly Ca²⁺ release from the sarcoplasmic reticulum and extracellular Ca²⁺ influx, respectively (11, 17, 23). Furthermore, vanadate, which inhibits protein-tyrosine phosphatases, also caused an increase in [Ca²⁺]_i. Of relevance, no additional increase in Ca²⁺ influx could be observed by adding 1,25(OH)₂D₃ or vanadate at the plateau level of the [Ca²⁺]_i response induced by either agent. The lack of effects of genistein and herbimycin, added when the sustained phase in [Ca²⁺]_i has been already triggered by 1,25(OH)₂D₃, suggests that once the TK-dependent Ca²⁺-signaling mechanism is activated by the hormone, subsequent events unrelated to protein tyrosine phosphorylation are responsible for keeping muscle cell cytosolic Ca²⁺ levels elevated.

The fact that pretreatment of muscle cells with genistein and herbimycin completely suppressed the changes in intracellular Ca²⁺ induced by 1,25(OH)₂D₃ indicates that tyrosine kinases mediate hormone stimulation of Ca²⁺ influx both through voltage-dependent and store-operated calcium channels. Sensitivity to TK inhibitors has been previously observed in various cell types with Ca²⁺-mobilizing agonists other than 1,25(OH)₂D₃ for either the VDCC (33, 34, 63, 64) or SOC channel (35-37)-mediated Ca²⁺ entry. The finding that 1,25(OH)₂D₃ rapidly (within 1 min) stimulates tyrosine phosphorylation of PLC (Fig. 8) strongly suggests that activation of this PLC isoform mediates, at least in part, inositol 1,4,5-trisphosphate-dependent Ca²⁺ release from inner stores, causing in turn the entry of extracellular Ca²⁺ through SOC channels. In addition, enhancement of PLC activity by 1,25(OH)₂D₃ may result, through diacylglycerol generation, in stimulation of PKC, which mediates sterol regulation of muscle cell VDCC (20). We have recently shown that hormone treatment of skeletal muscle cells induces a very fast increase in the activity of the non-receptor tyrosine kinase Src (65), a proximate activator of PLC (66, 67).

Altogether, these results suggest that protein tyrosine phosphorylation is a previously unrecognized mechanism that functions in concert with other membrane-signaling pathways (11, 16) to increase 1,25(OH)₂D₃-dependent intracellular Ca²⁺ levels in skeletal muscle cells. Further investigations are required to elucidate how interaction of 1,25(OH)₂D₃ at its primary site of action couples to the TK-mediated release of Ca²⁺ from intracellular stores and the influx through membrane Ca²⁺ channels. The intracellular vitamin D receptor itself may mediate the fast enhancement of tyrosine kinase activity in muscle cells. We have recently shown that 1,25(OH)₂D₃ significantly increases tyrosine phosphorylation of the vitamin D receptor, which is paralleled by association to and stimulation by tyrosine dephosphorylation of the non-receptor tyrosine kinase Src (65). The activation of Src and, in turn, of PLC by 1,25(OH)₂D₃ has been shown in rat colonocytes, but the intervention of the vitamin D receptor in the hormone effects was not demonstrated (62). Alternatively, we proposed (65) that a possible mechanism by which 1,25(OH)₂D₃ stimulates Src activity in muscle cells requires binding of 1,25(OH)₂D₃ to its cognate receptor, thus inducing a conformational change on this protein, which is then sensed by the receptor-associated Src.

This study demonstrates in addition that tyrosine kinase activity also plays a key role in the stimulation of skeletal muscle cell division by 1,25(OH)₂D₃. Anti-phosphotyrosine immunoblot analysis revealed that 1,25(OH)₂D₃ rapidly stimulates tyrosine phosphorylation of various muscle cell proteins, among which three major targets of the hormone of 42/44, 65, and 127 kDa could be identified as the growth-related proteins MAP kinase (extracellular signal-regulated kinase 1/2), c-myc, and PLC, respectively, on the basis of their immunoreactivity with corresponding selective antibodies. In the case of MAPK, the increase in phosphotyrosine content by 1,25(OH)₂D₃ was accompanied by an elevation of its enzymatic activity. In line with these observations, it has been recently reported that 1,25(OH)₂D₃ induces a rapid stimulation of MAP kinase phosphorylation in promyelocytic NB4 leukemia cells (40) and enterocytes (41).

Stimulation of the MAP kinase cascade may occur through activation of receptor tyrosine kinases or G protein-coupled receptors by stimulation of non-receptor Src kinases or by direct signaling to Raf via PKC (31, 32, 68-70). There is evidence that the effect of 1,25(OH)₂D₃ on the MAPK pathway in chick skeletal muscle cells involves a rapid increase in Src activity (65). Also, PKC partially mediates hormone stimulation of MAPK (71). This is in keeping with previous studies involving PKC in 1,25(OH)₂D₃ regulation of muscle cell proliferation (28, 30). The data obtained on sterol-induced protein tyrosine phosphorylation further imply that phospholipase C participates at least in part in the PKC-dependent mitogenic effect of 1,25(OH)₂D₃ in muscle. In our study, the fact that both genistein and the specific MEK inhibitor PD98059 blocked the ability of the hormone to stimulate DNA synthesis is consistent with the importance of the MAPK cascade in mediating the proliferative activity of 1,25(OH)₂D₃ in skeletal muscle cells. Upon activation by various extracellular stimuli, MAPK translocates to the nucleus, where it induces the expression of transcription factors involved in DNA synthesis and cell division such as the proto-oncogenes c-Fos and c-Jun (72).

The immunochemical identification of the 65-kDa protein phosphorylated in tyrosine in response to 1,25(OH)₂D₃ as the c-myc oncoprotein, which plays an essential role in cell cycle progression from G₁ into the S phase (73), represents a novel feature of this work not reported heretofore for any other agonist and cell type. In agreement with this experimental finding, by using the Prosite data base for protein consensus motifs (74), we detected within the sequence of chick (Gallus gallus) c-myc a putative tyrosine phosphorylation site corresponding to amino acids 15-21 (KNYDYDY), a region located within the N-terminal end of the oncogene transcriptional activation domain. Similar putative tyrosine phosphorylation sites were conservatively found in goat, mouse, and Xenopus laevis. The contribution of this phosphotyrosine residue to the functional activity of c-myc and its role in 1,25(OH)₂D₃ modulation of muscle cell growth remains to be determined.

The fact that the MAPK inhibitor PD98059 did not suppress the sterol-induced increase in c-Myc phosphotyrosine content, whereas the specific Src inhibitor PP1 completely abolished the effects of the hormone, indicates that tyrosine phosphorylation of c-myc by 1,25(OH)₂D₃ is independent of the MAPK pathway but involves Src kinase.

In conclusion, the results obtained, revealing that protein tyrosine phosphorylation is linked to 1,25(OH)₂D₃ regulation of muscle intracellular calcium homeostasis and cell proliferation, provide a new basis for understanding abnormalities in muscle contractility and growth associated with various vitamin D-related disorders such as renal osteodistrophy, chronic renal failure, and osteomalacia.

	FOOTNOTES

^* This research was supported by grants from the Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET), Agencia Nacional de Promocion Cientifica y Tecnologica, Comision de Investigaciones Cientificas de la Provincia de Buenos Aires (CIC), and the Universidad Nacional del Sur, Argentina.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. Biologia, Bioquimica y Farmacia, Universidad Nacional del Sur, San Juan 670, (8000) Bahia Blanca, Argentina. Tel.: 54-291-4595101; Fax: 54-291-4595130; E-mail: rboland@criba.edu.ar.

Published, JBC Papers in Press, August 29, 2000, DOI 10.1074/jbc.M002025200

	ABBREVIATIONS

The abbreviations used are: 1, 25(OH)₂D₃, 1,25-dihydroxy-vitamin D₃; TK, tyrosine kinase; PLC, phospholipase C; VDCC, voltage-dependent Ca²⁺ channels; SOC, store-operated Ca²⁺; MAPK, mitogen-activated protein kinase; PKC, protein kinase C; PAGE, polyacrylamide gel electrophoresis.

	REFERENCES

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