Nongenomic action of 1a,25(OH)2-vitamin D3

Activation of muscle cell PLCc through the tyrosine kinase c-Src and PtdIns 3-kinase

Claudia Buitrago, Veronica Gonzalez Pardo and Ana Russo de Boland

Department Biologıa, Bioquımica & Farmacia. Universidad Nacional del Sur, San Juan Bahia Blanca, Argentina

We have previously demonstrated that the steroid hormone1a,25(OH)2-vitamin D3 [1a,25(OH)2D3] stimulates theproduction of inositol trisphosphate (InsP3), the breakdownproduct of phosphatidylinositol 4,5-biphosphate (PtdInsP2)by phospholipase C (PtdIns-PLC), and activates the cyto-solic tyrosine kinase c-Src in skeletal muscle cells. In thepresent study we examined whether 1a,25(OH)2D3 inducesthe phosphorylation and membrane translocation of PLCcand the mechanism involved in this isozyme activation. Wefound that the steroid hormone triggers a significant phos-phorylation on tyrosine residues of PLCc and induces arapid increase in membrane-associated PLCc immunoreac-tivity with a time course that correlates with that of phos-phorylation in muscle cells. Genistein, a tyrosine kinaseinhibitor, blocked the phosphorylation of PLCc. Inhibitionof 1a,25(OH)2D3-induced c-Src activity by its specificinhibitor PP1 or muscle cell transfection with an antisenseoligodeoxynucleotide directed against c-Src mRNA, pre-vented hormone stimulation of PLCc tyrosine phosphory-lation. The isozyme phosphorylation is also blocked by both

wortmannin and LY294002, two structurally differentinhibitors of phosphatidyl inositol 3-kinase (PtdIns3K), theenzyme that produces PtdInsP3 known to activate PLCcisozymes specifically by interacting with their SH2 andpleckstrin homology domains. The hormone also increasesthe physical association of c-Src and PtdIns3K with PLCcand induces a c-Src-dependent tyrosine phosphorylation ofthe p85 regulatory subunit of PtdIns3K. The time course ofhormone-dependent PLCc phosphorylation closely corre-lates with the time course of its redistribution to the mem-brane, suggesting that phosphorylation and redistribution tothe membrane of PLCc are two interdependent events.1a,25(OH)2D3-induced membrane translocation of PLCcwas prevented to a great extent by c-Src and PtdIns3Kinhibitors, PP1 and LY294002. Taken together, the presentdata indicates that the cytosolic tyrosine kinase c-Src andPtdIns 3-kinase play indispensable roles in 1a,25(OH)2D3

signal transduction cascades leading to PLCc activation.

Keywords: c-Src; 1a,25(OH)2D3; PtdIns3K; PLCc.

Studies with animal models and cultured muscle cells haveshown that the hormonal form of vitamin D, 1a,25(OH)2-vitamin D3 [1a,25(OH)2D3], exerts direct effects on skeletalmuscle Ca2+ metabolism, contractility and growth [1–3]. Asin other target cells [4–7], 1a,25(OH)2D3 elicits responses inmuscle cells both through nuclear receptor-mediated genetranscription and a fast mechanism independent of newRNAandprotein synthesis [3,8]. The nongenomic actions of1a,25(OH)2D3 in muscle cells involve G protein-coupledstimulationof adenylyl cyclase andphospholipasesC,DandA2 and activation of protein kinases A and C which in turn

regulate the activity of voltage-dependent Ca2+ channels[9–14]. The hormone also promotesCa2+ mobilization fromintracellular stores and modulates store-operated Ca2+

channels as part of the 1a,25(OH)2D3-induced Ca2+ entryacross the plasma membrane of skeletal muscle cells [15,16].The hormone rapidly stimulates the hydrolysis of phospha-tidylinositol 4,5-biphosphate (PtdInsP2) by activation ofphospholipase C (PtdIns-PLC) to yield the two secondmessengers inositol trisphosphate (InsP3) and diacylglycerol[9,10,13]. Both products of PLC catalysis mediate release ofintracellular calcium and were associated with activation ofthe Ca2+-dependent a isoform of PKC [17]. The rapidnature and specificity by which 1a,25(OH)2D3 activatesthese second messenger pathways suggest that interactionwith a plasma membrane receptor is responsible for theinitiation of its effects. The existence of a novel membranereceptor received first experimental support in enterocytes[18] and more recently in chondrocytes [19]. The presence ofmembrane binding sites for 1a,25(OH)2D3 in skeletal muscle[20] aswell as for other steroid hormones in various cell typeshas been described [21].

Ten mammalian phosphoinositide-specific PLC isozymeshave been reported: PLCb1–4, PLCc1–2 and PLCd1–4 [22].These isoforms are distinguished by their mode of regula-tion. Association with heterotrimeric G protein subunitsstimulates the b-class of isozymes while the c-class isregulated by tyrosine phosphorylation, phosphatidylinositol3-kinase (PtdIns3K), or other signalling pathways [23].

Correspondence to A. Russo de Boland, Department Biologia,

Bioquimica & Farmacia, Universidad Nacional del Sur, San Juan

670, (8000) Bahia Blanca, Argentina.

Fax: + 54 291 595130, Tel.: + 54 291 595100 ext 2430,

E-mail: aboland@criba.edu.ar

Abbreviations: 1a,25(OH)2D3, vitamin D3; PLC, phospholipase C;

InsP3, inositol trisphosphate; PtdInsP2, phosphatidylinositol

4,5-biphosphate; PtdIns3K, phosphatidylinositol 3-kinase;

DMEM, Dulbecco’s modified Eagle’s medium; ECL, enhanced

chemiluminiscence; DAPI, 4,6-diamidino-2-phenylindole; ODN,

oligodeoxynucleotides; PKA, cAMP-dependent kinase.

Enzymes: phospholipase Cc (EC 3.1.4.3); c-Src (EC 2.7.1.112);

phosphatidyl inositol 3-kinase (EC 2.7.1.137).

(Received 9 October 2001, revised 14 February 2002,

accepted 4 April 2002)

Eur. J. Biochem. 269, 2506–2515 (2002) � FEBS 2002 doi:10.1046/j.1432-1033.2002.02915.x

Regulation of the d-class of isoforms has not beencharacterized fully [24].

In this study, we investigated the effects of 1a,25(OH)2D3

on the tyrosine phosphorylation of PLCc and the potentialrole of the nonreceptor tyrosine kinase c-Src and PtdIns3Kin the hormone signalling cascade leading to this isozymeactivation.

M A T E R I A L S A N D M E T H O D S

Chemicals

1a,25(OH)2D3 was supplied by H. Bachmann (Hoffmann-La Roche Ltd, Basel, Switzerland). Protein A–Sepharose,Dulbecco’s modified Eagle’s medium (DMEM), and fetalbovine serum were from Sigma Chemical Co. Wortmanninand LY294002 were from Alomone Laboratories (Jerusa-lem, Israel). Lipofectin was from Gibco BRL. PP1 was fromCalbiochem. Rabbit polyclonal anti-phosphotyrosine Ig,anti-PLCc Ig, anti-(c-Src) Ig and anti-PtdIns3K Ig (p85)were from Santa Cruz Biotechnology Inc. The secondaryantibody, horse radish peroxidase-conjugated goat anti-(rabbit IgG) Ig and the Super Signal CL-HRP substratesystem for enhanced chemiluminiscence (ECL) were fromAmersham Corp. 4,6-diamidino-2-phenylindole (DAPI)and secondary antibody Alexa 546-conjugated goat anti-(rabbit IgG) Ig were a generous gift of L. E. Politti(INIBIBB, Argentina), and were from Molecular ProbesInc. Antisense oligodeoxynucleotides were synthesized bythe DNAgency (Malvern, PA, USA). All other reagentswere of analytical grade.

Cell culture

Chick skeletal muscle cells (myoblasts) were obtained from13-day-old chick embryo breast muscles by stirring in Earl’sbalanced salt solution containing 0.06% trypsin for 30 minessentially as described previously [25]. The freed cells werecollected by centrifugation and the pellet was resuspended inDMEM supplemented with 10% fetal bovine serum andantibiotic–antimycotic solution. The suspension was dis-persed by pipetting, filtered through nylon mesh andpreplated on gelatin-coated Petri dishes to remove contam-inating fibroblasts. The unadsorbed cells were seeded at anappropriate density (120 000 cellsÆcm)2) in Petri dishes(100 mm diameter) and cultured at 37 �C under a humid-ified atmosphere (air/5% CO2). Cells were allowed to growuntil confluence before use. Under these conditions, musclecells proliferate within the first 48 h and at day 4 becomedifferentiated into myotubes expressing both biochemicaland morphological characteristics of adult skeletal musclefibrer [17].

Immunoprecipitation

After treatment, muscle cells were lysed (15 min at 4 �C) in50 mM Tris/HCl pH 7.4, 150 mM NaCl, 2 mM EGTA,25 mM NaF, 0.2 mM sodium orthovanadate, 1 mM

phenylmethanesulfonyl fluoride, 2 lgÆmL)1 leupeptin,2 lgÆmL)1 pepstatin, 2 lgÆmL)1 aprotinin, 0.25% sodiumdeoxycholate and 1% NP-40. Insoluble material waspelleted in a microcentrifuge at 18 000 g for 10 min at4 �C. The protein content of the clear lysates was

determined according to Lowry [26]. Aliquots (500–700 lgprotein) were incubated overnight at 4 �C with the corres-ponding antibody, followed by precipitation of the com-plexes with protein A–Sepharose. The immune complexeswere washed four times with cold immunoprecipitationbuffer (10 mM Tris/HCl pH 7.4, 150 mM NaCl, 1 mM

EGTA, 1 mM EDTA, 0.2 mM phenylmethanesulfonylfluoride, 0.2 mM sodium orthovanadate, 1% Triton X-100and 1% NP-40) and once with NaCl/Pi.

SDS/PAGE and immunoblotting

Immunoprecipitated proteins (or lysate proteins) dissolvedin Laemmli sample buffer were separated by SDS/PAGE(8% acrylamide) [27], and electrotransferred to polyviny-lidene difluoride membranes. The membranes were blockedfor 1 h at room temperature in TBST buffer (50 mM Tris/HCl pH 7.4, 200 mM NaCl, 1% Tween-20) containing 1%dry milk. For the detection of proteins, membranes weresubjected to immunoblotting using a rabbit anti-phospho-tyrosine Ig. Next, the membranes were washed three timesin TBST, incubated with a 1 : 10 000 dilution of peroxi-dase-conjugated anti-(rabbit IgG) Ig for 1 h at roomtemperature and washed three additional times with TBST.The membranes were then visualized using ECL accordingto the manufacturer’s intructions. Images were obtainedwith a model GS-700 Imaging Densitomer from Bio-Radby scanning at 600 d.p.i. and printing at the same resolu-tion. Bands were quantified using the MOLECULAR ANALYST

program (Bio-Rad).

Co-immunoprecipitation

Co-immunoprecipitation assays were performed undernative conditions in order to preserve protein–proteinassociations, and were conducted essentially as describedpreviously [28,29], with minor modifications. Briefly, afterhormone treatment, cells were lysed (15 min at 4 �C) in50 mM Tris/HCl pH 7.4, 150 mM NaCl, 3 mM KCl,0.5 mM EDTA, 0.2 mM sodium orthovanadate, 1 mM

NaF, 1 mM phenylmethanesulfonyl fluoride, 6 lgÆmL)1

leupeptin, 8 lgÆmL)1 aprotinin, 1% Tween-20. Lysates wereclarified by centrifugation (14 000 g, 10 min at 4 �C) andimmunoprecipitation on the supernatants was performedwith the indicated antibodies as described above, except thatprecipitated immunocomplexes were washed four timeswith NaCl/Pi.

Immunocytochemistry assays

Cultured cellswere fixedwith 2%paraformaldehyde for 2 h.After two washes with NaCl/Pi, the cells were permeabilizedwith 0.1% Triton for 15 min, washed with NaCl/Pi andincubated with anti-PLCc Ig for 1 h at room temperature.The cells were then washed three times with NaCl/Pi andincubated with the secondary antibodyAlexa 546-conjugat-ed goat anti-(rabbit IgG) Ig for 1 h at room temperature.Integrity of the nuclei was determined by fluorescencemicroscopy, using DAPI in the last 20 min of the incubationwith the secondary antibody.Analysiswas carried outwith aNicon Eclipse 600 mounted on a fluorescence microscopeequipped with a 40· pan fluor objective. High-resolutionfluorescence images were obtained by epifluorescence.

� FEBS 2002 1a,25(OH)2-Vitamin D3 stimulation of PLCc (Eur. J. Biochem. 269) 2507

Cell fractionation

Cells were lysed in 10 mM Tris/HCl pH 7.4; 0.3 M sucrose,1 mM EGTA, 1 mM NaF, 1 mM NaVO4, 1 mM phenyl-methylsulfonyl fluoride, 5 mM dithiothreitol, 0.2 mM

sodium orthovanadate, 20 lgÆmL)1 leupeptin and20 lgÆmL)1 aprotinin. The lysate was sonicated for 15 s,centrifuged at 1000 g for 10 min in a Sorvall refrigeratedcentrifuge; the pellet obtained was discarded, the superna-tant was centrifuged at 110 000 g for 60 min in a Beckmanultracentrifuge. The pellet (membranes) was washed twice inthe same buffer and the protein content of the pellet and thecytosolic fraction was measured according to Lowry [26].

Cell transfection

Transfection with oligodeoxynucleotides (ODNs) usingLipofectin was performed according to manufacturer’sinstructions. As in previous studies [30,31], ODNs wereincubated with Lipofectin in DMEM for 15 min at roomtemperature. Plates of subconfluent cells were washed toremove serum before addition of ODN–Lipofectin mixturesand incubation was performed for 12 h at 37 �C. The ODNsolution was removed, DMEM (1% serum) was added andthe plates were placed into a metabolic incubator for afurther 36 h. Control treatments included DMEM orLipofectin only. Dose- and time-response curves for Lipo-fectin and ODN were previously optimized (not shown).

The following ODN sequence with phosphorothioatelinkages throughout the entire molecule was used: anti-(c-Src), 5¢-CACCACCATGGGGAGCAGCA-3¢ (antisenseagainst the 95–114 nucleotide sequence containing the AUGregion from Gallus gallus c-Src mRNA).

Statistical analysis

Statistical significance of the data was evaluated usingStudent’s t-test [32] and P < 0.05 was considered signifi-cant. Results are expressed as means ± SD from theindicated set of experiments.

R E S U L T S A N D D I S C U S S I O N

PLCc is a cytoplasmic enzyme that, in order to hydrolysePtdInsP2 needs to both translocate to the membrane whereits substrate resides and undergo an increase in its intrinsiccatalytic potential [33,34]. Tyrosine phosphorylation ofPLCc is an obligatory step that augments its catalyticactivity [33]. In this study, we investigated the mechanisminvolved in the tyrosine phosphorylation of this isozyme by1a,25(OH)2D3. To evaluate the time-dependence of thehormone effects on PLCc tyrosine phosphorylation, pro-teins in lysates from muscle cells treated with up to 10)9

M

1a,25(OH)2D3 for 30 s)10 min were immunoprecipitatedwith anti-PLCc Ig, separated by SDS/PAGE and immu-noblotted with anti-phosphotyrosine Ig. As shown inFig. 1, we found, in agreement with a previous observation[35], that treatment of muscle cells with 1a,25(OH)2D3

markedly increased PLCc-tyrosine phosphorylation. Theeffect was already significant at 30 s (+100%), andreached a maximum at 1 min (+400%). When the orderof antibody addition was reversed and anti-phosphotyro-sine Ig immunoprecipitates were probed with anti-PLCc Ig,

a similar time-course of PLCc tyrosine phosphorylationwas detected (data not shown). To verify that the observedchanges in tyrosine phosphorylation did not reflect differ-ences in the amounts of PLCc precipitated by the anti-PLCc Ig, in the above experiments the band correspondingto IgG heavy chains was quantified. Equal amounts of IgGwere shown to be precipitated by the antibody at each ofthe time points. In addition, dose–response studies evi-denced higher effectiveness of 10)9

M 1a,25(OH)2D3 toincrease PLCc phosphorylation whereas decreasing theconcentration of the hormone to 10)10 and 10)11

M

diminished its potency after 1 min of treatment (Fig. 2).Pretreatment of muscle cells with the tyrosine kinaseinhibitor genistein (100 lM) completely prevented1a,25(OH)2D3-induced phosphorylation of PLCc, whiledaidzein, an inactive analogue of genistein, at concentra-tions as high as 100 lM did not block the increase in PLCcphosphorylation caused by 1a,25(OH)2D3 (Fig. 3). At theconcentrations used or higher (up to 370 lM) genistein hasbeen previously shown not to alter cAMP-dependentkinase (PKA), PKC and phosphorylase kinase in othercell types [36–38]. Moreover, herbimycin, an irreversibleand select protein tyrosine kinase inhibitor, at concentra-tions of 10–50 lM completely prevented any subsequentresponse to 1a,25(OH)2D3 (data not shown).

Two structurally related PLCc isozymes PLCc1 andPLCc2 have been identified [39]. Both isozymes have been

Fig. 1. Time course of 1a,25(OH)2D3-induced PLCc phosphorylation.

Skeletal muscle cells were treated with 10)9M 1a,25(OH)2D3 for

30 s)10 min. After cell lysis and immunoprecipitation with anti-PLCcIg, immunoprecipitated proteins were separated by SDS/PAGE

followed by Western blotting with anti-phosphotyrosine Ig as

described in Materials and methods. (A) Representative immunoblot.

(B) Quantification by scanning volumetric densitometry of blots from

three independent experiments; means ± SD are given. *P < 0.05,

**P < 0.01 with respect to control unstimulated cells.

2508 C. Buitrago et al. (Eur. J. Biochem. 269) � FEBS 2002

found in association with several signalling molecules,including kinases of the Src and Syk families [40–42]. Wehave recently demonstrated that 1a,25(OH)2D3 rapidlystimulates the enzymatic activity of c-Src tyrosine kinase inembryonic skeletal muscle cells [43]; the hormone alsoactivates c-Src in rat colonocytes [41] and human keratino-cytes [44]. In avian skeletal muscle cells 1a,25(OH)2D3

stimulates c-Src kinase activity, at least in part, by alteringthe tyrosine phosphorylation state of the enzyme [41] and inrat colonocytes the hormone activates c-Src by bothphosphorylation-dependent and -independent mechanisms[44]. Despite the large number of molecules shown tointeract with PLCc isozymes, the mechanism of PLCcactivation by 1a,25(OH)2D3 is not known in avianskeletal muscle cells, and is incompletely understood inother systems. To evaluate whether c-Src is part of1a,25(OH)2D3 signalling mechanism involving PLCc inavian skeletal muscle cells, we first investigated the effect ofthe Src family tyrosine kinase-selective inhibitor PP1 [45] onPLCc phosphorylation induced by the hormone. To thatend, cells were pretreated with 10 lM PP1 (the concentra-tion required to inhibit c-Src activity in skeletal muscle cells[43]), followed by exposure to 10)9

M 1a,25(OH)2D3 for1 min. Under these conditions, the effects of the hormoneon PLCc phosphorylation were abolished (Fig. 4). It hasbeen reported that rat colonocyte PLCc associates withc-Src upon 0.5 min treatment of cells with 1a,25(OH)2D3

[41]. Therefore, we next examined the physical associationof PLCc and the tyrosine kinase c-Src. To that end, PLCcwas immunoprecipitated from cell lysates obtained from

Fig. 2. 1a,25(OH)2D3 stimulates PLCc phosphorylation in a dose-

dependent fashion.Muscle cells were exposed for 1 min to 10)8)10)11M

1a,25(OH)2D3. The cells were then lysed and immunoprecipitated with

anti-PLCc Ig and protein A–Sepharose. The immunoprecipitates were

analysed by SDS/PAGE followed by anti-phosphotyrosine Ig immu-

noblotting as described in Materials and methods. (A) Representative

immunoblot. (B) Quantification by scanning volumetric densitometry

of blots from three independent experiments; means ± SD are given.

*P < 0.05, **P < 0.01 with respect to control unstimulated cells.

Fig. 3. Effect of genistein and daidzein on 1a,25(OH)2D3-induced

PLCc phosphorylation. Muscle cells were exposed for 1 min to 10)9M

1a,25(OH)2D3 in the absence or presence of genistein (100 lM) or

daidzein (100 lM). Cell lysates were immunoprecipitated and

immunoblotted as described in Fig. 1. (A) Representative immuno-

blot. (B) Quantification by scanning volumetric densitometry of blots

from three independent experiments; means ± SD are given.

*P < 0.05, **P < 0.01 with respect to the corresponding control.

Fig. 4. 1a,25(OH)2D3-dependent PLCc phosphorylation is suppressed

by the specific c-Src inhibitor PP1. Muscle cells were exposed for 1 min

to 10)9M 1a,25(OH)2D3 in the absence or presence of PP1 (10 lM).

Cell lysates were immunoprecipitated and immunoblotted as described

in Fig. 1. (A) Representative immunoblot. (B) Quantification by

scanning volumetric densitometry of blots from three independent

experiments; means ± SD are given. *P < 0.01 with respect to the

corresponding control.

� FEBS 2002 1a,25(OH)2-Vitamin D3 stimulation of PLCc (Eur. J. Biochem. 269) 2509

either 1a,25(OH)2D3-treated or untreated cells with anti-PLCc Ig, resolved in SDS/PAGE gels under denaturingconditions, and then immunoblotted with anti-(c-Src) Ig. Asshown in Fig. 5, the hormone increased the physicalassociation of c-Src with PLCc, suggesting that this PLCisoform is a direct substrate of c-Src in skeletal muscle cells.Similar results were obtained when control assays werecarried out adding the antibodies in reverse order: celllysates were precipitated with anti-(c-Src) Ig and blottedwith the anti-PLCc Ig (Fig. 5). These results are inagreement with those previously reporting in vitro tyrosinephosphorylation of PLCc by c-Src [46], and with agonist-induced activation and coprecipitation of this tyrosinekinase with PLCc [47,48].

To answer the question to what extent complete abolitionof 1a,25(OH)2D3-stimulated PLCc by c-Src inhibitionreflects an absolute requirement of c-Src for the hormone-dependent PLCc phosphorylation, we blocked the expres-sion of c-Src by transfecting cells with an antisenseoligodeoxynucleotide against c-Src mRNA from Gallusgallus. As observed in Fig. 6, in cells transfected with thec-Src antisense-ODN the phosphorylation of PLCc inducedby 1a,25(OH)2D3 was abolished completely. Moreover,immunoblotting of cell lysates with anti-(c-Src) Ig provedthe ability of the antisense ODN to suppress the targetedc-Src protein (data not shown). Thus, the data from the setof experiments above described provide strong evidence toindicate that activation of c-Src is part of the mechanismthrough which 1a,25(OH)2D3 stimulates PLCc phosphory-lation in avian skeletal muscle cells.

The 10 mammalian PLC isozymes identified to date aresingle polypeptides that contain a pleckstrin homology (PH)domain in their amino-terminal region [22]. The c-typeisozymes differ from the b- and d-types in that they containtwo SH2 domains, one SH3 domain and an additional PH

domain. PLCc can also be activated through a tyrosinekinase path involving PtdIns3K. PtdIns3K, a lipid kinasecomposed of a regulatory subunit (p85) and a 110-kDa(p110) catalytic subunit, phosphorylates the D-3 position ofPtdIns(4,5)P2 to produce PtdIns(3,4,5)P3, which activatesPLCc isozymes specifically by interacting with their SH2domains [49] and the PH domain [50]. 1a,25(OH)2D3 hasbeen shown to increase PtdIns3K activity and differenti-ation in myeloid cells [51]. In avian skeletal muscle cells, littleis known about function and signalling pathways ofPtdIns3K. To investigate whether PtdIns3K plays a rolein PLCc activation stimulated by 1a,25(OH)2D3, we testedthe effect of two structurally unrelated inhibitors ofPtdIns3K, wortmannin [52] and LY294002 [53]. Pretreat-ment with 100 nM wortmannin (Fig. 7A) or 10 lM

LY294002 (Fig. 7B) reduce by 40% 1a,25(OH)2D3-inducedPLCc phosphorylation. Wortmannin has been used toinhibit phospholipase D [54], but whether it does so inmuscle cells has not been determined. Wortmannin is nowused primarily as an inhibitor of PtdIns3K. By binding tothe p110 catalytic subunit of PtdIns3K, it irreversibly

Fig. 5. 1a,25(OH)2D3 increases the physical association of c-Src with

PLCc. Muscle cells were exposed to 1 nM 1a,25(OH)2D3 or vehicle

(ethanol <0.05%) for 1 min. (A) Left panel: cell lysates were obtained,

PLCc was immunoprecipitated with monoclonal anti-PLCc Ig under

native conditions, resolved onto 10% SDS/PAGE gels and then

immunoblotted with monoclonal anti-Src Ig as described in Materials

and methods; right panel: As above, but immunoprecipitation was

performed with monoclonal anti-Src Ig and immunoblotting was with

the anti-PLCc Ig. (B) Quantification by scanning volumetric densito-

metry of blots from three independent experiments; means ± SD are

given. *P < 0.01 with respect to the corresponding control.

Fig. 6. Effect of an antisense oligodeoxynucleotide against c-Src mRNA

on 1a,25(OH)2D3-induced PLCc phosphorylation. Chick skeletal

muscle cells were transfected with an antisense (ODN-AS) oligode-

oxynucleotide against c-Src mRNA, and 48 h later the cells were

exposed to 1 nM 1a,25(OH)2D3 for 1 min followed by immunopre-

cipitation with anti-PLCc Ig and Western blotting with anti-

phosphotyrosine Ig as described in Materials and methods.

(A) Representative immunoblot. (B) Quantification by scanning

volumetric densitometry of blots from two independent experiments;

means ± SD are given. *P < 0.01 with respect to the corresponding

control.

2510 C. Buitrago et al. (Eur. J. Biochem. 269) � FEBS 2002

inactivates the enzyme at nanomolar concentrations [52].The wortmannin and LY294002 sensitivity supports thenotion tyrosine phosphorylation PLCc by the steroidhormone requires upstream PtdIns3K activity. The involve-ment of multiple molecules in PLCc phosphorylationsuggests the presence of a complex molecular networkregulating PLCc activation.

To examine further whether PLCc associates withPtdIns3K upon 1a,25(OH)2D3 stimulation, aliquots fromanti-PLCc Ig and anti-PtdIns3K Ig (p85) immunoprecip-itates of hormone-treated muscle cells (10)9

M, 1 min)were subjected to SDS/PAGE and Western blotting. Blotswere probed with anti-PtdIns3K Ig (p85) and anti-PLCcIg, respectively, and developed by ECL. The resultsshown in Fig. 8 indicate that PLCc associates withPtdIns3K after 1a,25(OH)2D3 exposure. One well-estab-lished mechanism for activation of PtdIns3K is known toinvolve interaction of the Src homolgy 2 domain of thep85 regulatory subunit with tyrosine phosphorylatedproteins, including both receptor and nonreceptor proteintyrosine kinases [55]. Because the potential role of c-Srctyrosine kinase in 1a,25(OH)2D3-induced activation ofPtdIns3K remains unknown, the requirment of c-Src fortyrosine phosphorylation of the p85 subunit of PtdIns3Kwas examined. As shown in Fig. 9 1a,25(OH)2D3 induced,within 1 min, a threefold increase of p85 tyrosinephosphorylation, an effect that was effectively suppressedby the c-Src inhibitor PP1. These results suggest thatPLCc activation events in avian skeletal muscle cells arepreceded by phosphorylation of the regulatory subunit ofPtdIns3K.

PLCc is well known to be recruited to the plasmamembrane by activated receptor tyrosine kinases. There isevidence indicating that the PH domain of PLCc will bindto PtdIns(3,4,5)P3 targeting the enzyme to the plasmamembrane, in response to serum and growth factorstimulation [50]. Moreover, the SH2 and SH3 domainsof the two PLCc isozymes have been also found to

Fig. 7. Effect of the PtdIns3K inhibitors, wortmannin and LY294002

on 1a,25(OH)2D3-induced PLCc phosphorylation. Muscle cells were

exposed for 1 min to 10)9M 1a,25(OH)2D3, in the absence or presence

of wortmannin (100 nM) or LY294002 (10 lM). Cell lysates were

immunoprecipitated and immunoblotted as described in Fig. 1.

(A) Representative immunoblot. (B) Quantification by scanning

volumetric densitometry of blots from three independent experiments;

means ± SD are given. *P < 0.05 with respect to the corresponding

control.

Fig. 8. 1a,25(OH)2D3 increases the physical association of PtdIns3K

with PLCc. (A) Muscle cells were exposed to 1 nM 1a,25(OH)2D3 or

vehicle (ethanol <0.05%) for 1 min. Left panel: cell lysates were

obtained, PLCc was immunoprecipitated with monoclonal anti-PLCcIg under native conditions, resolved by SDS/PAGE (10% gels) and

then immunoblotted with monoclonal anti-PtdIns3K (p85) Ig as

described in Materials and methods. Right panel: as above, but

immunoprecipitation was performed with monoclonal anti-PtdIns3K

Ig (p85) and immunoblotting with the anti-PLCc Ig. (B) Quantifica-

tion by scanning volumetric densitometry of blots from two

independent experiments; means ± SD are given. *P < 0.01 with

respect to the corresponding control.

� FEBS 2002 1a,25(OH)2-Vitamin D3 stimulation of PLCc (Eur. J. Biochem. 269) 2511

contribute to PLCc membrane anchoring to bring theactive enzyme into the proximity of its substrate [56].Therefore, the role of 1a,25(OH)2D3 in PLCc membranetranslocation was explored first by immunofluorescencestudies using anti-PLCc Ig. In unstimulated cells, themajority of PLCc has a diffuse cytosolic distribution, withsome labelling of the plasma membrane (Fig. 10B). Upon1 min of treatment with 1a,25(OH)2D3 (10)9

M), a portionof PLCc localized to the cell membrane, as indicated bystrong reinforcement of the fluorescence signal (Fig. 10A).Results obtained by fluorescence microscopy were con-firmed by cell fractionation experiments. Western blots ofmembranes isolated from 1a,25(OH)2D3-stimulated cellsshowed an increase in PLCc as compared with unstimulatedcontrol cells (Fig. 11A). The time course of hormone-dependent PLCc phosphorylation closely correlates to thetime course of its redistribution to the membrane (Fig. 11B),suggesting that phosphorylation and redistribution to themembrane of PLCc are two interdependent events.

We next examined the possible role of c-Src andPtdIns3K in 1a,25(OH)2D3-dependent localization ofPLCc. Inhibition of c-Src activity by PP1, or suppressionof PtdIns(3,4,5)P3 generation by LY294002, under the sameexperimental conditions as the ones described aboveprevented, to a great extent (by 80%), PLCc membranetraslocation upon hormone treatment (Fig. 11C). Thisresult is in agreement with those previously reporting thatinhibition of c-Src tyrosine kinase limits membrane

translocation of PLCc in 1a,25(OH)2D3-stimulated ratcolonocytes [41]. Our results indicate also that PtdInsP3,the product of PtdIns3K, is also necessary to activate andrecruit the phospholipase to the lipid bilayer. PtdInsP3 islikely to provide a membrane anchoring site for the PHdomain of PLCc [50]. The involvement of multiple mole-cules in hormone PLCc activation suggest the presence of acomplex molecular network regulating PLCc translocationand phosphorylation in muscle cells.

In summary, the present data indicate that the steroidhormone 1a,25(OH)2D3 causes phosphorylation and mem-brane translocation of PLCc in avian skeletal muscle cellsand that the cytosolic tyrosine kinase c-Src works in concertwith PtdIns3K to fully activate PLCc in 1a,25(OH)2D3-stimulated cells.

The mode of signal transduction from the putative1a,25(OH)2D3 receptor to nonreceptor c-Src tyrosine kinase

Fig. 9. 1a,25(OH)2D3 stimulates the tyrosine phosphorylation of the

regulatory subunit (p85) of PtdIns3K: effect of the c-Src inhibitor PP1.

Muscle cells were exposed for 1 min to 10)9M 1a,25(OH)2D3, in the

absence or presence of PP1 (10 lM). Cell lysates were immunopre-

cipitated with anti-PtdIns3K (p85) Ig, immunoprecipitated proteins

were separated by SDS/PAGE followed by Western blotting with anti-

phosphotyrosine Ig as described in Materials and methods. (A) Rep-

resentative immunoblot. (B) Quantification by scanning volumetric

densitometry of blots from three independent experiments;

means ± SD are given. *P < 0.05 with respect to the corresponding

control.

Fig. 10. 1a,25(OH)2D3-induced translocation of PLCc to the muscle

cell membrane. Muscle cells were treated with 1a,25(OH)2D3 (1 min,

10)9M) or ethanol (control), fixed with paraformaldehyde, permea-

bilized, reacted with anti-PLCc Ig and with the secondary antibody

Alexa 546-conjugated goat anti-(rabbit IgG) Ig as described in Mate-

rials and methods. (A) Upon cell stimulation with 1a,25(OH)2D3,

green immunofluorescence condensed into the periphery showing

translocation of cytosolic PLCc to the plasma membrane (arrows).

(B) Untreated cells (control) show normal cytosolic distribution of

PLCc determined by diffuse and uniform green immunofluorescence

(arrows). (C, D) Micrographs showing an intense nuclear blue stain

and spherical nucleus shape indicating nuclear integrity and cell

viability determined by the DAPI nuclear marker. (E, F) Phase-con-

trast micrographs. Micrographs of (A), (C) and (E) show the same field

[1a,25(OH)2D3-treated cells] as do panels (B), (D) and (F) (control

cells). Bar ¼ 2.6 lm.

2512 C. Buitrago et al. (Eur. J. Biochem. 269) � FEBS 2002

and PtdIns3K is not known at present. Recent studies inother cell types have implicated both the a subunit [57] andthe bc subunits [58] of G-protein-coupled receptors in themediation of activation of Src family of nonreceptortyrosine kinases, and in myeloid-derived cells the bcsubunits have been shown to activate PtdIns3K [59].Further studies are necessary to evaluate whether1a,25(OH)2D3 uses similar pathways in avian skeletalmuscle cells.

A C K N O W L E D G E M E N T S

This research was supported by grants from the Agencia Nacional de

Promocion Cientıfica y Tecnologica, Consejo Nacional de Investigac-

iones Cientificas y Tecnicas (CONICET) and Universidad Nacional del

Sur, Argentina. We thank L. E. Politti (INIBBIB) for kindly providing

reagents and for his assistance with the immunofluorescence analysis.

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