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

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 1α,25(OH)2-vitamin D3 (1α,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 1α,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 Cγ (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 1α,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 1α,25(OH)2D3 regulation of Ca2+homeostasis in hormone target cells. In addition, the data involve tyrosine kinase cascades in the mitogenic effects of 1α,25(OH)2D3 on skeletal muscle cells.

1␣,25-Dihydroxy-vitamin D 3 (1␣,25(OH) 2 D 3 ) 1 in addition to its classical role in the regulation of extracellular calcium homeostasis, modulates cell proliferation and differentiation and the immune system (1)(2)(3)(4)(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) 2 D 3 (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 Ca 2ϩ metabolism, contractility, and growth (9 -11). As in other target cells (12)(13)(14)(15), 1␣,25(OH) 2 D 3 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) 2 D 3 in muscle cells involve G protein-coupled stimulation of adenylyl cyclase and phospholipases C, D, and A 2 and activation of protein kinases A and C which, in turn regulate the activity of voltage-dependent Ca 2ϩ channels (VDCC) (17)(18)(19)(20)(21)(22). The hormone also promotes Ca 2ϩ mobilization from intracellular stores and modulates store-operated Ca 2ϩ (SOC) channels as part of the 1␣,25(OH) 2 D 3 -induced Ca 2ϩ entry across the plasma membrane of skeletal muscle cells (23,24). The rapid nature and specificity by which 1␣,25(OH) 2 D 3 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) 2 D 3 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) 2 D 3 , 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 Ca 2ϩ entry both through the VDCC (33,34) and SOC channel (35)(36)(37) pathways. Variations in cytosolic Ca 2ϩ 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) 2 D 3 -dependent modulation of nongenomic responses, such as fast increases in cytosolic Ca 2ϩ and MAPK stimulation (39). Accordingly, it has been recently reported that 1␣,25(OH) 2 D 3 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) 2 D 3 regulates cytoplasmic Ca 2ϩ and exerts mitogenic effects in skeletal muscle cells and investigated hormone-dependent related changes in protein tyrosine phosphorylation.

EXPERIMENTAL PROCEDURES
Chemicals-1␣,25(OH) 2 D 3 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-dayold 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/ cm 2 ) in Petri dishes (80 mm in diameter) for tyrosine phosphorylation and MAPK assays or onto glass coverslips (24 ϫ 6 mm) for intracellular calcium measurements and cultured at 37°C under a humidified atmosphere (air, 5% CO 2 ). 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 Ci of [ 3 H]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 Ca 2ϩ changes were monitored using the Ca 2ϩ -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 MgCl 2 , 5 mM glucose, 10 mM Hepes, pH 7.4, 1.5 mM CaCl 2 plus 0.1% bovine serum albumin, 4 M 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 ϭ 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 [Ca 2ϩ ] 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 M ionomycin plus 3 mM Ca 2ϩ and 10 mM EGTA, pH 9.0, respectively. Under these conditions of measurement, the dissociation constant (K d ) for the Ca 2ϩ -Fura-2 complex was assumed to be 224 nM, and [Ca 2ϩ ] i according to the algorithm of Grynkiewicz et al. (44) derives from [Ca 2ϩ ] i ϭ K d (R Ϫ R min )/(R max Ϫ R) ϫ ␤, where R is the ratio of Fura-2 fluorescence at the selected wavelengths, R max and R min represent ratios from Ca 2ϩ -saturated and Ca 2ϩ -free intracellular dye, respectively, and ␤ is the ratio between the specific fluorescence of the Ca 2ϩ -free and Ca 2ϩ -bound forms of the dye at the longer wavelength (Sf 2 / Sb 2 ).
In some experiments, a Ca 2ϩ -free extracellular medium was used. In such situations, the absence of Ca 2ϩ in the medium means free Ca 2ϩ concentration near 1 nM, which is accomplished by preparing a nominally Ca 2ϩ -free buffer B (see composition above) plus 1 mM EGTA. Free Ca 2ϩ levels were calculated by using the WinMaxc program, version 1.7 (45). All buffers and saline solutions used were prepared with deionized water.
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.
Statistical Analysis-Statistical significance of the data was evaluated using Student's t test (48), and probability values below 0.05 (p Ͻ 0.05) were considered significant. Results are expressed as means Ϯ S.D. from the indicated set of experiments.

RESULTS
Stimulation of chick embryonic skeletal muscle cells with 1␣,25(OH) 2 D 3 triggers a rapid (30 s) and sustained increment in intracellular calcium concentration ([Ca 2ϩ ] i ) that persists elevated as long as the cells are exposed to the hormone (Fig. 1). We have previously shown that the rapid initial [Ca 2ϩ ] i response to the sterol mainly results from inositol 1,4,5-trisphosphate-mediated mobilization of Ca 2ϩ from a thapsigargin-sensitive store, whereas the plateau phase is entirely due to Ca 2ϩ 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 [Ca 2ϩ ] i regulation by 1␣,25(OH) 2 D 3 , we first examined the effect of TK inhibition on the hormone-generated variations in intracellular Ca 2ϩ . Pretreatment of myoblasts with the tyrosine kinase inhibitors genistein (50 -100 M) and herbimycin (10 -50 M) completely prevented any subsequent response to 1␣,25(OH) 2 D 3 ( Fig. 2A). The effects of both inhibitors on hormone-Ca 2ϩ responses are likely to be due to suppression of TK activity. At the concentrations employed or higher (up to 370 M), 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 M did not block the increase in muscle [Ca 2ϩ ] i caused by 1␣,25(OH) 2 D 3 (153 Ϯ 10 and 162 Ϯ 7 nM, for 1␣,25(OH) 2 D 3 -treated cells in the absence and presence of daidzein, respectively, at the peak of the [Ca 2ϩ ] i response; basal values were 98 Ϯ 11 nM). Herbimycin inhibits proteintyrosine 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 [Ca 2ϩ ] i had been reached did not affect further intracellular Ca 2ϩ levels (Fig. 2B).
The effect of tyrosine phosphatase inhibition on the muscle cell [Ca 2ϩ ] i response to 1␣,25(OH) 2 D 3 was assayed using sodium orthovanadate (vanadate). In the presence of Ca 2ϩ in the extracellular medium, 1 mM vanadate alone caused a more gradual increase in [Ca 2ϩ ] 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 Ca 2ϩ -free medium was used (Fig. 3A). These observations suggest that in skeletal muscle cells, inhibition of tyrosine phosphatase activity promotes influx of Ca 2ϩ 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) 2 D 3 [Ca 2ϩ ] i response produced no modification in the level of Ca 2ϩ 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 Ca 2ϩ -ATPase of plasma membrane (54). However, the possibility that the vanadate-induced increase in muscle cytosolic Ca 2ϩ may be due to Ca 2ϩ -ATPase inhibition is unlikely under our experimental conditions as genistein pretreatment of cells markedly reduced the vanadate-dependent increase in [Ca 2ϩ ] i (data not given).
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) 2 D 3 (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) 2 D 3 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) 2 D 3 -induced increment of protein phosphorylation could be suppressed by the tyrosine kinase inhibitor genistein (50 -100 M).
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) 2 D 3 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) 2 D 3 ; 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 M) blocked to a great extent the maximal response observed at a hormone concentration of 1 nM (Fig. 5B).
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) 2 D 3 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) 2 D 3 decayed. Immunoblotting with anti-MAPK antibody confirmed that equivalent amounts of MAPK were present in samples from control and 1␣,25(OH) 2 D 3 -treated cells (Fig. 6C).
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) 2 D 3 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) 2 D 3 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) 2 D 3 ; the hormone effects on enzyme activity fell between 2 and 10 min to control levels. Similarly to MAP kinase phosphorylation, the 1␣,25(OH) 2 D 3 -dependent increase in enzyme activity was completely abolished by the tyrosine kinase inhibitor genistein (Fig. 7B).
As revealed by the experiments of Fig. 4, one of the proteins that underwent a significant 1␣,25(OH) 2 D 3 -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) 2 D 3 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).

FIG. 3. Effect of the tyrosine phosphatase inhibitor vanadate on [Ca 2؉ ] i in skeletal muscle cells under basal conditions (A) and after 1␣,25(OH) 2 D 3 treatment (B).
A, cultured chick embryo skeletal muscle cells loaded with Fura-2 were exposed to 1 mM vanadate (arrow) in Ca 2ϩ -containing (1.5 mM, continuous trace) or Ca 2ϩ -free (Ͻ1 nM, dotted trace) medium, and [Ca 2ϩ ] i was measured as described under "Experimental Procedures." Shown are representative time-trace curves from 4 (ϩCa 2ϩ ) and 3 (ϪCa 2ϩ ) independent [Ca 2ϩ ] i recordings. B, the cells were stimulated with 10 Ϫ9 M 1␣,25(OH) 2 D 3 , and the hormonedependent [Ca 2ϩ ] i response was monitored until the plateau (Ca 2ϩ entry) phase was reached. Then 1 mM vanadate was added, and the measurement proceeded for an additional period of 5 min. The amplitude (%) of Ca 2ϩ entry changes after the addition of the tyrosine phosphatase inhibitor was compared with the initial condition (hormoneinduced plateau, 100%). The number of experiments for each group is given in the graph bars. ns, statistically not significant.

FIG. 4. 1␣,25(OH) 2 D 3 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) 2 D 3 in the absence or presence of genistein (50 -100 M). 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 tyrosinephosphorylated proteins was run in parallel as the positive control (Control ϩ). 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) 2 D 3 was involved in the mitogenic effects of the hormone in proliferating skeletal muscle cells. Fig. 9 shows that both genistein (100 M) and compound PD98059 (10 M), 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) 2 D 3 during a 6 -24-h treatment interval.
It has been previously shown that the proliferative effects of 1␣,25(OH) 2 D 3 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) 2 D 3 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 ϳ65 kDa, similar to that of the oncoprotein. Lysates from muscle cells incubated with 1␣,25(OH) 2 D 3 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 Ϯ S.D. ϭ 65.1 Ϯ 1.3), its phosphorylation being markedly increased by 1␣,25(OH) 2 D 3 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 M, completely suppressed hormone-dependent tyrosine phosphorylation of c-myc, whereas compound PD98059 was without effects (data not shown).

DISCUSSION
The results of the present investigation provide the first direct evidence involving TK activity in the regulation of intracellular Ca 2ϩ homeostasis by 1␣,25(OH) 2 -vitamin D 3 . In colonocytes, this has been only indirectly suggested by the finding that tyrosine phosphorylation mediates sterol activation of PLC␥ (62), known to increase [Ca 2ϩ ] i via inositol 1,4,5trisphosphate 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) 2 D 3 [Ca 2ϩ ] i response, which reflect mainly Ca 2ϩ release from the sarcoplasmic reticulum and extracellular Ca 2ϩ influx, respectively (11,17,23). Furthermore, vanadate, which inhibits protein-tyrosine phosphatases, also caused an increase in [Ca 2ϩ ] i . Of relevance, no additional increase in Ca 2ϩ influx could be observed by adding 1␣,25(OH) 2  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 Ϯ S.D. are given. *, p Ͻ 0.001; **, p Ͻ 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. triggered by 1␣,25(OH) 2 D 3 , suggests that once the TK-dependent Ca 2ϩ -signaling mechanism is activated by the hormone, subsequent events unrelated to protein tyrosine phosphorylation are responsible for keeping muscle cell cytosolic Ca 2ϩ levels elevated.
The fact that pretreatment of muscle cells with genistein and herbimycin completely suppressed the changes in intracellular Ca 2ϩ induced by 1␣,25(OH) 2 D 3 indicates that tyrosine kinases mediatehormonestimulationofCa 2ϩ influxboththroughvoltagedependent and store-operated calcium channels. Sensitivity to TK inhibitors has been previously observed in various cell types with Ca 2ϩ -mobilizing agonists other than 1␣,25(OH) 2 D 3 for either the VDCC (33,34,63,64) or SOC channel (35-37)mediated Ca 2ϩ entry. The finding that 1␣,25(OH) 2 D 3 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 Ca 2ϩ release from inner stores, causing in turn the entry of extracellular Ca 2ϩ through SOC channels. In addition, enhancement of PLC␥ activity by 1␣,25(OH) 2 D 3 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) 2 D 3 -dependent intracellular Ca 2ϩ levels in skeletal muscle cells. Further investigations are required to elucidate how interaction of 1␣,25(OH) 2 D 3 at its primary site of action couples to the TK-mediated release of Ca 2ϩ from intracellular stores and the influx through membrane Ca 2ϩ 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) 2 D 3 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) 2 D 3 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) 2 D 3 stimulates Src activity in muscle cells requires binding of 1␣,25(OH) 2 D 3 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) 2 D 3 . Anti-phosphotyrosine immunoblot analysis revealed that 1␣,25(OH) 2 D 3 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) 2 D 3 was accompanied by an elevation of its enzymatic activity. In line with these observations, it has been recently reported that 1␣,25(OH) 2 D 3 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) 2 D 3 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) 2 D 3 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) 2 D 3 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) 2 D 3 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) 2 D 3 as the c-myc oncoprotein, which plays an essential role in cell cycle progression from G 1 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) 2 D 3 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) 2 D 3 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) 2 D 3 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.