641
The sarcoplasmic–endoplasmic reticulum Ca2C ATPase 2b regulates the Ca2C transients elicited by P2Y2 activation in PC Cl3 thyroid cells Luca Ulianich2,3, Maria Giovanna Elia1, Antonella Sonia Treglia1, Antonella Muscella1, Bruno Di Jeso1, Carlo Storelli1 and Santo Marsigliante1 1
Dipartimento di Scienze e Tecnologie Biologiche e Ambientali, Universita` degli Studi di Lecce, Ecotekne, Via Provinciale per Monteroni, 73100 Lecce, Italy
2
Istituto di Endocrinologia e Oncologia Sperimentale G Salvatore, CNR, Naples, Italy
3
Dipartimento di Biologia e Patologia Cellulare e Molecolare L Califano, Naples, Italy
(Requests for offprints should be addressed to B D Jeso; Email:
[email protected] or S Marsigliante; Email:
[email protected])
Abstract In PC Cl3 cells, a continuous, fully differentiated rat thyroid cell line, P2Y2 purinoceptor activation provoked a transient increase of [Ca2C]i, followed by a decreasing sustained phase. The a and b1 protein kinase C (PKC) inhibitor Go¨ 6976 decreased the rate of decrement to the basal [Ca2C]i level and increased the peak of Ca2C entry of the P2Y2-provoked Ca2C transients. These effects of Go¨ 6976 were not caused by an increased permeability of the plasma membrane, since the Mn2C and Ba2C uptake were not changed by Go¨ 6976. Similarly, the NaC/Ca2C exchanger was not implicated, since the rate of decrement to the basal [Ca2C]i level was equally decreased in physiological and NaC-free buffers, in
the presence of Go¨ 6976. On the contrary, the activity of the sarcoplasmic–endoplasmic reticulum Ca2C ATPase (SERCA) 2b was profoundly affected by Go¨ 6976 since the drug was able to completely inhibit the stimulation of the SERCA 2b activity elicited by P2-purinergic agonists. Finally, the PKC activator phorbol myristate acetate had effects opposite to Go¨ 6976, in that it markedly increased the rate of decrement to the basal [Ca2C]i level after P2Y2 stimulation and also increased the activity of SERCA 2b. These results suggest that SERCA 2b plays a role in regulating the sustained phase of Ca2C transients caused by P2Y2 stimulation.
Introduction
2002). The activation of this signalling cascade stimulates H2O2 production (Bjorkman & Ekholm 1992) and iodide efflux (Okajima et al. 1988), two important events in the biological function of thyroid gland, namely thyroglobulin (Tg) iodination and subsequent T3/T4 synthesis. However, we have previously shown that P2-purinergic agonists also stimulate Tg secretion, another crucial event for Tg iodination and thyroid hormone production (Di Jeso et al. 1993). Therefore, P2 purinergic effectively stimulates many important steps of thyroid hormonogenesis. It has also been shown that even an exclusive Ca2C response, elicited in thyroid cells by the Ca2C ionophore A23187, is able to induce H2O2 production and iodide efflux (Weiss et al. 1984, Bjorkman & Ekholm 1988), suggesting that the Ca2C branch of the signalling of P2-purinergic receptor agonists plays an important role in the regulation of thyroid hormonogenesis. In addition, a Ca2C response could have more general effects, ranging from proliferation to apoptosis (Berridge 2004). In all cells, a Ca2C response is ended by the contribution of several Ca2C-extruding proteins, i.e. the NaC/Ca2C exchanger and the sarcoplasmic–endoplasmic reticulum Ca2C ATPase (SERCA) and plasma-membrane Ca2CATPase (PMCA; Lytton et al. 1991). We have recently characterized the presence and regulation of the SERCA in
P2-purinergic agonists have important biological functions on several tissues, due to the wide distribution of their receptors and their ubiquitous nature, since they derive from the cytosol of damaged cells or exocytotic vesicles and/or granules contained in many types of secretory cells (Dubyak & el-Moatassim 1993). There are two families of P2-purinergic receptors: the P2X ligand-gated ionotropic channel family and the P2Y metabotropic G-protein-coupled receptor family (Vassort 2001). Most P2Y receptors are coupled to phospholipase Cb (PLCb) and their engagement by ligands causes phosphoinositide hydrolysis, raise of the [Ca2C]i and protein kinase C (PKC) activation (Vassort 2001). They mediate a vast variety of effects ranging from regulation of epithelial transport (Bucheimer & Linden 2004) to platelet aggregation (Hechler et al. 2005). The signal transmission pathway and physiological effects of P2-purinergic agonists have been studied in the thyrocyte, an epithelial cell. P2-purinergic agonists act on thyroid cells through a P2Y2 receptor, via PLC stimulation, causing PKC activation and a Ca2C response composed of Ca2C store depletion, capacitative Ca2C entry and L-type voltagedependent Ca2C channels activation (Marsigliante et al.
Journal of Endocrinology (2006) 190, 641–649
Journal of Endocrinology (2006) 190, 641–649 0022-0795/06/0190–641 q 2006 Society for Endocrinology Printed in Great Britain
DOI: 10.1677/joe.1.06455 Online version via http://www.endocrinology-journals.org
642
L ULIANICH
and others $ SERCA 2b regulation of Ca2C in thyroid cells
thyroid (Pacifico et al. 2003, Ulianich et al. 2004) and demonstrated that P2Y2 receptor stimulation activates several signal transmission pathways, mainly PKCs, extracellular signalregulated kinases (ERKs) and fos (Elia et al. 2005). In this study, our aim has been to determine if and how the Ca2C response elicited in thyroid cells by P2-purinergic agonists is regulated. We have focussed our attention on the activation of the PKC pathway by P2-purinergic agonists by studying if PKC modulates the dynamics of the P2purinergic-induced Ca2C response.
Materials and Methods Fetal bovine serum (FBS) and glutamine were from Euroclone (Paignton, Devon, UK). Fura 2-AM, thapsigargin and pluronic F-127 were from Molecular Probes (Eugene, OR, USA). Hydrocortisone, transferrin, L-glycyl-histidyllysine and somatostatin were from ICN Biomedicals (Costa Mesa, CA, USA). Coon’s modified Ham’s F12 medium, BSA, Go¨ 6976, GF109203X and other reagents were from Sigma-Aldrich Co.
Cell culture Several different batches of the PC Cl3 cells were used in the experiments and the cells were grown for approximately 40 passages. No difference was observed in the responsiveness of the cells to ATP and UTP during those passages. PC Cl3 rat thyroid cells were grown as previously reported (Pacifico et al. 2003, Ulianich et al. 2004, Elia et al. 2005). Fresh medium with 0$5% FBS was always added 24 h prior to an experiment.
Determination of [Ca2C]i The medium was aspirated and the cells were harvested with 0$05% trypsin–EDTA solution. After washing the cells three times by pelleting (100 g for 5 min), the cells (4!106) were incubated with 5 mM fura-2-AM and 0$2% pluronic F-127 for 45 min at 37 8C with continuous shaking (100 cycles/ min). Following the loading period, the cells were washed twice with a modified Krebs–Ringer buffer in which the bicarbonate was replaced by 20 mM Hepes (pH 7$4), incubated again for at least 10 min at room temperature to facilitate hydrolysis of the esterified probe and washed once again. The cells were resuspended in 2 ml of the same buffer containing 0$1% BSA and 20 ml cell suspension was added to a 2 ml fluorescence cuvette kept at 37 8C, and stirred throughout the experiment. The fluorescence intensity was measured with a JASCO FP 750 fluorimeter (Jasco Corporation, Hachioji, Tokyo, Japan). The excitation wavelengths were 340 and 380 nm and the emission was measured at 510 nm. The maximal fluorescence was determined at the end of the assay by adding 20 ml, 10% SDS and the minimal fluorescence by adding 20 ml, 0$5 M EGTA solution (pH 9$0). The cytoplasmic Ca2C concentration at time t was calculated using the software of the Journal of Endocrinology (2006) 190, 641–649
fluorimeter and assuming a Kd for the fura-2–Ca2C complex of 224 nM, according to the Grynkiewicz equation (Grynkiewicz et al. 1985) ½Ca2C; nMt Z 224ðFt K Fmin ÞFmin380 =ðFmax K Ft ÞFmax380 where F denotes the time-course of the fluorescence at 510 nm after dual excitation at 340/380 nm and F380, the fluorescence at 510 nm after excitation at 380 nm. Ba2C rendered fura-2 fluorescence spectra similar to Ca2C with a distinct increase and an accompanying decrease in fluorescence intensity at excitation wavelengths near 340 and 380 nm respectively, whereas Mn2C strongly quenched the fura-2 fluorescence at 360 nm. The results are expressed as relative fluorescence quenching with respect to the maximal quenching induced by the addition of digitonin (80 mg/ml).
Assay of Ca2C-dependent ATPase activity of SERCAs We previously developed a method to determine the SERCA activity (Pacifico et al. 2003) based on the use of the specific SERCAs-inhibitor thapsigargin (Lytton et al. 1992), which is sensitive enough to be used not only on purified microsomal fractions but also on total cellular lysates. In this study, we used total cellular lysates to analyze the regulated expression of SERCA 2b in thyroid, prepared as previously reported (Ulianich et al. 2004). ATPase activities were determined at 37 8C (Ottolenghi 1975) by measuring inorganic phosphate (Pi) production by a modification of the method of Fiske and Subbarrow (Higgins 1987). This method is based on the reaction between phosphate and molybdate to give the yellow molybdate phosphoric acid, which contains a molybdate, Mo (VI), which is then reduced to Mo (V) present in a bluecoloured heteropolyacid compound. This blue compound is directly measured by reading the absorbance at 700 nm. Typically, 100 mg total cell lysates were incubated in 25 mM KMOPS, 100 mM KCl, 5 mM MgCl 2, 4 mM ATP, 0$11 mM EGTA, 107 mM CaCl2 (pH 7$0), [Ca2C]free 5 mM, for 10 min at 37 8C. The free Ca2C concentration has been calculated using the CHELATOR program (implemented in Turbo Pascal 5$5 for IBM PC; Pacifico et al. 2003). The phosphate produced in the presence or absence of 25 nM thapsigargin was determined directly by reading the absorbance at 700 nm of a standard curve. Activity in the presence of 25 nM thapsigargin (Lytton et al. 1992) was subtracted. Negative controls for each experimental condition were set by boiling cell lysates for 15 min before starting the reactions in the presence and absence of 25 nM thapsigargin (the activity of the negative controls was always less than 5% of the activity of the samples). When the assay was performed on purified microsomal fractions (Pacifico et al. 2003), the activities in the presence of EGTA and thapsigargin were comparable and inclusion in the buffer of sodium azide or p-trifluoromethoxyphenylhydrazone, potent inhibitors of mitochondrial Ca2C uptake, did not modify the results. Reactions were linear with respect to time and protein concentrations. www.endocrinology-journals.org
SERCA 2b regulation of Ca2C in thyroid cells $
Statistical analysis Experimental points represent the meanGS.D. of three to six replicates. Statistical analysis was carried out using Student’s t-test for unpaired samples and ANOVA. When indicated, post hoc tests (Bonferroni and Dunn) were also performed. A P value of !0$05 was considered to be statistically significant.
Results Effect of PKC inhibitors on [Ca2C]i in PC Cl3 cells stimulated by UTP/ATP To assess the involvement of PKC in the modulation of the ATP/UTP-induced [Ca2C]i transients (Marsigliante et al. 2002), the calcium-dependent PKC inhibitor Go¨ 6976 and the calcium-dependent and -independent PKC inhibitor GF109203X were used (Martiny-Baron et al. 1993, Qatsha et al. 1993). In the absence of PKC inhibitors, the addition of ATP or UTP evoked an early peak rise in [Ca2C]i with maximal
L ULIANICH
and others 643
increases occurring at approximately 30 s, followed by a progressively decreasing level (over 6 min) to the initial, pre-stimulated level (Fig. 1A). Pre-incubation of cells with both Go¨ 6976 and GF109203X (10 and 1000 nM for 15 min) had no effects on the early peak rise in [Ca2C]i, but changed the rate of decrement to the basal [Ca2C]i level significantly (Fig. 1A and B). More precisely, these inhibitors increased the D[Ca2C]i after 4 min from approximately 130 to 400 nM, thus retaining the time needed to reach the pre-stimulated [Ca2C]i level. To measure Ca2C release from the intracellular stores, cells were stimulated with UTP/ATP in the absence of extracellular Ca2C, using calcium-free buffers, which were previously passed through a Chelex 100 column. Subsequently, 2 mM Ca2C was added to the medium to allow Ca2C influx to occur. As shown in Fig. 1C and D, when PC Cl3 cells were stimulated with 100 mM UTP/ATP in Ca2C-free medium, there was no significant difference in Ca2C release from the intracellular stores, but large differences occurred in Ca2C influx between cells incubated or not with PKC inhibitors. Precisely, the peak of extracellular Ca2C entry in the presence of PKC inhibitors increased from 300G20 to 500G25 nM.
Figure 1 (A) Effect of ATP/UTP on Ca2C mobilization in PC Cl3 cells in the absence (:) or presence of Go¨ 6976 10 nM (&) and 1000 nM (*). (B) [Ca2C]i values obtained from A after 30 s (early peak rise) and 4 min stimulation with ATP/UTP. The data are meansGS.D. of four different experiments run in triplicate. Values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests. a vs b P!0$001; a vs c P!0$05. (C) Extracellular Ca2C entry into PC Cl3 cells pre-incubated (10, & and 1000 nM, *) or not (:) with Go¨ 6976. In nominal Ca2C-free medium, 100 mM ATP/UTP was added followed by 2 mM CaCl2 after 260 s. (D) [Ca2C]i values obtained from C representing the mobilization of internal calcium (peak) and the extracellular Ca2C entry (CCaCl2) after stimulation with ATP/UTP in the presence or absence of Go¨ 6976. The data are meansGS.D. of four different experiments run in triplicate. Values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests. a vs b P!0$01; a vs c P!0$001. Arrow indicates time point at which ATP was added. Results shown are representative of at least four experiments. www.endocrinology-journals.org
Journal of Endocrinology (2006) 190, 641–649
644
L ULIANICH
and others $ SERCA 2b regulation of Ca2C in thyroid cells
Effect of UTP/ATP on Mn2C quenching and Ba2C uptake in PC Cl3 cells To test whether the changes of the Ca2C response caused by UTP and ATP were due to modulation of the plasma membrane permeability, we measured Mn2C and Ba2C influx after the addition of ATP and UTP. Mn2C and Ba2C are good Ca2C-entry tracers, since they are not pumped out of the cell (Hallam et al. 1988, Merritt & Hallam 1988, Suh et al. 1996). Mn2C uptake was estimated by the quenching of fura-2 fluorescence when excited at 360 nm, which is an isosbestic wavelength and is insensitive to variations in Ca2C concentration. Ba2C uptake was estimated by the increase in the fura-2 fluorescence ratio when excited at 340 and 380 nm. Figure 2A shows the fluorescence quenching by Mn2C influx when cells were stimulated with 100 mM UTP/ATP. The presence or absence of PKC inhibitors Go¨ 6976 did not change the Mn2C influx (Fig. 2C and E). This result was also supported by the Ba2C uptake. To measure Ba2C influx, cells were stimulated with UTP/ATP in the absence of external Ca2C. When the Ba2C was added to the medium, it caused an increase in the
fluorescence intensity reflecting Ba2C uptake (Fig. 2B). The influx of Ba2C elicited by ATP and UTP was similar in the presence or absence of Go¨ 6976 (Fig. 2D and F). The same results were obtained with the use of GF109203X (data not shown). These results suggest that the UTP/ATP-activated PKCs modulate the activity of one or more Ca2C transporters without interfering with the plasmalemma Ca2C permeability. To study the involvement of a NaC/Ca2C exchanger in the PKC-controlled [Ca2C]i changes, the effects of PKC inhibitors on the ATP/UTP-evoked Ca2C changes were investigated using buffers made at 0 and 140 mM NaC. The replacement of extracellular NaC with choline significantly increased the resting levels of [Ca2C]i in fura-2-loaded cells (from 69$5G5$3 to 92G7 nM, Student’s t-test, P!0$01, nZ4). The effects of ATP/UTP were significantly dependent on the NaC concentration, since in the absence of NaC, the early peak rise in D[Ca2C]i changed from 421G30 to 528G28 (ANOVA; P!0$01, nZ4), while the D[Ca2C]i (D[Ca2C]i represents the difference between the pre-stimulatory and the transient levels of [Ca2C]i) after 4 min did not change (Fig. 3A and B).
Figure 2 (A, C, E) Cells were kept in nominally Ca2C-free medium, stimulated with 100 mM UTP/ATP and cation entry was initiated by addition of MnCl2. Mn2C entry was measured by the quenching of fluorescence with an excitation wavelength of 360 nm and an emission wavelength of 510 nm. The curves represent fluorescence relative to total quenching attained by the addition of digitonin to saturate fura-2 with the indicated quenching cation. Since 2 mM Mn2C was applied to the medium (arrow), it entered cells with the same velocity, regardless of the presence of Go¨ 6976. (B, D, F) UTP/ATP-induced entry of Ba2C in PC Cl3 cells. Cells, in nominally Ca2Cfree medium, were first stimulated with 100 mM ATP/UTP in the presence or absence of Go¨ 6976 and cation entry was initiated by cumulative addition of Ba2C at the final concentrations of 2, 5 and 10 mM. Arrows indicate the time point at which cations were added. Each tracing is representative of five separate experiments. Journal of Endocrinology (2006) 190, 641–649
www.endocrinology-journals.org
SERCA 2b regulation of Ca2C in thyroid cells $
L ULIANICH
and others 645
Figure 3 (A) Effects of UTP/ATP on Ca2C influx using buffers with NaC concentrations of 0 and 140 nM in cells preincubated (C and D) or not (A and B) with 10 nM Go¨ 6976. Results shown are representative of four experiments. (B) [Ca2C]i values obtained from A after 30 s (early peak rise) and 4-min stimulation with ATP/UTP in normal and NaC-free buffers in the absence of Go¨ 6976. The data are meansGS.D. of four different experiments run in triplicate. a vs b P!0$0001; a vs c P!0$01; c vs d P!0$001. (C) Effects of UTP/ATP on Ca2C influx using buffers with NaC concentrations of 0 and 140 nM in cells preincubated with 10 nM Go¨ 6976. (D) [Ca2C]i values obtained from C after 30 s (early peak rise) and 4-min stimulation with ATP/UTP in normal and NaC-free buffers in the presence of Go¨ 6976. The data are meansGS.D. of four different experiments run in triplicate. Values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests.
Both Go¨ 6976 and GF109203X pre-incubations (10 nM for 15 min) had no effects on the early peak rise in [Ca2C]i evoked by ATP/UTP regardless of the NaC concentration, but significantly changed the D[Ca2C]i to the same rate of decrement to the basal [Ca2C]i level in both physiological and NaC-free buffers (Fig. 3A and B), suggesting that Go¨ 6976 and GF109203X have no effects on the NaC/Ca2C exchanger activity.
P2-purinergic agonists stimulate the activity of SERCA 2b via a PKC a and b1 activation As shown above, the plasma membrane channels and the NaC/Ca2C exchanger were not involved in the PKC-mediated regulation of the [Ca2C]i-sustained phase following the UTP/ATP stimulation. Therefore, we investigated if the SERCA, that actively pump Ca2C from the cytosol to the ER www.endocrinology-journals.org
lumen and, as such, should be able to modulate the Ca2C response, are actually involved in this regulation. As a first step, we studied if SERCA activity is UTP/ATP responsive. With this aim, we employed a very sensitive and reproducible method previously developed by the authors (Pacifico et al. 2003). Furthermore, since thyroid and PC Cl3 cells express almost exclusively SERCA 2b (Pacifico et al. 2003), the measured activity reflects that of this SERCA isoform. PC Cl3 cells were left untreated or exposed for the indicated times to 100 mM UTP and Ca2C-ATPase activity was measured. As shown in Fig. 4A, UTP stimulated SERCA 2b activity by about 50%. The increase was evident at 10 min (the shortest time investigated) and was sustained until 30 min (the longest time studied). As expected, very similar results were obtained with 100 mM ATP (Fig. 4B), as both are ligands of the P2Y2 receptors (Sak & Webb 2002). Journal of Endocrinology (2006) 190, 641–649
646
L ULIANICH
and others $ SERCA 2b regulation of Ca2C in thyroid cells
Effects of phorbol myristate acetate (PMA) on the [Ca2C]i response to UTP/ATP in PC Cl3 cells
Figure 4 The effects of (A) UTP and (B) ATP on the activity of SERCA 2b in PC Cl3 cells. Cells were incubated with a single dose of 100 mM UTP/ATP for 10 or 30 min and activity was assayed. The data are meansGS.D. of six different experiments run in triplicate and are presented as nmol Pi/h per mg protein. ANOVA for (A) and (B), P!0$0001. Values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests.
Therefore, SERCA 2b activity is stimulated by P2Y2 receptor occupancy, strongly suggesting its role in the PKC-mediated regulation of the [Ca2C]i-sustained phase following a P2purinergic stimulation. Thus, we tested if UTP/ATP stimulates SERCA 2b activity via PKC. PC Cl3 cells were stimulated by UTP/ATP in the absence or presence of the a and the b1 PKC inhibitor Go¨ 6976, added to cells 30 min before UTP/ATP. As shown in Fig. 5, 10 and 1000 nM Go¨ 6976 completely abolished the UTP/ATP stimulation of SERCA 2b activity, implying SERCA 2b activity as a determinant of the [Ca2C]i-plateau phase following UTP/ATP stimulation. The broader PKC inhibitor, GF109203X, gave results highly similar to Go¨ 6976 (not shown), indicating a specific role of a and b1 PKC in mediating the UTP/ATP effect on SERCA 2b.
To further examine the role of PKC in the regulation of [Ca2C]i, the effect of PMA on the ATP/UTP-induced changes in [Ca2C]i was studied. As shown in Fig. 6A and B, pre-incubation of PC Cl3 cells with 100 nM PMA for 10 min, followed by 100 mM ATP/UTP stimulation, markedly increased the rate of decrement to the basal [Ca2C]i level from 80G6 nM [Ca2C]i/min in the absence of PMA to 160G 22 nM [Ca2C]i/min in the presence of 100 nM PMA (nZ4). The Ca2C release from the intracellular stores and the extracellular Ca2C entry was assessed in PC Cl3 cells preincubated with 100 nM PMA. PC Cl3 cells were stimulated with UTP/ATP in the absence of extracellular Ca2C and 2 mM Ca2C was added to the medium subsequently. As shown in Fig. 6C and D, there was a significant decrease in extracellular Ca2C entry in PMA-treated cells from 300G6 to 210G15 nM. Since PMA has an opposite effect with respect to PKC inhibitors on the rate of decrement to the basal [Ca2C]i level following UTP/ATP stimulation, we verified if PMA, at the same concentrations used in the [Ca2C]i experiments, stimulated SERCA 2b activity. PC Cl3 cells were starved as described above and stimulated by 100 nM PMA for the indicated times. As shown in Fig. 7A, PMA caused a significant increase in the SERCA 2b activity at 10 and 30 min. Moreover, we intentionally extended the incubation time with PMA to 3 h, to try to show a biphasic effect of PMA, indicative of an early stimulation and a following downregulation of PKC activity, as reported in many systems (Cabot et al. 1989, Thompson et al. 1997, Hsu et al. 1998, Komati et al. 2004) and also in differentiated thyroid cells in continuous culture (Gupta et al. 1995). As shown in Fig. 7A, long treatments of PC Cl3 cells with 100 nM PMA reveal inhibition of SERCA 2b activity, strongly suggesting that 100 nM PMA provoked an early stimulation and a late downregulation of PKC. Furthermore, a low dose of PMA was stimulatory at all times investigated (Fig. 7B). These results further strengthened the SERCA 2b role in the [Ca2C]i-sustained phase following a P2-purinergic stimulation.
Discussion
Figure 5 The calcium-dependent PKC inhibitor Go¨ 6976 prevents UTP/ATP-induced SERCA activity but does not affect basal activity. PC Cl3 cells were treated with 10 and 1000 nM Go¨ 6976 and then with either control medium or medium containing 100 mM UTP/ATP for 10 min and activity was assayed. The data are meansG S.D. of six different experiments run in triplicate and are presented as nmol Pi/h per mg protein. ANOVA for (A) and (B), P!0$0001. Values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests. Journal of Endocrinology (2006) 190, 641–649
Thyroid cells in primary (Raspe et al. 1991a,b) and continuous culture (Okajima et al. 1989) respond to P2-purinergic agonists through P2Y receptors. In the PC Cl3 cell line, P2Y2 receptor stimulation activates PLC, which in turn, hydrolyzes phosphatidylinositol 4,5-bisphosphate to diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (InsP3). InsP3 mobilizes Ca2C from InsP3-sensitive intracellular stores, which is followed by a Ca2C capacitative influx, while DAG activates PKCs (Marsigliante et al. 2002, Elia et al. 2005). Data showed here suggest that this signal transmission pathway has an internal regulatory loop that, through PKC and SERCA 2b, regulates the rate of decrement to the basal [Ca2C]i level. www.endocrinology-journals.org
SERCA 2b regulation of Ca2C in thyroid cells $
L ULIANICH
and others 647
Figure 6 (A) Effect of ATP/UTP on Ca2C mobilization in PC Cl3 cells pre-incubated (:) or not (&) with 100 nM PMA. (B) [Ca2C]i values obtained from A after 30 s (early peak rise) and 4-min stimulation with ATP/UTP. The data are meansGS.D. of four different experiments run in triplicate. Values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests. (C) Extracellular Ca2C entry into PC Cl3 cells pre-incubated (:) or not (&) with 100 mM PMA. In nominal Ca2C-free medium, 100 mM UTP/ATP was added followed by 2 mM CaCl2 after 260 s. (D) [Ca2C]i values obtained from C representing the mobilization of internal calcium (peak) and the extracellular Ca2C entry (CCaCl2) after stimulation with ATP/UTP in the presence or absence of PMA. The data are meansGS.D. of four different experiments run in triplicate. Values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests. Arrow indicates the time point at which ATP was added. Results shown are representative of at least four experiments.
Precisely, the present results suggest a role for conventional PKCs in thyroid cells, i.e. the control of intracellular Ca2C homeostasis after purinergic stimulation, based, at least in part, upon the increase in SERCA 2b activity, which may be of physiological importance. We base our conclusion on the following observations obtained after conventional PKC inhibition: first, the P2Y2-evoked plateau level in [Ca2C]i increased; second, no effects on the P2Y2-evoked entry of both manganese and barium in the cells was noticed; and third, the P2Y2-stimulated SERCA 2b activity decreased to the basal, unstimulated, level. We show that an inhibition of PKCs decreased the rate of recovery to basal [Ca2C]i level following a Ca2C peak evoked by P2Y2 receptor activation. Hence, PKCs promote the recovery phase of the Ca2C transient (Fig. 1). As shown previously, P2Y2 activation provoked in PC Cl3 cells a cytosol-to-membrane translocation of PKC-a, -bI and -3; of these, the novel PKC-3 does not seem to be involved in the modulation of Ca2C transient shown here, since inhibition of www.endocrinology-journals.org
Ca2C-dependent or both Ca2C-dependent and -independent PKCs gave similar results. Altogether, PKC-3 was shown to be implicated in the P2Y2 activation of the mitogen-activated protein kinase (MAPK)/ERK pathway (Elia et al. 2005). In theory, the modulation of the sustained phase of the Ca2C transient could be achieved by several mechanisms, such as changes in plasma membrane permeability to Ca2C, activity of SERCA or plasma membrane Ca2C ATPase (PMCA), or of the NaC/Ca2C exchanger. We have excluded the possibility that PKC regulates plasma membrane Ca2C permeability since Mn2C and Ba2C cell entry (both entering the cytosol from the extracellular space through the same pathways as Ca2C) were unaffected by Go¨ 6976 (Fig. 2). As far as the NaC/Ca2C exchange is concerned, it is evident that the Ca2C response driven by UTP/ATP stimulation also resulted in the involvement of this antiporter, which appeared to be operative in control conditions (unstimulated cells). In fact, the replacement of extracellular NaC with choline significantly increased resting levels of Journal of Endocrinology (2006) 190, 641–649
648
L ULIANICH
and others $ SERCA 2b regulation of Ca2C in thyroid cells
Figure 7 The stimulation of an cells with the PKC activator phorbol 12-myristate acetate (100 and 10 nM) induced an increase in SERCA 2b activity in PC Cl3 cells. Cells were incubated with a single dose of UTP/ATP (A) 100 nM or (B) 10 nM for the times indicated and activity was assayed. Results are meansGS.D. of three different experiments run in triplicate and are presented as nmol Pi/h per mg protein. Values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests. P!0$0001.
[Ca2C]i, and the ATP-induced transient increase in [Ca2C]i also increased (Fig. 3). PKC has no effect on NaC/Ca2C exchanger activity since Go¨ 6976 has the same effect on the sustained phase of the Ca2C-transient elicited by ATP/UTP in both physiological and NaC-free buffers (Fig. 3). On the other hand, we have shown that P2Y2 agonists stimulate SERCAs activity and that Go¨ 6976 was able to completely prevent such activation (Figs 4 and 5). Therefore, the same PKC isoforms, a and b1, were involved in the regulation of the plateau phase of the P2Y2-evoked Ca2C transient and in the purinergic-stimulated SERCA activity. This strongly suggests that, indeed, SERCAs are involved, at least in part, in the regulation of the Ca2C transient in thyroid cells. We have also shown that PMA increases the rate of recovery to basal [Ca2C]i level following a Ca2C peak evoked by P2Y2 receptor activation (Fig. 6). At the same time, PMA stimulates SERCA 2b activity (Fig. 7A). Therefore, PMA acts in an exactly opposite way with respect to Go¨ 6976. A biphasic effect of a high dose of PMA and an exclusive stimulatory effect of a low dose of Journal of Endocrinology (2006) 190, 641–649
PMA on SERCA activity was evident (Fig. 7A and B). This suggests that the predominant effect of PMA, under our experimental conditions, is the stimulation of PKC, since it is well documented that low PMA doses correlate with PKC membrane translocation, while high doses correlate with PKC downregulation in a variety of systems (Cabot et al. 1989, Thompson et al. 1997, Hsu et al. 1998, Komati et al. 2004) and also in thyroid cells in culture (Gupta et al. 1995). Obviously, Ca2C is actively extruded out of the cell through PMCA, the involvement of which, in these phenomena, was not investigated at this stage. These results also extend our previous findings on expression and regulation of SERCAs in thyroid cells. We have shown that thyroid expresses the 2b form of SERCAs exclusively and that, in thyroid, its expression and activity is regulated by neoplastic transformation (Pacifico et al. 2003) and thyroid-stimulating hormone through the cAMP–PKA pathway (Ulianich et al. 2004). Here, it is shown that SERCA 2b activity is stimulated by P2-purinergic agonists through the PLC–PKC pathway. These results strengthen the concept that SERCA 2b, in highly differentiated cells, such as thyroid (Pacifico et al. 2003) or enamel cells (Franklin et al. 2001), is regulated by various signal transduction pathways. This new standpoint extends our understanding of SERCA 2b function, considered until recently housekeeping at variance with that of SERCA 3 that varies with differentiation in several systems (Papp et al. 1993, Gelebart et al. 2002). In the thyroid, the physiological effects of P2-purinergic receptors stimulation are remarkable. They stimulate H2O2 production (Bjorkman & Ekholm 1992) and iodide efflux (Okajima et al. 1988), two necessary events for Tg iodination. In addition, we have previously shown that P2-purinergic agonists also stimulate the secretion of Tg, which constitutes the molecular site of synthesis of thyroid hormones (Di Jeso et al. 1993). Since Tg must be secreted in order to be iodinated, its secretion constitutes another crucial event for thyroid hormone production. Therefore, P2-purinergic stimulation of thyroid cells could be very important for hormonogenesis. Of the two branches of the signal transduction pathway elicited by P2-purinergic agonists, the PKC branch and the Ca2Cresponse branch, the latter seems to be more important than the former for the stimulation of H2O2 production and iodide efflux (Weiss et al. 1984, Bjorkman & Ekholm 1988). It is conceivable that the internal loop of the P2-purinergicsignalling cascade described here could have some effect in the regulation of these physiological responses. In fact, it is well known that the spatiotemporal aspects of Ca2C signalling are as important as its amplitude (Berridge 1993). In addition, a Ca2C response could have general effects on cells, such as regulation of proliferation and apoptosis (Berridge 2004). Studies are in progress in our laboratories to understand if the Ca2C response elicited by P2Y2 receptorsignalling cascade and its regulation shown here, plays a role in crucial cellular responses, such as proliferation and apoptosis, particularly in PC Cl3 cells transformed by several oncogenes that are available in our laboratories. www.endocrinology-journals.org
SERCA 2b regulation of Ca2C in thyroid cells $
Acknowledgements The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
References Berridge MJ 1993 Cell signalling. A tale of two messengers. Nature 365 388–389. Berridge MJ 2004 Calcium signal transduction and cellular control mechanisms. Biochimica et Biophysica Acta 1742 3–7. Bjorkman U & Ekholm R 1988 Accelerated exocytosis and H2O2 generation in isolated thyroid follicles enhance protein iodination. Endocrinology 122 488–494. Bjorkman U & Ekholm R 1992 Hydrogen peroxide generation and its regulation in FRTL-5 and porcine thyroid. Endocrinology 130 393–399. Bucheimer RE & Linden J 2004 Purinergic regulation of epithelial transport. Journal of Physiology 555 311–321. Cabot MC, Welsh CJ, Zhang ZC & Cao HT 1989 Evidence for a protein kinase C-directed mechanism in the phorbol diester-induced phospholipase D pathway of diacylglycerol generation from phosphatidylcholine. FEBS Letters 245 85–90. Di Jeso B, Laviola L, Liguoro D, Formisano S & Consiglio E 1993 P2 purinergic agonists and 12-O-tetradecanoylphorbol-13-acetate, as well as protein kinase A activators, stimulate thyroglobulin secretion in FRTL-5 cells. Biochemical and Biophysical Research Communications 191 385–391. Dubyak GR & el-Moatassim C 1993 Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides. American Journal of Physiology 265 C577–C606. Elia MG, Muscella A, Romano S, Greco S, Di Jeso B, Verri T, Storelli C & Marsigliante S 2005 Effects of extracellular nucleotides in the thyroid: P2Y2 receptor-mediated ERK1/2 activation and c-Fos induction in PC Cl3 cells. Cellular Signalling 17 739–749. Franklin IK, Winz RA & Hubbard MJ 2001 Endoplasmic reticulum Ca2CATPase pump is up regulated in calcium-transporting dental enamel cells: a non-housekeeping role for SERCA 2b. Biochemical Journal 358 217–224. Gelebart P, Kovacs T, Brouland JP, Van Gorp R, Grossmann J, Rivard N, Panis Y, Martin V, Bredoux R, Enouf J et al. 2002 Expression of endomembrane calcium pumps in colon and gastric cancer cells. Induction of SERCA 3 expression during differentiation. Journal of Biological Chemistry 277 26310–26320. Grynkiewicz G, Poenie M & Tsien RY 1985 A new generation of Ca2C indicators with greatly improved fluorescence properties. Journal of Biological Chemistry 260 3440–3450. Gupta S, Gomez-Munoz A, Matowe WC, Brindley DN & Ginsberg J 1995 Thyroid-stimulating hormone activates phospholipase D in FRTL-5 thyroid cells via stimulation of protein kinase C. Endocrinology 136 3794–3799. Hallam TJ, Jacob R & Merritt JE 1988 Evidence that agonists stimulate bivalent-cation influx into human endothelial cells. Biochemical Journal 255 179–184. Hechler B, Cattaneo M & Gachet C 2005 The P2 receptors in platelet function. Seminars in Thrombosis Hemostasis 31 150–161. Higgins A 1987 Separation and analysis of membrane lipid components. In Biological Membranes, A Practical Approach, pp 103–137. Eds JBC Findlay & WH Evans. Oxford, Washington, DC: IRL Press. Hsu SL, Chou YH, Yin SC & Liu JY 1998 Differential effects of phorbol ester on growth and protein kinase C isoenzyme regulation in human hepatoma Hep3B cells. Biochemical Journal 333 57–64. Komati H, Minasi A, Naro F, Lagarde M, Prigent AF, Adamo S & Nemoz G 2004 Phorbol ester-induced differentiation of L6 myogenic cells involves phospholipase D activation. FEBS Letters 577 409–414. Lytton J, Westlin M & Hanley MR 1991 Thapsigargin inhibits the sarcoplasmic and endoplasmic reticulum Ca-ATPase family of calcium pumps. Journal of Biological Chemistry 266 17067–17071. www.endocrinology-journals.org
L ULIANICH
and others 649
Lytton J, Westlin M, Burk SE, Shull GE & MacLennan DH 1992 Functional comparisons between isoforms of the sarcoplasmic or endoplasmic reticulum family of calcium pumps. Journal of Biological Chemistry 267 14483–14489. Marsigliante S, Elia MG, Di Jeso B, Greco S, Muscella A & Storelli C 2002 Increase of [Ca2C]i via activation of ATP receptors in PC-Cl3 rat thyroid cell line. Cellular Signalling 14 61–67. Martiny-Baron G, Kazanietz MG, Mischak H, Blumbergs PM, Kochs G, Hug H, Marme` D & Schachtele C 1993 Selective inhibition of protein kinase C isozymes by the indolocarbazole GO 6976. Journal of Biological Chemistry 268 9194–9197. Merritt JE & Hallam TJ 1988 Platelets and parotid acinar cells have different mechanisms for agonist-stimulated divalent cation entry. Journal of Biological Chemistry 263 6161–6164. Okajima F, Sho K & Kondo Y 1988 Inhibition by islet-activating protein, pertussis toxin, of P2-purinergic receptor-mediated iodide efflux and phosphoinositide turnover in FRTL-5 cells. Endocrinology 123 1035–1043. Okajima F, Sato K & Kondo Y 1989 P2-purinergic agonists activate phospholipase C in a guanine nucleotide- and Ca2C-dependent manner in FRTL-5 thyroid cell membranes. FEBS Letters 253 132–136. Ottolenghi P 1975 The reversible delipidation of a solubilized sodium-pluspotassium-ion-dependent adenosine triphosphatase from the salt gland of the spiny dogfish. Biochemical Journal 151 61–66. Pacifico F, Ulianich L, De Micheli S, Treglia S, Leonardi A, Vito P, Formisano S, Consiglio E & Di Jeso B 2003 The expression of the sarco/endoplasmic reticulum Ca2C-ATPases in thyroid and its down-regulation following neoplastic transformation. Journal of Molecular Endocrinology 30 399–409. Papp B, Corvazier E, Magnier C, Kovacs T, Bourdeau N, Levy-Toledano S, Bredoux R, Levy B, Poitevin P & Lompre AM 1993 Spontaneously hypertensive rats and platelet Ca2C-ATPases: specific up-regulation of the 97 kDa isoform. Biochemical Journal 295 685–690. Qatsha KA, Rudolph C, Marme D, Schachtele C & May WS 1993 Go 6976, a selective inhibitor of protein kinase C, is a potent antagonist of human immunodeficiency virus 1 induction from latent/low-level-producing reservoir cells in vitro. PNAS 15 4674–4678. Raspe E, Laurent E, Andry G & Dumont JE 1991a ATP, bradykinin, TRH and TSH activate the Ca2C-phosphatidylinositol cascade of human thyrocytes in primary culture. Molecular and Cellular Endocrinology 81 175–183. Raspe E, Laurent E, Corvilain B, Verjans B, Erneux C & Dumont JE 1991b Control of the intracellular Ca2C-concentration and the inositol phosphate accumulation in dog thyrocyte primary culture: evidence for different kinetics of Ca2C-phosphatidylinositol cascade activation and for involvement in the regulation of H2O2 production. Journal of Cellular Physiology 146 242–250. Sak K & Webb TE 2002 Retrospective of recombinant P2Y receptor subtypes and their pharmacology. Archives of Biochemistry and Biophysics 397 131–136. Suh BC, Song SK, Kim YK & Kim KT 1996 Induction of cytosolic Ca2C elevation mediated by Mas-7 occurs through membrane pore formation. Journal of Biological Chemistry 271 32753–32759. Thompson MG, Mackie SC, Thom A & Palmer RM 1997 Regulation of phospholipase D in L6 skeletal muscle myoblasts. Role of protein kinase c and relationship to protein synthesis. Journal of Biological Chemistry 272 10910–10916. Ulianich L, Secondo A, De Micheli S, Treglia S, Pacifico F, Liguoro D, Moscato F, Marsigliante S, Annunziato L, Formisano S et al. 2004 TSH/cAMP up-regulate sarco/endoplasmic reticulum Ca2C-ATPases expression and activity in PC Cl3 thyroid. European Journal of Endocrinology 150 851–861. Vassort G 2001 Adenosine 5 0 -triphosphate: a P2-purinergic agonist in the myocardium. Physiological Review 81 767–806. Weiss SJ, Philp NJ & Grollman EF 1984 Effect of thyrotropin on iodide efflux in FRTL-5 cells mediated by Ca2C. Endocrinology 114 1108–1113.
Received in final form 11 May 2006 Accepted 26 May 2006 Journal of Endocrinology (2006) 190, 641–649
