733Research Article

IntroductionThe steroid hormone testosterone controls a vast number ofcellular processes including cell growth and differentiation(Beato, 1989; Mooradian et al., 1987). In neurons, thishormone can induce changes at the cellular level, leading tochanges in behavior (Kelly et al., 1999). As a neurosteroid,testosterone can influence sleep, the reaction to stress, moodand memory (McEwen, 1991; Naghdi and Asadollahi, 2004).The genomic responses to testosterone are mediated throughthe intracellular androgen receptor (iAR), a 110 kDa proteinwith domains for androgen binding, nuclear localization, DNAbinding and transactivation (Mooradian et al., 1987). Theseresponses occur following a delay measured in hours. Bycontrast, several reports indicate that androgens are capable ofproducing rapid, seconds to minutes, non-genomic effects(Benten et al., 1999a; Lieberherr and Grosse, 1994). The non-genomic actions of androgens are diverse, but common to theseearly effects is a rapid intracellular Ca2+ increase andsubsequent activation of Ca2+-dependent signaling cascades(Benten et al., 1999b; Estrada et al., 2000; Lieberherr andGrosse, 1994).

By altering the intracellular Ca2+ concentration incharacteristic ways the cell can use the same signalingmolecule to specifically regulate different cellular functions(Carafoli et al., 2001). For example, the rapid turn on and offof Ca2+ signals often produces oscillations. The key

quantifiable properties of oscillations are frequency andamplitude, and modulation of either property can controlcellular processes (Aizman et al., 2001; Dolmetsch et al., 1998;Li et al., 1998). Initial reports showed that the rapid, steroid-induced Ca2+ increases were single Ca2+ transients (Losel andWehling, 2003). More recently, it has been shown in skeletalmuscle cells that testosterone induces intracellular Ca2+

oscillations (Estrada et al., 2005; Estrada et al., 2003; Estradaet al., 2000), which may encode more precise information thanalterations in the amplitude of a single Ca2+ transient.

An important component of the Ca2+ signaling toolkit whichis essential for regulation of Ca2+ oscillations is the inositol1,4,5-trisphosphate receptor [Ins(1,4,5)P3R], a Ca2+ permeablechannel located in the membrane of the endoplasmic reticulum.The Ins(1,4,5)P3R has several properties that make it ideal formodulating oscillatory patterns: Ins(1,4,5)P3-gated channelactivity can be regulated by Ca2+ with a bell-shapeddependence (Bezprozvanny et al., 1991), by a number of Ca2+

binding proteins (Berridge et al., 2003; Choe et al., 2004) andby phosphorylation (Tang et al., 2003). Interestingly, theIns(1,4,5)P3R is also found inside the nucleus, associated withthe nucleoplasmic reticulum (Echevarria et al., 2003), and thisIns(1,4,5)P3R can release Ca2+ independently of cytoplasmicreceptors (Echevarria et al., 2003; Leite et al., 2003; Pusl et al.,2002). It has been suggested that Ca2+ levels in the nucleus areresponsible for processes related to gene transcription and cell

Testosterone has short- and long-term roles in regulatingneuronal function. Here, we show rapid intracellularandrogen receptor-independent effects of testosterone onintracellular Ca2+ in neuroblastoma cells. We identifiedtestosterone-induced Ca2+ signals that began primarily atthe neurite tip, followed by propagation towards thenucleus, which was then repeated to create an oscillatorypattern. The initial transient depended upon production ofinositol 1,4,5-trisphosphate [Ins(1,4,5)P3], but subsequenttransients required both extracellular Ca2+ influx and Ca2+

release from intracellular stores. Inhibition of pertussistoxin-sensitive G-protein receptors or the use of siRNA forthe Ins(1,4,5)P3 receptor type 1 blocked the Ca2+ response,whereas inhibition or knock-down of the intracellularandrogen receptor was without effect. Cytosolic andnuclear Ca2+ were buffered with parvalbumin engineeredto be targeted to the cytosol or nucleus. Cytoplasmic

parvalbumin blocked Ca2+ signaling in bothcompartments; nuclear parvalbumin blocked only nuclearsignals. Expression of a mutant parvalbumin did notmodify the testosterone-induced Ca2+ signal. Neuriteoutgrowth in neuroblastoma cells was enhanced by theaddition of testosterone. This effect was inhibited whencytosolic Ca2+ was buffered and was attenuated whenparvalbumin was targeted to the nucleus. Our results areconsistent with a fast effect of testosterone, involvingIns(1,4,5)P3-mediated Ca2+ oscillations and support thenotion that there is synergism in the pathways used forneuronal cell differentiation involving rapid non-genomiceffects and the classical genomic actions of androgens.

Key words: Androgens, Ca2+ signaling, Inositol 1,4,5-trisphosphatereceptor, Nongenomic response, Neurite outgrowth

Summary

Ca2+ oscillations induced by testosterone enhanceneurite outgrowthManuel Estrada1,2, Per Uhlen1,2 and Barbara E. Ehrlich1,2,*1Departments of Pharmacology, Cell and Molecular Physiology, Yale University, New Haven, CT 06520, USA2Neurosciences Institute of the Marine Biological Laboratory, Woods Hole, MA 02543, USA*Author for correspondence (e-mail: barbara.ehrlich@yale.edu)

Accepted 1 November 2005Journal of Cell Science 119, 733-743 Published by The Company of Biologists 2006doi:10.1242/jcs.02775

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growth whereas changes in cytoplasmic Ca2+ are used forprocesses such as secretion and motility.

In this study we found a new pathway for testosteroneactivity that has a rapid onset and leads to the generation oflong lasting Ca2+ oscillations. Within seconds after its addition,testosterone induces intracellular Ca2+ increases inneuroblastoma cells, which begin as Ca2+ transients initiated inthe cytosol and propagate as waves of Ca2+ in the cytoplasmand nucleus. These complex Ca2+ signals depend on aninterplay between Ins(1,4,5)P3-sensitive stores and influx fromthe extracellular medium. The Ca2+ transients develop intooscillatory patterns which have been shown to activate specificdownstream pathways (Dolmetsch et al., 1998; Li et al., 1998).The Ca2+ signals are independent of the iAR and are mediatedvia a pertussis toxin-sensitive plasma membrane receptor. Inaddition, we found that testosterone enhanced the neuriteoutgrowth and an increase in cytosolic Ca2+ was shown to berequired for this effect. These results demonstrate an importantphysiological mechanism for the action of testosterone inparallel, or prior, to androgen receptor-mediated genomicevents in neurons.

ResultsEffects of testosterone on intracellular Ca2+ inneuroblastoma cellsIntracellular Ca2+ changes were monitored in SH-SY5Yneuroblastoma cells loaded with Fluo-4/AM, a Ca2+-senstive,cell permeable fluorescent dye. Addition of testosteroneevoked a local intracellular Ca2+ increase and then apropagating wave within the cell. The initial Ca2+ rise resolvedand was followed by subsequent Ca2+ increases creating anoscillatory pattern (Fig. 1). Typically, the first increase inintracellular Ca2+ occurred at the extremity of the cell,approximately 30 seconds after addition of testosterone (Fig.1A). A Ca2+ wave then spread into the cytosol along the axisof the cell, invaded the nucleus and reached the oppositeextremity of the cell. An oscillatory Ca2+ response was definedwhen intracellular Ca2+ increases were repeated more thanthree times within a single cell. In some rare cases, Ca2+

oscillations were observed without propagating Ca2+ waves.The response to testosterone was concentration dependent. Wecharacterized the testosterone-induce Ca2+ response todifferent levels of the hormone by several parameters:percentage of cells responding, frequency of Ca2+ oscillations,and the amplitude of the Ca2+ peaks. Concentrations in the 10nM range evoked Ca2+ signals in 30% of the cells (14 of 48cells from six different cultures; Fig. 1B). When thetestosterone concentration was increased to 100 nM, thenumber of responsive cells increased to 68% (87 of 128 cells,from 16 different cultures) as well as the frequency ofoscillatory Ca2+ response (Fig. 1C). A similar intracellular Ca2+

response has previously been observed in neuroblastoma cellsusing agonists known to activate G-protein coupled receptors(Tovey et al., 2001).

To measure the regularity of the testosterone-induced Ca2+

oscillations we used power spectral analysis (Fig. 1D). Thiscomputational method reduces a complex signal into thedifferent sine wave components that contribute to form thecomplex signal and ranks the components by power so that themost dominant frequencies in the complex signal can beidentified (Uhlen, 2004). An added advantage of spectral

analysis is that irregular and stochastic contributions to thesignal are minimized. We found that the Ca2+ oscillations couldbe described adequately with one major peak (Fig. 1D),indicating a highly regular oscillatory response. Spectralanalysis of testosterone-induced Ca2+ oscillations generated anaverage frequency of 18±2 mHz, n=7 (periodicity of 56±5seconds) and 22±1 mHz, n=34 (periodicity of 46±3 seconds),for 10 nM and 100 nM of testosterone, respectively. Theconcentration dependence of the amplitude of the first peakshowed that elevating concentrations of testosteronesignificantly increased the Ca2+ peaks, reaching a maximumresponse at 100 nM (Fig. 1E). These concentrations oftestosterone are in the range found in normal human males(Kelly et al., 1999; Mooradian et al., 1987). In all subsequentexperiments, 100 nM testosterone was used.

The specificity of the response to testosterone wasinvestigated by applying several other steroids. Cortisol,progesterone, or dexamethasone (all at 100 nM) had no effecton intracellular Ca2+ levels (data not shown). Interestingly, 100nM 17�-estradiol (Fig. 1F, red line; n=58 of 86 cells, fourindependent cultures) produced Ca2+ transients whereas at thesame concentration the biologically inactive isomer 17�-estradiol (Fig. 1F, green line; n=46 of 46 cells, threeindependent cultures) did not induce any changes inintracellular Ca2+ levels. To eliminate the possibility that theeffects of testosterone were due to the activation of an estrogenreceptor pathway, cells were pre-incubated with 1 mMtamoxifen, an antagonist of the estrogen receptor, prior toaddition of testosterone. Under these conditions thetestosterone-induced Ca2+ oscillations were the same as in thepresence of testosterone alone (Fig. 1F, black line, n=28 of 37cells, three independent cultures), indicating that a functionalestrogen receptor is not needed.

Propagation of Ca2+ waves produced by testosteroneTypically the Ca2+ response was initiated in one discrete zoneat the tip of the neurite. Then, the Ca2+ transient spread fromthe point of origin and propagated along the axis of the cell(Fig. 1A). To improve the time resolution of this signalingevent we performed linescan experiments (Fig. 1G). Thefluorescence intensity was recorded in different regions of thecytosol and nucleus (Fig. 1G). Transient rises in Ca2+ wereobserved along the dendrite, producing a cytosolic Ca2+ wavethat propagated towards the nucleus, into the nucleus, and oftento the tip of the opposite dendrite (Fig. 1G,H). The propagationrate of the Ca2+ wave was 11.2±1.8 �m/second (Fig. 1G). Forcomparison, the diffusion constant (D) of Ca2+ ions in cytosolis estimated to be 13 �m2/second and that for Ins(1,4,5)P3 is280 �m2/second (Allbritton et al., 1992).

Cytosolic and nuclear Ca2+ increases induced bytestosteroneIn order to determine the contribution of cytosolic and nuclearCa2+ to the global Ca2+ response, Ca2+ was buffered in eachcompartment separately. To accomplish compartmentalizedbuffering, we heterologously expressed a fusion protein thatcontained the Ca2+ buffer parvalbumin (PV) tagged with a redfluorescent protein (DSR) and signal sequences which targetedthe fusion protein either to the cytosol or nucleus (Pusl et al.,2002). Cells were transfected with PV-DSR targeted to nucleus(PV-NLS-DSR), a Ca2+ insensitive form of PV-DSR targeted

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to nucleus (PV-NLS-CDEF-DSR), or PV-DSR targeted tocytosol (PV-NES-DSR). Western blot analysis of whole celllysates show the increase in the amount of PV in all threeconditions (Fig. 2A-C). The location of DSR and a nuclearstain was used to show that the fusion proteins were expressedin the expected subcellular location (Fig. 2D-F). Basal levelsof Ca2+ were not changed in cells expressing PV-NLS-DSR orPV-NES-DSR, relative to control cells, as described previously(Pusl et al., 2002). Transfection with an empty vectorcontaining DRS did not modify the effects of testosterone (datanot shown). Cells transfected with the PV-NLS-DSR did notexhibit any nuclear Ca2+ increase after testosterone exposure,but the cells showed normal cytosolic Ca2+ oscillations (Fig.2G, n=21 of 21 cells, four independent cultures). To testwhether the inhibition of the nuclear Ca2+ signal was due tothe Ca2+-chelating properties of PV, cells were transfected withPV-NLS-CDEF-DSR, which has a double mutated EF-handmotif that prevents the protein from binding Ca2+ (Fig. 2H). In

cells expressing PV-NLS-CDEF-DSR, the addition oftestosterone induced cytosolic and nuclear Ca2+ oscillations(Fig. 2H, n=27 of 27 cells, four independent cultures), showingthat the Ca2+-buffering capacity of the PV is required. Bycontrast, cells expressing PV-NES-DSR did not exhibit anyCa2+ increase in either the cytosol or nucleus (Fig. 2I; n=31 of31, three independent cultures), indicating that thetestosterone-induced Ca2+ increase began in the cytosol andthen propagated into the nucleus.

Testosterone-induced Ca2+ signals are independent ofthe intracellular androgen receptorThe iAR is present in neuroblastoma cells suggesting that thesecells are a target for the physiological action of testosterone(Maggi et al., 1998; Yerramilli-Rao et al., 1995). Using anantibody directed against the N-terminal domain of the humaniAR, the iAR was found throughout the cell, but with highestexpression in the nucleus (Fig. 3A). In some cells the highest

Fig. 1. Single cell imaging oftestosterone-induced intracellularCa2+ oscillations. (A) Fluorescencesequence over the time periodindicated (images in pseudocolor).The Ca2+ signal began at the extremetip of the cell and propagated as aCa2+ wave. A few seconds later a newCa2+ wave was produced, generatingCa2+ oscillations. (B) NormalizedCa2+ changes induced by 10 nMtestosterone. (C) Cells stimulated with100 nM testosterone. The time ofaddition of the hormone is indicated(arrows in B and C). (D) Powerspectral analysis shows that thedominant peak of testosterone-induced Ca2+ oscillations occurs at 22mHz. (E) Concentration dependenceof the amplitude of the testosterone-induced Ca2+ transient, with amaximum response produced at 100nM testosterone. (F) 17�-estradiol(red line) but not 17�-estradiol (greenline) induces Ca2+ transients. Cell pre-incubated with 1 mM tamoxifen, anantagonist of estrogen receptor, didnot modify the testosterone-inducedCa2+ oscillations, indicating that theestrogen pathway is not required andthe response was specific for thetestosterone effect. (G) The linescanmode of recording fluorescenceintensity was used to monitordifferent cellular regions. The x axisshows time, read from left to right.The y axis is the distance within thecell shown on the far left. The dashedlines, labeled 1 to 3, are the representative regions plotted in panel H. (H) Transient rises in Ca2+ were observed at different times, showing acytosolic Ca2+ wave that propagated towards the nucleus, into the nucleus, and often to the opposite end of the cell. *P<0.05.

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expression of the iAR was at the extreme tip of the neurites(Fig. 3A, upper panel). Upon stimulation with testosterone, thecytosolic iAR translocated to the nucleus after 1 hour, showingthat the system was functional (Fig. 3A, lower panel). Toevaluate whether the rapid Ca2+ response seen after theaddition of testosterone was independent of the iAR, we used

both a pharmacological blocker and genetic knock-down of theiAR. Cells transfected with small interfering RNA for the iAR(iAR-siRNA) showed a significantly reduced proteinexpression. Western blot analysis revealed that iAR-siRNAdownregulated the protein by 80% (Fig. 3B,C). Neither theknock-down (Fig. 3D, n=25 of 25 cells, five independent

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Fig. 2. The effect of targetedparvalbumin fusion proteins ontestosterone-induced cytosolicand nuclear Ca2+ responses.Western blots of PV-DSR fusionproteins. Cells were transfectedwith expression vectors for PV-NLS (A), PV-NLS-CDEF (B) orPV-NES (C). Total cell lysateswere separated by SDS-PAGEand parvalbumin protein wasvisualized with a monoclonalanti-PV antibody; �-actin isshown as a loading control. In allthree conditions the expressionof PV was increased in thetransfected cells, as comparedwith the untreated cells.Experiments are representativeof three independentexperiments. The subcellulardistribution of the PV constructswas examined using confocalmicroscopy. Red indicates thelocation of DSR, blue indicatesnuclear staining with TO-PRO3.(D) PV-NLS-DSR and (E) PV-NLS-CDEF-DSR (targeted tonucleus) are restricted to thenucleus. (F) Expression of PV-NES-DSR (targeted to cytosol) is uniformly distributed throughout the cytosol but excluded from the nucleus.(G) Cells transfected with the PV-NLS-DSR did not exhibit any nuclear Ca2+ increase after testosterone exposure, but the cells showed normalcytosolic Ca2+ oscillations. (H) In cells expressing PV-NLS-CDEF-DSR, a mutated PV that does not bind Ca2+, testosterone induced cytosolicand nuclear Ca2+ oscillations. (I) Cells expressing PV-NES-DSR did not exhibit any cytosolic or nuclear Ca2+ increase.

Fig. 3. The effect of inhibition ofintracellular androgen receptor ontestosterone-induced Ca2+

oscillations. (A) Fluorescence imagesof iAR localization weresuperimposed on bright-field imagesof single cells. (Upper panel) Incontrol cells, iAR was distributedthroughout the cell, but there was ahigher relative abundance in thenucleus. Note also the clusters of iARin the neurites. (Lower panel) Upontestosterone (100 nM) stimulation for1 hour, the cytoplasmic iARtranslocated to the nucleus.(B,C) Cells were transientlytransfected with AR-siRNA and theexpression of the protein was reduced~80% with respect to controlconditions, *P<0.05 versus basal(n=4). (D,E) Neither iAR-siRNA norcyproterone modify the ability of testosterone to induce Ca2+ oscillations. (F) To show that internalization of testosterone is not required, theplasma membrane-impermeable testosterone bound to albumin (T-BSA) was tested and it mimicked the effects of the free hormone.

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cultures) nor the treatment with thespecific inhibitor of the iAR,cyproterone (Fig. 3E, n=29 of 35,four independent cultures),modified the ability of testosteroneto induce intracellular Ca2+

oscillations. To evaluate whetherthe effect of the hormone wasmediated by an extracellularmembrane receptor, the effect oftestosterone covalently bound toalbumin (T-BSA) was tested. Thiscompound does not cross theplasma membrane making itunable to act on iAR. The responsewas similar when cells wereexposed to testosterone or T-BSA(100 nM; Fig. 3F, n=32 of 40 cells,four independent cultures).Albumin alone (0.1%) does notproduce any change in theintracellular Ca2+ (data not shown).In addition, the peak frequenciescalculated from spectral analysis ofthe Ca2+ oscillations in all threetest conditions and untreated cellswere similar. Taken together theseresults indicate that testosterone-induced intracellular Ca2+

oscillations were independent ofthe iAR-mediated genomicresponse.

Sources of Ca2+ involved intestosterone-induced Ca2+

oscillationsExperiments were done todetermine whether the action oftestosterone on intracellular Ca2+

was due to an influx of Ca2+ fromthe extracellular medium and/ormobilization of Ca2+ fromintracellular stores. Pre-treatmentof the cells with nifedipine (5 �M),a L-type voltage-dependent Ca2+

channel blocker, did not modifythe ability of testosterone to induce Ca2+ oscillations (Fig. 4A,n=16 of 19 cells, three independent cultures). When cells wereincubated in Ca2+-free medium for 5 minutes prior totestosterone stimulation, the hormone induced a single Ca2+

transient after a delay of 33±14 seconds (n=38 of 72 cells from12 different cultures). No oscillations were produced underthese conditions (Fig. 4B). The response in Ca2+-free mediumresembled the first Ca2+ peak generated in Ca2+-containingmedium with a similar delay and magnitude of response(compare Fig. 1C with Fig. 4B, 3.2±0.6 and 3.0±0.4 F/F0,Ca2+-containing or Ca2+-free medium, respectively). Theseresults suggest that the testosterone-induced regenerative Ca2+

oscillations require both extracellular and intracellular Ca2+

mobilization. In control experiments, the basal levels ofintracellular Ca2+ in cells not treated with the hormone were

unaffected by the removal of extracellular Ca2+ during the timeof data acquisition (data not shown). In addition, pre-incubation of the cells with Gd3+ (1 �M), a nonspecific plasmamembrane Ca2+ channel blocker, in Ca2+ containing medium,suppressed the Ca2+ oscillations without affecting the first Ca2+

peak (Fig. 4C, n=28 of 35, four independent cultures). Theseresults indicate that the extracellular Ca2+ influx was due toactivation of plasma membrane Ca2+ channels that are distinctfrom the L-type voltage-dependent Ca2+ channels. Moreimportantly, extracellular Ca2+ influx is necessary to maintainCa2+ oscillations in response to testosterone.

Depletion of intracellular Ca2+ stores by pre-treating thecells with the endoplasmic reticulum Ca2+-ATPase inhibitorthapsigargin, completely blocked the hormone-triggered Ca2+

response (data not shown, n=25 of 25 cells) indicating that the

Fig. 4. The source of the intracellular Ca2+ signal induced by testosterone. Cells were stimulatedwith testosterone in the presence of intracellular Ca2+ release inhibitors. (A) Pre-incubation of cellswith nifedipine did not alter the Ca2+ response. (B) In Ca2+-free medium (0.5 mM EGTA) only thefirst peak was produced and no oscillations were observed. (C) Incubation with 1 mM Gd3+ (a non-specific Ca2+ channel blocker) mimicked the response seen in Ca2+-free medium. (D) Ryanodinehad no effect, whereas U73122 and 2APB abolished the Ca2+ signal (E,F).

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Ca2+ increase produced by testosterone also requiredthapsigargin-sensitive intracellular Ca2+ stores. Pre-treatmentof neuroblastoma cells with ryanodine, at 20 �M, aconcentration known to inhibit the RyR channel (Ehrlich et al.,1994), had no effect on testosterone-induced Ca2+ oscillations,suggesting that Ca2+ mobilization did not require the RyR (Fig.4D, n=23 of 23, from four different cultures). By contrast, thetestosterone-induced Ca2+ response was completely abolishedby the phospholipase C (PLC) inhibitor, U-73122 (Fig. 4E,n=26 of 28, four independent cultures) showing that generationof Ins(1,4,5)P3 is necessary. The amount of Ca2+ in theintracellular stores was not affected by U-73122, as determinedby the magnitude of the release after addition of thapsigargin(Fig. 4E). Cells pre-treated with 2-aminoethyl diphenylborate(2-APB; 10 �M), a plasma membrane permeable inhibitor ofthe Ins(1,4,5)P3R, did not respond to testosterone (data notshown, n=20). To confirm the participation of theIns(1,4,5)P3R in the testosterone-induced Ca2+ oscillations,two additional types of experiments were done. First, 2-APBwas added to testosterone-treated cells that were in the processof producing Ca2+ oscillations. Immediately after 2-APB wasapplied to the cells, the Ca2+ oscillations were abolished (Fig.4F, n=12 of 16 cells, from three independent cultures). Second,the amount of Ins(1,4,5)P3R was knocked-down by transientlytransfecting the cells with siRNA for the Ins(1,4,5)P3R type 1.This isoform was chosen because the cell line used in thesestudies (SH-SY5Y) expresses mainly the Ins(1,4,5)P3R type 1(Wojcikiewicz, 1995). There was a significant reduction in theimunosignal for the Ins(1,4,5)P3R type 1 in transfected cells(~80%, P<0.05; Fig. 5A,B). Interestingly, we observed twotypes of Ca2+ response to testosterone in the Ins(1,4,5)P3R type

1 knocked-down cells (Fig. 5C). In one sub-group (n=26 of 46cells, from five different cultures), the testosterone responsewas completely abolished. In the other sub-group (n=20 of 46cells), cells exhibited a fast and transient Ca2+ increase, similarto the peak Ca2+ increase observed in the Ca2+-free medium.However, this peak response was reduced by 68% (P<0.05compared with the control), as compared with the initial Ca2+

peak in non-transfected cells (Fig. 5D). It is important to notethat no Ca2+ oscillations were observed in Ins(1,4,5)P3R type1 knock-down cells. These results suggest that the loss offunctional Ins(1,4,5)P3R impedes the ability of the initial Ca2+

response to trigger a regenerative Ca2+ oscillation. Takentogether, these results imply that testosterone-inducedintracellular Ca2+ oscillations are the result of the coordinatedactions of Ca2+ mobilization from Ins(1,4,5)P3-sensitive Ca2+

stores and Ca2+ influx through plasma membrane Ca2+

channels.

A pertussis toxin-sensitive G protein coupled receptormediates the rapid Ca2+ response to testosteroneActivation of PLC at the plasma membrane could involveeither tyrosine kinase or G-protein coupled receptors. Toinvestigate the early events involved in generating the Ca2+

signals produced by addition of testosterone, cells wereincubated with genistein (50 �M, 20 minutes), a tyrosinekinase receptor inhibitor. Genistein modified neither the initialintracellular Ca2+ increase nor the Ca2+ oscillations induced bytestosterone (Fig. 6A, n=16 of 16, two independent cultures).To test for the involvement of a G-protein-coupled receptor,cells were permeabilized for 5 minutes with saponin in thepresence of guanosine 5�-O-(2-thiodiphosphate) (GDP�S; 100

nM), a non-hydrolysable analog of GTP.Permeabilization did not modifytestosterone-induced Ca2+ responses (Fig.6B, dashed line; n=12 of 16 cells, twoindependent cultures), whereas GDP�Sdid suppress the Ca2+ increases (Fig. 6B,solid line; n=21 of 21; four differentcultures). When cells were pre-incubatedwith pertussis toxin (PTX, 1 �g/ml, 6hours) before the addition of testosteronethere was no Ca2+ signal produced by thehormone (Fig. 6C n=38 of 38 cells; sixdifferent cultures). These results suggestthat testosterone action requires PTX-sensitive G-protein coupled receptors toactivate PLC and generate Ins(1,4,5)P3 toproduce Ca2+ signals.

Testosterone induces neuriteoutgrowthNeuroblastoma cells grown under controlconditions have short neurites. Todetermine the effect of testosterone onmorphology, cells were incubated withthe hormone for 3 days. To visualize themorphological changes, cells were loadedwith Cell Tracker (Fig. 7A), a fluorescentdye that does not affect the neuronalviability (Ang et al., 2003). For theanalysis of neurite outgrowth, cells with a

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Fig. 5. Effect of Ins(1,4,5)P3R type 1 knock-down on testosterone-induced Ca2+

oscillations. (A,B) Cells transiently transfected with Ins(1,4,5)P3R type 1 siRNA (InsP3R1),showed a significant reduction (~80%) of the immunosignal. (C) In Ins(1,4,5)P3R type 1-siRNA transfected cells two types of Ca2+ response to testosterone were observed. Themagnitude of the transient was reduced by 68% compared with untransfected cells, orabolished. (D) Control trace in cells not expressing the Ins(1,4,5)P3R siRNA.

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neurite that was longer than the soma were included. Tonormalize for variations in cell size, neurite outgrowth wascalculated as the ratio of the neurite length to soma length inthe neurite projection. When neuroblastoma cells were exposedto testosterone, smooth and elongated neurites were observed

(Fig. 7B). Testosterone treatment increased the neuriteoutgrowth 2- to 2.5-fold (n=108; P<0.01) when compared withuntreated cells (n=99) (Fig. 7C). Next, we examined the Ca2+

requirements for this testosterone-induced phenomenon usingthe parvalbumin constructs targeted to cytosol (PV-NES-DSR)

Fig. 6. Ca2+ signals induced by testosterone involve a pertussis toxin-sensitive G protein. (A) Cells were incubated for 20 minutes with 50�M genistein, a tyrosine kinase inhibitor, and then stimulated withtestosterone (100 nM). The use of genistein did not modify the Ca2+

increases produced by the hormone. (B) Cells were permeabilizedwith saponin, the saponin was removed to allow the membrane toreseal, and then cells were stimulated with testosterone (dashed line);using this treatment neuroblastoma cells maintained their capacity torespond to the hormone. Permeabilization in the presence of GDP�S(100 nM; solid line) blocked the testosterone-induced Ca2+ increases.(C) When cells were incubated with PTX (1 �g/ml) for 6 hours andthen stimulated with testosterone, the Ca2+ increases were blocked.The time of addition of the hormone is indicated (arrows).

Fig. 7. Testosterone enhances neurite outgrowth. Morphologicalchanges in neuroblastoma cells were monitored after 3 days oftreatment with testosterone. Cells were incubated with Cell Trackergreen and visualized by confocal microscopy. (A) Neuroblastomacells grown under control conditions have short neurites. (B) Cellstreated with testosterone exhibit an increase in the neurite outgrowthcompared with non-stimulated control cells. Bar, 30 �m. (C) Neuriteoutgrowth was normalized by calculating the ratio of the neuritelength to the soma length (neurite/soma). Testosterone-inducedneurite outgrowth was ~2.5-fold higher than control cells. Neuriteelongation induced by testosterone was inhibited in cells with thecytosolic Ca2+ buffered by the expression of PV-NES-DSR.Buffering the nuclear pool of Ca2+ with PV-NLS-DSR induced asmaller increase in the neurite outgrowth compared with non-transfected cells. Cells expressing the mutated form of the nuclearlocalized parvalbumin showed a response to testosterone similar tothe response observed in control cells. (D) Neurite elongation wassmaller after inhibition of the iAR pathway (siRNA-AR orcyproterone) or when using T-BSA compared with the response withtestosterone alone. These results suggest that genomic and non-genomic mechanisms for testosterone-induced neurite outgrowth inneuroblastoma cells are inter-dependent. Values are mean ± s.e.m.,*P<0.05; **P<0.01.

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or nucleus (PV-NLS-DSR). Expression of the parvalbuminproteins did not modify the normal growth or cell morphologyof the control cells. Testosterone-induced neurite outgrowthwas inhibited in cells expressing the cytosolic Ca2+ bufferprotein, PV-NES-DSR (Fig. 7C; n=78 of 78 transfected cells;P<0.01). When the nuclear pool of Ca2+ was buffered with PV-NLS-DSR, an increase in the neurite elongation produced bytestosterone was observed (Fig. 7C; n=86 of 86 transfectedcells, P<0.05). This enhanced neurite outgrowth, however, wasreduced by 40% with respect to non-transfected stimulatedcells. Cells expressing the nuclear localized mutatedparvalbumin (PV-NLS-CDEF-DSR) showed a rise in theneurite outgrowth in response to testosterone as observed incontrol cells (n=36 of 36 transfected cells, P<0.01). These datademonstrate that targeted cytosolic expression of parvalbumininhibits testosterone-induced neurite outgrowth and that anuclear Ca2+ increase is also needed for the neurite elongation.Under conditions that block the iAR, the increase in neuriteoutgrowth was smaller than the response obtained with theaddition of testosterone alone (Fig. 7D). To determine thecontribution of the genomic pathway in testosterone-inducedneurite elongation, we silenced the iAR by use of siRNA-ARor by treatment with the iAR antagonist cyproterone. Cellswere co-transfected with siRNA-AR and DSRed-pCMV sothat transfected cells (those with DSRed) could be visualizedand compared with non-transfected cells in the sameexperiment. After these cells were stimulated with testosterone(100 nM) for 24 hours, the testosterone-mediated neuriteoutgrowth was decreased 70% relative to non-transfected cellsin the siRNA-iAR treated cells (Fig. 7D; n=78 of 78 cells,P<0.05, four independent cultures) or 59% in cyproterone-treated cells (Fig. 7D; n=46 of 46, P<0.05, three independentcultures). Treatment with the membrane-delimited testosterone(T-BSA) decreased the neurite elongation by 60% (Fig. 7D;n=36 of 36 cells, P<0.05, three independent cultures). In allthese comparisons, the magnitude of the neurite outgrowth inthe absence of testosterone is defined as control. The responsesafter silencing the iAR show testosterone-induced neuriteoutgrowth and activation of the iAR allows an enhancement ofthis response. Together these results suggest that full activationof the testosterone-induced neurite outgrowth requires both aCa2+-mediated signaling pathway as well as the transcriptionalactivity associated with the iAR.

DiscussionThe goal of this study was to investigate the mechanisms usedin the generation of rapid testosterone-induced Ca2+ signals inneurons. Rapid, non-genomic effects of androgens have beendescribed (Benten et al., 1999b; Estrada et al., 2000; Lieberherrand Grosse, 1994), but their biological implication wasunresolved. To investigate these effects of testosterone, wemonitored the temporal and spatial characteristics oftestosterone-induced Ca2+ signals and began a dissection of themolecular basis of the signals. Our data show that testosteroneinduces rapid intracellular Ca2+ increases, which begin asdiscrete Ca2+ transients and develop into propagating waves inthe cytosol, a process that repeats to become an oscillatorypattern. These Ca2+ signals are mediated via a plasmamembrane receptor rather than the iAR, and they depend on aninterplay between Ins(1,4,5)P3-sensitive Ca2+ stores and influxfrom the extracellular medium. This new pathway for the

action of testosterone leads to the modulation of the neuriteoutgrowth. These rapid changes in Ca2+ precede the traditionalgenomic effects, but most probably they are criticalcomponents of the overall physiological response to thishormone.

Intracellular Ca2+ oscillations are a common signaling eventobserved in many cell types (Berridge et al., 2003). Thesesignaling cascades are not random events, but rather specificsignals that depend on the stimuli and the cell type (Aizman etal., 2001; Dolmetsch et al., 1998; Estrada et al., 2005). Forexample, in the cells we investigated, the initial Ca2+ releasenever began in the nucleus, implying that the signal needs topropagate through the cell to reach the nucleus. Ca2+ ions havebeen shown to travel slower than Ins(1,4,5)P3 and generally,Ca2+ does not diffuse more than 1 �m before it is sequesteredin the cell (Allbritton et al., 1992). The distance between theplace where the Ca2+ transient began and the nucleus in thesecells is approximately 10 �m and hence too long for Ca2+ totravel by simple diffusion. It is also unlikely that thepropagated Ca2+ wave in testosterone-treated neuroblastomacells can be explained purely by diffusion of Ins(1,4,5)P3because the signal observed traveled 10 �m/second, which isconsiderably slower than the diffusion constant for Ins(1,4,5)P3(280 �m2/second). These data suggest that the testosterone-induced Ca2+ wave observed in these experiments is a dynamicconsequence of the diffusion of both Ca2+ and Ins(1,4,5)P3.

We found that the oscillatory pattern induced by testosteroneexhibits a constant frequency of ~20 mHz. The requirement foroscillations, rather than single Ca2+ transients or prolongedelevations in intracellular Ca2+, to encode information has beenreported and specific frequencies were shown to activatespecific genes (Dolmetsch et al., 1998; Li et al., 1998; Sneydet al., 2004). Basically, by exploiting the two key features ofoscillatory signals – frequency and amplitude – the cell can useCa2+ as a second messenger to generate a large variety ofintracellular signals. This is an efficient way to use the samesecond messenger to activate many different processes. Thetestosterone-induced Ca2+ increase was evident in both cytosoland nucleus of neuroblastoma cells. Nuclear Ca2+ signals candirectly modify gene expression, fertilization, meiosis andapoptosis (Hardingham et al., 1997), representing a pivotalconnection point between extracellular and intracellularstimuli. Several reports show that nuclear Ca2+ elevations aredue to changes in the cytosolic Ca2+ concentration (Genka etal., 1999) whereas other reports indicate that the nuclear Ca2+

changes can be regulated independently of cytosolic Ca2+

(Leite et al., 2003). In order to choose between thesepossibilities we selectively inhibited the Ca2+ signaling ineither the nucleus or cytosol by expressing location-specificCa2+ buffer protein, parvalbumin. Although we cannot rule outtestosterone-induced intranuclear Ca2+ release, our datasuggests that the response to testosterone is initiated in thecytoplasm and that subsequent Ca2+ signals are generated bydiffusion of Ca2+ and Ins(1,4,5)P3 in the cytoplasm and intothe nucleus. In addition, aromatase, an enzyme that convertstestosterone into the estrogen (17�-estradiol) has been reportedin the CNS and neuroblastoma cells (Wozniak et al., 1998). Wefound that 17�-estradiol but not 17�-estradiol induced Ca2+

signals. The estrogen-induced Ca2+ rises have a differenttemporal pattern than those measured after addition oftestosterone. To rule out the possibility that testosterone acts

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via the estrogen pathway, experiments were done in thepresence of tamoxifen, an antagonist of the intracellularestrogen receptor. As shown in Fig. 1F (black line), theinhibitor did not have any effect on the testosterone-inducedCa2+ oscillations, suggesting that the response was specific fora direct testosterone action and not due to its metabolization toestrogen.

Ca2+ oscillations induced by testosterone only occurred inthe presence of extracellular Ca2+. Stimulation of cells in Ca2+-free conditions evoked a localized Ca2+ increase which did notpropagate throughout the cell, but resembled the initial peak inCa2+-containing conditions. Similar results were obtainedwhen Ca2+ was blocked with Gd3+. These findings suggest thatthe initial Ca2+ rise requires mobilization from internal stores,but Ca2+ influx is required to generate a propagated Ca2+ waveand subsequent Ca2+ oscillations. In several cell models, Ca2+

oscillations have been reported to be initiated by Ins(1,4,5)P3-induced release of Ca2+ from intracellular Ca2+ stores (Aizmanet al., 2001; Berridge et al., 2003; Estrada et al., 2005).Maintenance of these waves, however, required Ca2+ influxthrough Ca2+ channels in the plasma membrane (Sneyd et al.,2004).

Testosterone exerts its genomic effects through binding to,and activation of, a iAR which translocates to the nucleus andfunctions as a transcription factor (Beato, 1989). The iARtranslocation from the cytosol to nucleus upon hormonebinding is necessary for its activation and subsequent action ontranscriptional machinery (Lucas and Granner, 1992). Wefound that testosterone-evoked Ca2+ oscillations were notblunted by cyproterone, an antagonist of the iAR, themembrane-impermeant testosterone conjugate (T-BSA)induced effects that were similar to the free hormone, andknock-down of the iAR did not modify the oscillatory patterninduced by testosterone. Taken together, these results suggestthat the rapid effects of testosterone are mediated by a receptorrestricted to the plasma membrane. Recently, several reportshave shown that androgens can activate PTX-sensitive Gproteins (Benten et al., 1999b; Estrada et al., 2003). We useda pharmacological approach to determine whether testosteroneactivated G proteins and found evidence to support theinvolvement of a plasma membrane receptor in this signalingevent. The presence of membrane binding sites for androgenshas been previously suggested (Estrada et al., 2003; Lieberherrand Grosse, 1994), even in macrophages, which lack a classicaliAR (Benten et al., 1999b). Our results expand this concept toa neuronal cell line. Thus, a transient increase in Ca2+ appearsto be a response used by many cell types to directly altercellular processes through a nongenomic pathway, without theneed for slower, genomic processes.

A major question about the rapid effects of steroidhormones is whether there is a physiological role for thesesignals. In neurons, Ca2+ oscillations have been shown to beessential to migration (Spitzer et al., 2000), differentiation andneurite outgrowth (Gomez and Spitzer, 1999; Lautermilch andSpitzer, 2000). Our observations are in agreement withprevious studies showing the ability of androgens to increaseneurite outgrowth in PC12 cells (Lustig, 1994) and in motorneuron cells (Marron et al., 2005). These effects were assumedto be a consequence of activation of the iAR, but theparticipation of second messenger cascades was not clear.Cytosolic kinase activity, such as PKA and PI 3-kinase, have

been shown to be important for the initial steps of neuriteelongation in SH-SY5Y cells directly stimulated by additionof a membrane permeable activator of PKA, cyclic AMP(Sanchez et al., 2001). In addition, it has been reported thatstimulation of ERK pathways regulates neuronal growth andsurvival (Bonni et al., 1999). Interestingly, in neurons, thesekinase pathways can be activated and modulated byintracellular Ca2+ signaling (Doherty et al., 2000). Thus, Ca2+

oscillations induced by testosterone could represent an earlykey regulator of these events. To investigate this suggestion,we buffered intracellular Ca2+ signaling in specific locationsin the cell. We found that the testosterone-induced elongationsof the neurites were inhibited in cells in which the cytosolicCa2+ was buffered, whereas buffering the nuclear Ca2+ poolinhibited the neurite outgrowth by only 40%. Our datastrongly suggest that cytosolic and nuclear Ca2+ increases arecritical mechanisms controlling the neurite outgrowth inducedby testosterone. Androgens stimulate the differentiation ofdifferent neuronal cell types via the activation of the iAR andgene expression (Kelly et al., 1999). We found that themagnitude of the neurite elongation was less in cells that hadbeen modified to inactivate the iAR pathway than in cellsstimulated with testosterone alone. In both cases the neuriteelongation was significantly more than in cells that had notbeen treated with testosterone. These data show that the initialCa2+ signaling as well as an intact iAR are required tomaximize neurite elongation. Nongenomic actions can play aphysiological role in events prior to iAR activation. At thecytosolic level, Ca2+ increases stimulate the binding ofandrogens to their receptors (Cabeza et al., 2004). Moreover,androgens can activate Ca2+-dependent kinase pathways, suchas ERK, PI 3-kinase or Src (Estrada et al., 2003; Migliaccioet al., 2000), which could phosphorylate the iAR and enhanceits activity. It has also been suggested that both the temporaland spatial changes of the nuclear Ca2+ signal (Echevarria etal., 2003; Leite et al., 2003) and cytosolic Ca2+ signal(Dolmetsch et al., 1998; Li et al., 1998) are involved in thecontrol of gene expression. Our results lead to the conclusionthat the genomic and non-genomic pathways of testosteroneaction are inter-linked and that a concerted action is requiredfor normal cell function.

In neurons, androgens can induce changes at the cellularlevel, which can lead to changes in behavior such as sleep, thereaction to stress, mood and memory (Kelly et al., 1999).Although testosterone is an important neurotrophic andneuroprotective agent, which protects cells against death andinjury, it also has its negative side. The concentration oftestosterone used in this study (100 nM) is on the high end ofthe normal range measured in human males. However, anumber of physiological circumstances including age, sex, orphysical condition, can cause plasma levels to increase,reaching values similar to those studied here (Kelly et al., 1999;Mooradian et al., 1987). It is likely then, that the signals wereport here correspond to normal responses of the neuronalcells to transient levels of these hormones that may be reachedunder some particular physiological condition. Testosteroneincreases aggressive behavior (Kelly et al., 1999) and highlevels have been linked to neuronal apoptosis and cell death(M.E. and B.E.E., unpublished observations). These negativeeffects of androgens are not limited to motor neurons butextend to many cell types. Surprisingly, these harmful effects

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of high levels of testosterone can occur rapidly, and may beinitiated by a dysregulation of the Ca2+ signals described in thisreport. With a more global view, these effects of testosteroneobserved at the single cell level can be shown to have long-term effects at the organ level.

In this study, we found that testosterone induces rapidintracellular Ca2+ increases, which are characterized aspropagated Ca2+ waves in the cytosol and nucleus generatingoscillatory Ca2+ patterns. The oscillatory nature of the Ca2+

signals depends on a concerted action between theIns(1,4,5)P3-sensitive intracellular Ca2+ stores and Ca2+ influxfrom the extracellular medium. Kinetic analysis andnuclear/cytosol-targeted parvalbumin fusion proteins showedthat the Ca2+ rise is initiated in the cytosol, principally in thetip of the neurite, and then is propagated to the nucleusproducing regenerative Ca2+ oscillations. Moreover, theseeffects were independent of the iAR and were mediated by aplasma membrane G protein-coupled receptor. Thetestosterone-induced neurite outgrowth required changes inintracellular Ca2+ signaling in the cytosol, presumably toinitiate translocation of factors that activate nuclear events.These data support the conclusion that there is cross-talk in thepathways used for neuronal cell differentiation involving rapidnon-genomic effects, such as Ca2+ oscillations, and theclassical genomic actions of androgens, suggesting a novel rolefor testosterone.

Materials and MethodsPlasmidsThe nuclear-targeted parvalbumin (PV) expression vector was constructed aspreviously described (Pusl et al., 2002), using the full-length parvalbumin gene,which was sub-cloned into the pCMV-Myc-Nuc vector (Invitrogen). DsRed (DSR)was substituted for the original GFP in each construct. The DSR coding sequencefrom pDsRed2-N1 (Clontech) was PCR-amplified to introduce 5� and 3� NotI sites.The DSR PCR product was digested with NotI and subcloned into each PVconstruct. For the cytosolic-targeted PV expression vector, the specific sub-cellularlocalization of the fusion protein was achieved by introducing the nuclear exclusionsignal (NES) sequence derived from MKK1. The NES sequence derived fromMKK1 encodes a short stretch of amino acids that represent residues 32-44 ofMEK1, which does not contain the putative ERK binding site. These primers alsointroduced SalI and XbaI sites, which were used to subclone this DNA fragmentinto pCMV-Myc-Cyto to generate PV-NES-DSR. The final resulting fusion proteinswere designated PV-NLS-DSR with nuclear localization and PV-NES-DSR withcytosolic localization. A site-directed mutagenesis was carried out with the nuclearPV to generate the mutant parvalbumin PV-NLS-CDEF coding for protein in whichboth functional Ca2+-binding sites (CD and EF domain) were inactivated bysubstituting a glutamate for a valine residue at position 12 of each Ca2+-bindingloop. This new plasmid is designated as PV-NLS-CDEF-DSR. The mutant PV wasused as a control for the Ca2+-buffering capacity of PV.

Cell cultures and transfectionThe human neuroblastoma cell line (SH-SY5Y; ATCC) was cultured in 1:1DMEM:Ham’s F12 medium supplemented with 10% (v/v) heat-inactivated fetalbovine serum, 5% non-essential amino acids, 100 IU penicillin and 50 �g/mlstreptomycin, in a 95% air-5% CO2 humidified atmosphere in an incubator at 37°C.Cells were grown on 22-mm gelatin-coated glass coverslips for the Ca2+

measurements or 60-mm Petri dishes for biochemical assays. Cultured cells werewashed once with PBS before stimulation with testosterone (from concentratedstocks made in ethanol). The final ethanol concentration (<0.01%) had no effect onintracellular Ca2+ concentration or biochemical determinations. For transienttransfections, neuroblastoma cells were grown in 60-mm dishes to 60% confluence.Before transfection, the cells were washed to remove serum and were transientlytransfected with empty vector: PV-NES-DSR, PV-NLS-DSR or PV-CDEF-DSR (2-4 �g) using Lipofectamine (Invitrogen) in Opti-MEM (Invitrogen) for 4 hoursfollowing the manufacturer’s instructions. A single plate of transfected cells wasthen used to set-up the experimental cultures required for each assay, ensuring equaltransfection efficiencies between different treatments, and cells were cultured for anadditional 24 hours before treatment with testosterone.

Ca2+ imagingCells were loaded with 5 �M Fluo-4/AM at 37°C for 30 minutes in a standardsolution (in mM): 135 NaCl, 5 KCl, 2 MgCl2, 2 CaCl2, 10 Hepes, 5.6 glucose, pH7.4. After loading, cells were washed twice with the standard solution and placedon an inverted microscope connected to a laser scanning imaging system (LSM 510META, Zeiss). Cells were stimulated with the hormone diluted in the standardsolution. A 488 nm excitation wavelength focused through a 40� Neo-Fluorobjective lens (NA 1.4; Zeiss) was provided by an argon laser. In order to identifythe cells expressing the PV construct with the DSR protein, transfected cells wereexamined using fluorescence excitation at 568 nm. Regions of interest (ROI) weremonitored within the nucleus and cytoplasm of the same cell; transfected and non-transfected cells in the same field were followed over time. Each experimentinvolved a single independent cell and whole cell fluorescence was measured. Agiven cell was considered to oscillate when at least three Ca2+ transients wererecorded over the time monitored, usually 10 minutes. ROIs with the same pixeldimensions, were identified and analyzed using ImageJ software (NIH, Bethesda,MA, USA). The inhibitors were added during the dye incubation; times andconcentrations are indicated in the Results. To assess the role of G proteins, cellswere incubated either with 1 �g/ml PTX for 6 hours or in a permeabilization solution[100 mM KCl, 20 mM NaCl, 5 mM MgSO4, 1 mM NaH2PO4, 25 mM NaHCO3, 3mM EGTA, 1 mM CaCl2, 20 mM Tris-HCl (pH 7.4), 0.1% BSA, 1 mM ATP, 0.1%glucose and 40 mg/ml saponin] for 5 minutes in the presence or absence of 100 nMGDP�S, a nonhydrolyzable analog of GDP. After permeabilization, but beforehormone stimulation, the cells were incubated for 1 hour in the imaging solutionbuffer to re-stabilize the membrane integrity (Estrada et al., 2003). Ca2+-inducedfluorescence intensity ratio (F/Fo) was plotted as a function of time. For linescanning, a single line (shown in the cell images as a solid white line) was chosenfrom the entire confocal section and repeatedly scanned every 20 mseconds. Thesuccessive lines were stacked horizontally to compile an image where timeincreased from left to right, and the spatial dimension was preserved in the verticalaxis. To perform power spectrum analysis, we used an algorithm written inMATLAB as described previously (Uhlen, 2004).

siRNAWe used siRNA for Ins(1,4,5)P3R type 1 in order to reduce the levels of the receptor(provided by F. Leite, Federal University of Minas Gerais, Belo Horizonte, Brazil).The siRNA template was obtained from Ambion and the sequence wasAAAGCACCAGCAGCTACAACTCCTGTCTC of the human Ins(1,4,5)P3R type1. The siRNA for the androgen receptor was obtained from Santa CruzBiotechnology (cat. # sc-29204). Transfection with siRNA was performed usingRNAiFect (Qiagen) and down-regulation of either Ins(1,4,5)P3R or iAR wasconfirmed by immunofluorescence and western blot analysis.

ImmunocytochemistryNeuroblastoma cells grown on coverslips were fixed in ice-cold methanol for 15minutes, blocked in PBS containing 1% BSA for 60 minutes, and incubated withprimary antibodies at 4°C overnight. Primary antibody against androgen receptor(N-20; Santa Cruz Biotechnology) was used at 1:250. The cells were then washedfive times with PBS/BSA and incubated with the appropriate Alexa-conjugated goatanti-rabbit secondary antibody (Molecular Probes) for 1 hour at room temperature(1:5000). After three washes, the coverslips were mounted in Prolong Antifade(Molecular Probes) to retard photo-bleaching. The samples were evaluated usingconfocal microscopy (LSM 510 META, Zeiss). To compare the confocal images ofcontrol and stimulated cells, the settings for the data acquisition and analysis werestandardized.

Western blotCells lysates containing 40 �g of protein were separated by SDS-PAGE in a 4-20%linear gradient for Ins(1,4,5)P3R-1 and iAR or 10% gel for parvalbumin proteinfollowed by electrophoretic transfer onto PVDF membranes for 2 hours at 400 mA.The following primary antibodies and their respective dilutions were used: anti-iAR(1:1000; Santa Cruz), anti-Ins(1,4,5)P3R type 1 (1:2000) (Choe et al., 2004), anti-parvalbumin (1:1000; Sigma), anti-actin (1:1000; Santa Cruz). Membranes wereincubated with primary antibodies overnight at 4°C. After incubation withhorseradish peroxidase-conjugated secondary antibodies (1:5000) for 2 hours atroom temperature, the bands were visualized by an enhanced chemiluminescencesystem (Pierce). Membranes were stripped and re-probed with �-actin antibody inorder to control the protein loaded. Blots were quantified by scanning densitometry.

Analysis of neurite outgrowthNeuroblastoma cells were cultured in growth medium for 24 hours. Next, themedium was replaced with a medium supplemented with or without testosteroneand cultured for an additional 3 days. To determine whether Ca2+ signaling wasnecessary for neurite outgrowth, cells were transfected with parvalbumin plasmidsthat were engineered to express either in the cytoplasm (PV-NES-DSR) or nucleus(PV-NLS-DSR or PV-NLS-DSR). Intracellular androgen receptor participation wasdetermined using siRNA-AR, cyproterone and T-BSA, as described above. Toquantify the morphological changes induced by testosterone, cells were incubated

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with the fluorescent dye Cell-Tracker (Molecular Probes) for 45 min. Cells werevisualized by confocal microscopy (LSM 510 META, Zeiss), and the acquiredfluorescence images were analyzed and compared with LSM Image Browser(Zeiss). For the analysis of neurite outgrowth, only cells with a neurite that waslonger than the soma were included. To normalize for various cell size, neuriteoutgrowth was calculated as the ratio of the neurite length to soma length in theneurite projection.

Statistical analysisData are expressed as mean ± s.e.m. or as representative traces. Statistical analysisof the differences between groups was performed using ANOVA, followed by theBonferroni post-test. P<0.05 was considered statistically significant.

This work was supported by NIH grants GM63496 and DK61747(B.E.E.), a Grass Foundation Fellowship (M.E.), and a grant fromVetenskapsrådet – the Swedish Research Council (P.U.). We aregrateful to A. Bennett and F. Leite for providing critical reagents andto M. Nathanson, P. Correa, A. Varshney and C. Gibson for thoughtfuldiscussions and comments on the manuscript.

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