Effect of Roscovitine on Intracellular Calcium Dynamics: Differential

Oct 29, 2013 - *Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Italy, Via Amendola 165/A, 70125 Bari, Italy. Tel...
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Article pubs.acs.org/molecularpharmaceutics

Effect of Roscovitine on Intracellular Calcium Dynamics: Differential Enantioselective Responses Grazia Tamma,*,†,‡ Marianna Ranieri,†,‡ Annarita Di Mise,† Alessia Spirlì,†,§ Annamaria Russo,† Maria Svelto,†,∥ and Giovanna Valenti†,∥ †

Department of Biosciences, Biotechnologies and Biopharmaceutics and ∥Centre of Excellence Genomic and Proteomics GEBCA, University of Bari, Italy § Department of Pharmacology & Toxicology, University of Lausanne, Switzerland ABSTRACT: Cyclin-dependent kinases (CDKs) inhibitors have emerged as interesting therapeutic candidates. Of these, (S)-roscovitine has been proposed as potential neuroprotective molecule for stroke while (R)-roscovitine is currently entering phase II clinical trials against cancers and phase I clinical tests against glomerulonephritis. In addition, (R)-roscovitine has been suggested as potential antihypertensive and antiinflammatory drug. Dysfunction of intracellular calcium balance is a common denominator of these diseases, and the two roscovitine enantiomers (S and R) are known to modulate calcium voltage channel activity differentially. Here, we provide a detailed description of short- and long-term responses of roscovitine on intracellular calcium handling in renal epithelial cells. Short-term exposure to (S)-roscovitine induced a cytosolic calcium peak, which was abolished after stores depletion with cyclopiazonic acid (CPA). Instead, (R)-roscovitine caused a calcium peak followed by a small calcium plateau. Cytosolic calcium response was prevented after stores depletion. Bafilomycin, a selective vacuolar H+-ATPase inhibitor, abolished the small calcium plateau. Long-term exposure to (R)-roscovitine significantly reduced the basal calcium level compared to control and (S)-roscovitine treated cells. However, both enantiomers increased calcium accumulation in the endoplasmic reticulum (ER). Consistently, cells treated with (R)-roscovitine showed a significant increase in SERCA activity, whereas (S)-roscovitine incubation resulted in a reduced PMCA expression. We also found a tonic decreased ability to release calcium from the ER, likely via IP3 signaling, under treatment with (S)- or (R)-roscovitine. Together our data revealed that (S)-roscovitine and (R)-roscovitine exert distinct enantiospecific effects on intracellular calcium signaling in renal epithelial cells. This distinct pharmacological profile can be relevant for roscovitine clinical use. KEYWORDS: roscovitine, drug targeting, cytoskeleton, calcium homeostasis, cyclin-dependent-kinases inhibitors, Rho-GTPases

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INTRODUCTION Roscovitine is an olomoucine related purine that was originally developed to inhibit the kinase activity of cyclin-dependent kinases (cdks),1 which are key players in controlling cell cycle and several cellular functions such as differentiation, cell death (especially apoptosis), cell migration and actin reorganization.2,3 In keeping with this, cyclin-dependent kinase inhibitors (CDKi) represent interesting therapeutic candidates. Of these, roscovitine exists as two optical isomers tested for their kinase inhibitory activity.4,5 Specifically, (S)-roscovitine displayed a slight lower activity than (R)-roscovitine which results almost similar respect to cdk2/cyclin-E.5 (R)-Roscovitine is currently entering phase II clinical trials against cancers and phase I clinical tests against glomerulonephritis and has emerged as potential antihypertensive and anti-inflammatory drug.6,7 (S)Roscovitine has been proposed as potential neuroprotective molecule for stroke.8,9 At molecular level, dysregulation of intracellular calcium homeostasis may be a common denominator of these pathological processes causing abnormal cell cycles, promoting cell differentiation and proliferation.10−13 Several studies, indeed, have demonstrated that roscovitine can © 2013 American Chemical Society

also affect neuronal voltage calcium channels activity, independently of its inhibitory action on cdks.14,15 In particular, (R)-roscovitine inhibits L-current by slowing activation and enhancing inactivation, whereas (S)-roscovitine enhances inactivation by binding different sites of voltage calcium channels.16 Therefore, the biological action of roscovitine is not restricted to the inhibition of cdks, providing new perspectives for its applications in the field of cancer therapy and in several disorders associated with alteration of intracellular calcium homeostasis. In this respect, it is still not clear how roscovitine modulates intracellular calcium signaling and if the two enantiomers may exert different responses on calcium homeostasis in epithelial cells. Elevation in intracellular free calcium ([Ca2+]i) is due to the entry from extracellular compartment and the release from intracellular pools: the endoplasmic reticulum (ER) and the Received: Revised: Accepted: Published: 4620

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lysosome-like acidic organelles (LLAO).17 Calcium release, from the ER, mainly occurs via ryanodine receptors (RyRs) and inositol-1,4,5-trisphosphate (IP3) signaling, whereas the one from LLAO via nicotinic acid adenine dinucleotide phosphate (NAADP).18,19 At resting, the physiological level of intracellular calcium is regulated by the simultaneous interplay of different counteracting mechanisms, including the involvement of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and the plasma membrane Ca2+-ATPase (PMCA). The former pumps Ca2+ back to the ER, while the latter removes Ca2+ from intracellular to extracellular environment, thus playing a central role in maintaining a very low intracellular calcium concentration.20 Therefore, the very low resting Ca2+ concentration in the cytosol, ideal for signaling transmission, is due to an orchestrated rapid diffusion of Ca2+ from extracellular to intracellular pools and between intracellular compartments, making calcium signal transduction pathway extremely complex.21 When cells are stimulated by various means, the resulting Ca2+ signal might be code as elementary spike, global wave or cytosolic oscillations highly organized in space, frequency and amplitude because the localization and the integrated release of free cytosolic Ca2+, over time, carry specific signaling information.22,23 Mutations or alterations in the expression and/or the activity of proteins involved in intracellular Ca2+ regulation may be responsible for a plethora of known and unknown diseases.24,25 Here, we provide a detailed description of short- and long-term responses of roscovitine on intracellular calcium handling in renal epithelial cells. We demonstrate that besides their ability to inhibit cdks,1,8,26 (R)- and (S)-roscovitine display different enantiospecific responses on intracellular calcium balance by regulating the activity and the protein expression of SERCA and PMCA respectively.

transferred onto Immobilon-P membranes (Millipore Corporate Headquarters, Billerica, USA) for Western blot analysis, blocked in TBS-Tween-20 containing 3% BSA and incubated with primary antibodies specific for PMCA1/4 diluted 1:300 in TBS-Tween-20 containing 1% BSA . Immunoreactive bands were detected with secondary antibody conjugated to horseradish peroxidase (HRP) (1:5000) obtained from Santa Cruz Biotechnologies (Tebu Bio, Milano, Italy). Membranes were developed using SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, USA) with Chemidoc System (Bio-Rad Laboratories, Milano, Italy). Band intensities were quantified by densitometric analysis using National Institutes of Health (NIH) ImageJ software. Actin Visualization. MCDK cells were grown on Ø12 mm glass coverslips and fixed with 4% paraformaldehyde in phosphate buffer saline (PBS) for 20 min. Cells were washed 3 times for 5 min in PBS and permeabilized with 0.1% Triton X-100 in PBS for 5 min. Actin cytoskeleton was visualized by incubation with phalloidin−TRITC (100 μg/mL) for 45 min. Coverslips were mounted on glass slides with Mowiol mounting medium. The actin cytoskeleton was visualized by a confocal microscope Leica TCS SP2 (Leica Microsystems, Heerbrugg, Switzerland). Actin Polymerization Assay. Actin polymerization was analyzed as described.27 Briefly, MDCK cells were seeded on a 24-well plate and grown to confluence. Cells were left under basal condition or treated O/N with (R)-roscovitine (100 nM) or (S)-roscovitine (100 nM) as described above. Treatments were stopped by adding 400 μL of 3.7% paraformaldehyde, 0.1% Triton X-100, 0.25 μM TRITC−phalloidin in 20 mM potassium phosphate, 10 mM PIPES, 5 mM EGTA and 2 mM MgCl2, pH 6.8. After staining for 1 h, cells were washed three times with PBS and 800 μL of methanol was added overnight. The fluorescence (540/565 nm) was read in a Shimadzu RF5301PC fluorometer. The obtained fluorescent values were normalized for the total protein content. Data were analyzed by one-way ANOVA followed by Newman−Keuls multiple comparison test with *p < 0.05 considered to be statistically different. Video Imaging Intracellular Calcium Measurements. For intracellular Ca2+ measurements, cells were grown on Ø40 mm glass coverslips. Cells were loaded with 4 μM Fura-2AM for 20 min at 37 °C in DMEM. During experiment, cell were perfused with Ringer’s solution containing 137 mM NaCl, 5.4 mM KCl, 0.5 mM MgCl2, 4.2 mM NaHCO3, 3 mM Na2HPO4, 0.4 mM KH2PO4, 10 mM Hepes sulfonic acid, 10 mM glucose and 1.3 mM CaCl2, pH 7.4. In fluorescence measurements, coverslips, with dye-loaded cells, were mounted in a perfusion chamber (FCS2 Closed Chamber System, BIOPTECHS, Butler, U.S.A.) and measurements were performed using an inverted microscope (Nikon Eclipse TE2000-S microscope) equipped for single cell fluorescence measurements and imaging analysis. The sample was illuminated through a 40× oil immersion objective (NA = 1.30). The Fura-2AM loaded sample was excited at 340 and 380 nm. Emitted fluorescence was passed through a dichroic mirror, filtered at 510 nm (Omega Optical, Brattleboro, VT, USA) and captured by a cooled CCD camera (CoolSNAP HQ, Photometrics). Fluorescence measurements were carried out using Metafluor Software (Molecular Devices, MDS Analytical Technologies, Toronto, Canada). Intracellular calcium level was calculated as described by Grynkiewicz.28 Briefly, calcium concentration was

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EXPERIMENTAL SECTION Materials. All chemicals were purchased from Sigma (Sigma-Aldrich, Milan, Italy). Fura-2AM was obtained from Molecular Probes (Life Technologies, Monza, Italy). (R)Roscovitine was bought from Selleck Chemicals (Munich, Germany) and (S)-roscovitine from Vinci Biochem (Vinci, FI, Italy). PMCA1/4 antibody was purchased from Santa Cruz Biotechnologies (Tebu Bio, Milano Italy). Anti-Na+/K+ATPase was bought from Millipore (Milano, Italy). Cell Culture. Madin-Darby canine kidney (MDCK) type I cells (kindly gift by Prof. Deen) were grown in Dulbecco’s modified Eagle’s medium (DMEM) high glucose, supplemented with 5% (v/v) fetal bovine serum, 1% (v/v) Lglutamine, 1% (v/v) nonessential amino acids and 0.5% ciprofloxacin, at 37 °C in 5% CO2. For long-term experiments, cells were left under basal condition or incubated overnight (O/N) with (R)-roscovitine (100 nM) and (S)-roscovitine (100 nM). Cell Fractionation. Cells were scraped and sonicated in a buffer containing 220 mM mannitol, 70 mM sucrose, 20 mM Tris-HCl in the presence of protease and phosphatase inhibitors. Nucleus and mitochondrion enriched fractions were removed by centrifugation at 800g and 8000g respectively for 20 min at 4 °C. Membrane enriched fractions were obtained by centrifugation for 1h at 4 °C at 17000g in a Beckman Allegra R-64 centrifuge. Gel Electrophoresis and Immunoblotting. Cellular proteins were separated on 10% bis-tris acrilamide gels under reducing conditions. Protein bands were electrophoretically 4621

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determined from the emission fluorescence ratio of the two excitation wavelengths accordingly to the formula (Ca2+)i = Kd*Q(R − Rmin)/(Rmax − R), where Kd (224 nM) indicates the dissociation constant of Fura-2AM for (Ca)i and Q indicates the ratio of the fluorescence intensities (F) at the minimum and the maximum calcium concentration at 380 nm. Each sample was calibrated by the addition of 5 μM ionomycin in the presence of 1 mM EGTA (Rmin) followed by 5 μM ionomycin in 5 mM CaCl2 (Rmax). Fluorescence Resonance Energy Transfer (FRET) Measurements. FRET experiments were performed as described.29 MDCK cells were transiently transfected with a plasmid encoding the D1ER cameleon (gift from Prof. Roger Tsien30) for FRET studies. FRET measurements were carried out using MetaMorph software (Molecular Devices, MDS Analytical Technologies, Toronto, Canada). ECFP and citrine were excited at 435 or 509 nm, respectively. FRET from ECFP to citrine was determined by excitation of ECFP and measurement of fluorescence emitted from citrine. Corrected nFRET values were determined accordingly to Tamma.29 Statistical Analysis. Data are reported as mean values ± SEM. Statistical analysis was performed by one-way ANOVA followed by Newman−Keuls multiple comparison test with *p < 0.05 considered statistically different. When applicable, paired t test (Student) was also used.

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RESULTS Short-Term Effect of Roscovitine on Intracellular Calcium Dynamics. MDCK was used as cell model because it is well characterized and commonly used to investigate the intracellular signaling at the molecular level. In addition, MDCK cells can form highly polarized cell monolayer mimicking apical versus basolateral in vivo situation. MDCK cells were cultured as described in the Experimental Section and loaded with Fura-2AM (4 μM) to measure changes in intracellular calcium under roscovitine treatment at shortterm (5 μM, time is indicated by the horizontal bars in figures). Cells were exposed to (S)- or (R)-roscovitine, and variations in intracellular calcium (Ca2+)i were evaluated by single-cell epifluorescence imaging. Representative traces showing the comparison between the effects on intracellular calcium concentration induced by roscovitine and those elicited by the classical calcium agonist ATP (100 μM) are shown in Figure 1. Treatment with (S)-roscovitine evoked a major calcium spike which is, occasionally, followed by smaller peaks ((S)-Rosc 43.07 ± 3.29% vs ATP 100%; *p < 0.0001, n = 55 cells), which was abolished by cyclopiazonic acid (CPA) pretreatment indicating that calcium was released from endoplasmic reticulum (ER) deposits (Figure 1A). A different profile of intracellular calcium mobilization was instead observed under (R)-roscovitine: a calcium spike ((R)-Rosc 43.64 ± 3.26% vs ATP 100%; *p < 0.0001, n = 35 cells; calcium variations calculated at peak) was followed by a prolonged calcium plateau, which disappeared after removing (R)roscovitine (Figure 1B). Preincubation with CPA selectively abolished the spike without affecting the plateau. To better analyze the characteristic of these distinct intracellular calcium responses, cells were prior exposed to Bafilomycin A1, a strong inhibitor of the vacuolar type H+-ATPase, and then to (R)roscovitine (Figure 2). Bafilomycin A1, by inhibiting the vacuolar type H+-ATPase, increases lysosomal pH thus reducing lysosomal calcium concentration.31 Under these conditions, (R)-roscovitine

Figure 1. (S)-Rosc and (R)-Rosc effect on cytosolic Ca2+ changes in MDCK cells. MDCK cells, loaded with Fura-2AM 4 μM, were perfused with ringer solutions containing ATP, (S)-roscovitine (A) or (R)-roscovitine (B) for the time indicated by the horizontal bars. Fluorescence ratio 340 nm/380 nm was recorded and responses to the roscovitine were compared to that obtained after stimulation with a maximal dose of the calcium-mediated agonist ATP 100 μM that was used as internal control. Each trace is representative of 3−4 different experiments. CPA pretreatment did not induce any increase in intracellular calcium under (S)-roscovitine stimulation (A), whereas a small calcium plateau was still detected after (R)-roscovitine addition (B).

elicited a single calcium spike. On the right of Figure 2, the area under calcium variations curve underlines a significant decrease of (R)-roscovitine-induced intracellular calcium mobilization under Bafilomycin A1 incubation ((R)-Rosc 1.00 ± 0.026 vs (R)-Rosc+Bafilomycin 0.948 ± 0.0029; *p < 0.0001, n = 15 cells), due to the loss of calcium plateau. Co-incubation with CPA and Bafilomycin A1 abolished completely (R)roscovitine induced intracellular calcium release (Figure 3), suggesting that a calcium pool was release from the ER (calcium spike) and a second pool from acidic compartment (plateau). Long-Term Effect of Roscovitine on Intracellular Calcium Signaling. Calcium is involved in controlling numerous vital processes, and every cell holds a calcium signaling “toolkit” equipped of many proteins and enzymes to keep cells in a signaling-competent condition.10 Therefore, intracellular calcium concentration is constantly maintained at a level of about 4 orders of magnitude lower than the extracellular environment or the lumen of the ER. We evaluated whether in vitro administration of roscovitine (100 nM O/N) alters the basal cytosolic calcium. At rest, cells preincubated 4622

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Figure 2. Effect of the inhibitor of vacuolar H+, Bafilomycin A1, on (R)-roscovitine-induced calcium changes. MDCK cells loaded with Fura-2AM were first stimulated with (R)-roscovitine for the time indicated by the horizontal bars. Treatment with Bafilomycin A1 abolished intracellular calcium plateau, without affecting calcium peak induced by (R)-roscovitine. On the right, histogram showing the calculated area under the curve obtained after (R)-roscovitine stimulation in the absence or in the presence of Bafilomycin A1.

Figure 3. Effect cotreatment with Bafilomycin A1 and CPA on (R)roscovitine dependent calcium variations MDCK cells loaded with Fura-2AM were first stimulated with (R)-roscovitine for the time indicated by the horizontal bars. Any intracellular calcium variations were observed after cotreatment with Bafilomycin A1 and CPA.

Figure 4. Effect of (S)- and (R)-roscovitine on free cytosolic calcium at steady state. MDCK cells were treated with (S)- or (R)-roscovitine overnight, and the free cytosolic [Ca2+]i was calculated according to the Grynkiewicz formula,28 as described in the Experimental Section. At resting, (R)-roscovitine incubation was associated with a significant decrease in intracellular calcium level compared with (S)-roscovitine and control cells.

with (R)-roscovitine showed a significant decrease in cytosolic calcium concentration compared with control and (S)roscovitine treated cells ((R)-Rosc 82.84 ± 3.37 nM, n = 124 cells; (S)-Rosc 131.4 ± 9.23 nM, n = 74 cells vs Ctrl 130.7 ± 4.05 nM, n = 136 cells, *p < 0.0001) (Figure 4). Next, calcium in the ER was determined by FRET experiments using the ERtargeted Cameleon (D1ER) probe30 because raising the calcium stored in the ER would explain the effect of (R)roscovitine on cytosolic calcium level. This probe contains an ER-retention motif and calmodulin motif (D1). When calcium binds to the calmodulin motif (D1), it causes an intramolecular rearrangement leading to energy transfer between the donor and acceptor molecules, resulting in FRET signal output. Cells treated with both enantiomers exhibited a higher FRET signal, compared with untreated cells (Figure 5) ((S)-Rosc 117.8 ± 2.40, n = 159 cells; (R)-Rosc 110.0 ± 4.01, n = 175 cells; vs Ctrl 100.0 ± 2.44, n = 132 cells, *p < 0.01) consistent with a significant increase in calcium stored in the ER. These observations suggest that both roscovitine enantiomers might modulate the expression and/ or the activity of pivotal calcium signaling proteins altering

intracellular calcium equilibrium. The efficiency to store calcium in the ER mainly depends on SERCA activity. Therefore, the activity of this pump was evaluated by dynamic FRET experiments (Figure 6). Specifically, calcium accumulated in the ER was depleted with CPA, and then the speed of calcium reuptake into the ER was measured using the FRETbased D1ER sensor.30,32 We found that cells preincubated with (R)-roscovitine showed a higher SERCA activity compared with control and (S)-roscovitine treated cells ((R)-Rosc 0.50 ± 0.12, n = 18 cells; (S)-Rosc 0.23 ± 0.03, n = 21 cells vs Ctrl 0.17 ± 0.02, n = 15 cells, *p < 0.001), as indicated by the quantification of the initial slope of the FRET ratio (histogram on the right panel Figure 6). Obtained data suggest that (R)roscovitine modulates SERCA activity increasing the ER calcium content. To better investigate the complex machinery regulating intracellular calcium homeostasis under (S)- and 4623

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roscovitine incubation was also detected in the membrane expression of Na+/K+-ATPase, used as internal control (Figure 7 on the bottom). Next, we evaluated the effect of the IP3-generating calcium efflux elicited the agonist ATP (Figure 8). To this end, cells were treated O/N with (S)- and (R)-roscovitine respectively. In the absence of extracellular calcium, acute ATP stimulation caused a significant decrease of calcium released from the ER under (S)- or (R)-roscovitine long-term treatments, compared to untreated cells ((S)-Rosc 1.46 ± 0.025, n = 113 cells; (R)Rosc 1.38 ± 0.017, n = 127 cells vs Ctrl 1.55 ± 0.026, n = 101 cells, *p < 0.001), suggesting that treatments with both enantiomers are associated with a tonic decreased ability to release calcium from the ER likely via IP3 signaling. Therefore, we might conclude that (S)- and (R)-roscovitine modulate, distinctly, the expression and the activity of PMCA and SERCA, which are both fundamental in controlling intracellular calcium compartmentalization. Long-Term Effect of Roscovitine on Rho Activity. The molecular architecture of intracellular calcium compartments is extremely complex and requires a correct interplay between several signaling proteins including the small GTPases of Rho family. Treatment with (S)- or (R)-roscovitine results in a significant decrease of Rho activity, as assessed by FRET experiments, using a probe containing a Rho-binding domain (RBD) sandwiched by YFP and CFP.33 Binding of endogenous GTP-RhoA to RBD displaced YFP and CFP, thereby decreasing FRET efficiency and monitoring the abundance of GTP-RhoA. The results of the FRET experiments are summarized in Figure 9A. Compared to control cells, (S)- or (R)-roscovitine significantly increased nFRET signal, consistent with a decrease in the activity of RhoA ((S)-Rosc 166.3 ± 14.43, n = 41; (R)-Rosc 210.2 ± 23.50, n = 54; vs Ctrl 100.0 ± 3.38, n = 56, *p < 0.001). Rho inhibition is, indeed, associated with actin depolymerization in renal collecting duct cells.34−36 Confocal studies (Figure 9B) and semiquantitative analysis of

Figure 5. Evaluation of [Ca2+]ER with ER-targeted cameleon (D1ER) probe after (S)- and (R)-roscovitine incubation. MDCK cells were transiently transfected with D1ER probe as described in the Experimental Section, and the experiments were executed 48 h after transfection. Cells were treated with (S)- or (R)-roscovitine overnight, and changes of nFRET ratio were calculated as described.29 Histogram compares changes of nFRET Ratio, between control, (S)- and (R)roscovitine treated cells indicating that both enantiomers increased the [Ca2+]ER.

(R)-roscovitine, the expression of the PMCA pump was also evaluated. Cells were left untreated or long-term incubated with (S)- or (R)-roscovitine. Plasma membrane enriched fractions were prepared and subjected (60 μg) to immunoblotting and probed with specific antibodies recognizing PMCA1/4 or Na+/ K+-ATPase α1 (Figure 7). Results showed that the expression level of PMCA was significantly reduced only in (S)-roscovitine treated cells ((S)-Rosc 0.55 ± 0.09 vs Ctrl 1.00 ± 0.08, n = 4, *p < 0.05). No significant change was detected under (R)roscovitine treatment ((R)-Rosc 1.23 ± 0.23 vs Ctrl 1.00 ± 0.08, n = 4). Any relevant alteration under (S)- or (R)-

Figure 6. Evaluation of SERCA expression and activity. On the left, dynamic FRET experiment. Calcium was depleted from the ER, and the speed of calcium reuptake into the ER was measured with a FRET-based D1ER sensor. Fluorescence ratio was recorded and calculated as ΔR/R0. Each trace represents the Ca2+ entry phase via SERCA following store depletion caused by CPA. On the right, statistical analysis of the SERCA activity was evaluated based on the initial slope of ratio increase after calcium readdition. Data indicate that (R)-roscovitine incubation was associated with a significant increase in SERCA activity compared with (S)-roscovitine and control cells. 4624

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Figure 7. PMCA expression. MDCK cells were left untreated or incubated with (S)- or (R)-roscovitine (O/N). Fractions enriched in plasma membrane were prepared, and equal amount of proteins (60 μg) from each condition were subjected to Western blotting analysis with anti-PMCA (1:300) or with anti-Na+/K+-ATPase (1:10000) and revealed with anti-mouse HRP-coupled secondary antibodies (1:5000). On the bottom, statistical analysis of the detectable bands revealed a significant decrease of PMCA abundance only in cells incubated with (S)-roscovitine.

calcium signaling. This specific signaling action might explain some of the diverse responses of these compounds and their potential clinical applications. Specifically, the main findings reported in this work are summarized as follows: (a) (S)- and (R)-roscovitine evoked a transient increase of cytosolic calcium; (b) basal intracellular calcium concentration was significantly lower in cells treated with (R)-roscovitine compared with control and (S)-roscovitine treated cells; (c) (S)- and (R)roscovitine elicited an increased calcium level stored in the ER, as assessed by FRET experiments with the D1ER probe, which detects (Ca2+)ER directly; (d) (R)-roscovitine increased SERCA activity whereas (S)-roscovitine decreased PMCA expression compared to untreated cells; (e) both enantiomers reduced the activity of Rho proteins resulting in a partial depolymerization of actin cytoskeleton. Together, these findings demonstrate that the two enantiomers exert distinct responses on calcium signaling in renal epithelial cells. The distinct effects are mediated by modulation of the expression and the activity of pivotal proteins involved in controlling intracellular calcium balance. Roscovitine has been generated as selective and reversible inhibitor of cdks although many studies demonstrated additional effects26 on different cell types displaying distinct sensitivity to this drug.37 Opposite responses have been found in renal tubular epithelial cells treated with low (∼10 μM) or high (∼50 μM) dose of (R)-roscovitine. Specifically, in renal cells, low roscovitine concentration induced cell senescence whereas apoptosis was observed at higher concentration.37 Independently of its inhibitory action on cdks,14,15 (R)-roscovitine can inhibit other kinases such as ERK1 and ERK2, at micromolar concentration.38 Moreover, (S)- and (R)-roscovitine affect the activity of voltage-dependent calcium channels at clinically relevant concentrations (10−50 μM). We show here that both enantiomers can modulate intracellular calcium balance differ-

Figure 8. Evaluation of IP3-generating agonist ATP on cytosolic calcium under (S)- and (R)-roscovitine treatment. MDCK cells were left untreated or incubated with (S)- or (R)-roscovitine (O/N). Fluorescence ratio 340 nm/380 nm was recorded. Responses to IP3generating agonist ATP (100 μM), under (S)- or (R)-roscovitine longterm treatment, were calculated as ΔF/F0. Statistical analysis revealed that both enantiomers significantly reduced the calcium peak amplitude compared to untreated cells (Ctrl).

F-actin content (Figure 9C), evaluated with the actin polymerization assay,27 confirmed that (S)- or (R)-roscovitine incubation significantly reduced F-actin (Figure 9) ((S)-Rosc 69.31 ± 8.73, n = 13; (R)-Rosc 77.46 ± 4.11, n = 38 vs Ctrl 100.0 ± 4.37, n = 34, *p < 0.001).

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DISCUSSION Roscovitine is a selective and reversible inhibitor of cdks. In the present study we demonstrate a functionally different effect elicited by the two optical isomers of roscovitine S and R on 4625

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calcium spike, the kinetics of intracellular calcium variations revealed that (R)-roscovitine caused calcium release from the ER, (similarly to (S)-roscovitine), and from acidic organelles (Figures 2 and 3). These observations might be relevant to explain different pharmacologic actions of these drugs. (R)-Roscovitine is known to ameliorate motor defect in Nieman-Pick type C mice by reducing the hyperphosphorylation of tau.39 The Nieman-Pick type C disease is due to a lysosomal storage defect caused by a significant attenuation of NAADP-evoked Ca2+ from acidic organelles.40 On the basis of our findings, it could be speculated that the beneficial effect of (R)-roscovitine39 might be associated with an increase in calcium release from lysosomes, thus ameliorating the observed lysosomal defect. Interestingly, roscovitine enantiomers control intracellular calcium compartmentalization differentially, also under long treatment. These effects resulted from integrated signaling actions including a tight modulation of selective proteins playing a role in controlling cellular calcium balance. In fact, in vitro administration of (R)-roscovitine is associated with a reduced intracellular calcium concentration (Figure 4) paralleled by a significant increase in calcium stored in the ER (Figure 5), which is maintained by higher SERCA activity. Instead, (S)-roscovitine caused a higher accumulation of calcium in the ER without affecting intracellular calcium level, which is tightly controlled by a reduced PMCA expression (Figure 7). Both enantiomers cause a significant decrease in Rho activity and a consequent partial depolymerization of actin filaments (Figure 9), crucially important to control cellular calcium balance via IP3R and TRPC1.41 In line with that, stimulation with the IP3-generating agonist ATP elicited a significant reduced calcium release in the presence of (S)- and (R)-roscovitine, possibly via IP3R signaling (Figure 8). IP3R is tightly regulated by reversible phosphorylation42 by several kinases including PKA43 and cdks.44,45 While we can exclude the possibility that roscovitine affect PKA activity by changing cAMP level in this cell model (unpublished data), it is worth to argue that roscovitine could reduce IP3R, directly, by inhibiting cdks which are known to activate this receptor44,45 and via Rho signaling.46−48 Based on the findings obtained in this work, we propose the following working model depicted in Figure 10: both (S)- and (R)-roscovitine cause a reduction of Rho activity which might cause a tonic decrease of the IP3R functionality. This would increase calcium stored in the ER. The significantly higher activity of SERCA associated with (R)-roscovitine treatment; further increase calcium stored in the ER reducing cytosolic calcium levels. Instead, under (S)-roscovitine treatment, despite a higher level of calcium stored in the ER, the intracellular calcium concentration is maintained to a physiological range due to a downregulation of PMCA expression only observed in (S)-roscovitine treated cells. Altogether, our data provide new inputs in explaining the molecular basis of the distinct therapeutic effect of (S)- and (R)-roscovitine, which might result in the different effect on calcium handling. While roscovitine has emerged as potential drug because of its ability to induce apoptosis by inhibiting cyclin dependent kinases, roscovitine also reduces L-type calcium current by direct and selective binding to voltage channels.6,7,14,16,38 Alternatively, the possibility that the enantioselective action on intracellular calcium level might play a role in controlling the functional role of several calcium regulated proteins such as the calcium-dependent phosphatases

Figure 9. (S)- and (R)-roscovitine effect on Rho signaling. (A) MDCK cells were transiently transfected with Rho-Raichu RBD33 as described in the Experimental Section, and the experiments were executed 48 h after transfection. Cells were treated with (S)- or (R)-roscovitine overnight, and changes of nFRET ratio were calculated as described.29 Histogram compares changes of nFRET Ratio, between control (Ctrl), (S)- and (R)-roscovitine treated cells indicating that both enantiomers significantly decreased Rho activity. (B) Cells were either left untreated (Ctrl) or stimulated with (S)- or (R)-roscovitine O/N. Factin was stained with TRITC-conjugated phalloidin and visualized by confocal microscopy. (C) F-actin quantization by actin polymerization assay. Confluent cells were either left untreated (Ctrl) or stimulated with (S)- or (R)-roscovitine O/N. After staining with TRITC− phalloidin, cells were extracted with cold methanol and the fluorescence absorbance of extracts was read (540/565 nm) in a RF5301PC fluorimeter. The values obtained were compared by one-way ANOVA and Newman−Keuls multiple comparison test.

entially, at very low concentration, in acute (5 μM) and under long-term treatment (100 nM). Both enantiomers evoked a transient increase in cytosolic calcium under acute administration (Figure 1). While (S)-roscovitine elicited a major 4626

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Figure 10. Schematic model. Proposed model of the effect of roscovitine enantiomers in renal epithelial cells. See Discussion for details.

PP2A and PP2B cannot be excluded.49,50 In fact, cytosolic calcium decrease, induced by (R)-roscovitine, might downregulate the activity of calcium-regulated phosphatases altering the phosphorylation level of specific calcium transporters and channels including L-type channels, which are known to be expressed in MDCK cells.51 Interestingly, abnormal intracellular calcium balance leading to prolonged and global elevation of cytosolic calcium is often observed during inflammation, tissue fibrosis or TRPC6-induced glomerulonephritis.7,52,53 (S)- and (R)-roscovitine, by controlling distinct pathway regulating intracellular calcium compartmentalization, might be differentiated as therapeutic tool due to their differential ability to control calcium homeostasis.

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AUTHOR INFORMATION

Corresponding Author

*Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Italy, Via Amendola 165/A, 70125 Bari, Italy. Tel: +39 080 5443414. Fax:+39 080 5443388. Email: [email protected]. Author Contributions ‡

G.T. and M.R. contributed equally to this work

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was funded by grants from University of Bari, Italy (Idea Giovani 2011), and from PRIN (Research Program of National Interest) projects to G.T. (Tamma01373409Prin). We thank Prof. Roger Tsien for the D1ER probe.

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