Spontaneous Calcium Oscillations Control c-fosTranscription via the Serum Response Element in Neuroendocrine Cells
2002; Elsevier BV; Volume: 277; Issue: 42 Linguagem: Inglês
10.1074/jbc.m200464200
ISSN1083-351X
AutoresAndrés D. Maturana, Goedele van Haasteren, Isabelle Piuz, Cyril Castelbou, Nicolas Demaurex, Werner Schlegel,
Tópico(s)interferon and immune responses
ResumoIn excitable cells the localization of Ca2+ signals plays a central role in the cellular response, especially in the control of gene transcription. To study the effect of localized Ca2+signals on the transcriptional activation of the c-fosoncogene, we stably expressed various c-fos β-lactamase reporter constructs in pituitary AtT20 cells. A significant, but heterogenous expression of c-fos β-lactamase was observed in unstimulated cells, and a further increase was observed using KCl depolarization, epidermal growth factor (EGF), pituitary adenylate cyclase-activating polypeptide (PACAP), and serum. The KCl response was almost abolished by a nuclear Ca2+ clamp, indicating that a rise in nuclear Ca2+ is required. In contrast, the basal expression was not affected by the nuclear Ca2+ clamp, but it was strongly reduced by nifedipine, a specific antagonist of l-type Ca2+ channels. Spontaneous Ca2+ oscillations, blocked by nifedipine, were observed in the cytosol but did not propagate to the nucleus, suggesting that a rise in cytosolic Ca2+ is sufficient for basal c-fos expression. Inactivation of the c-fos promoter cAMP/Ca2+ response element (CRE) had no effect on basal or stimulated expression, whereas inactivation of the serum response element (SRE) had the same marked inhibitory effect as nifedipine. These experiments suggest that in AtT20 cells spontaneous Ca2+ oscillations maintain a basal c-fos transcription through the serum response element. Further induction of c-fos expression by depolarization requires a nuclear Ca2+ increase. In excitable cells the localization of Ca2+ signals plays a central role in the cellular response, especially in the control of gene transcription. To study the effect of localized Ca2+signals on the transcriptional activation of the c-fosoncogene, we stably expressed various c-fos β-lactamase reporter constructs in pituitary AtT20 cells. A significant, but heterogenous expression of c-fos β-lactamase was observed in unstimulated cells, and a further increase was observed using KCl depolarization, epidermal growth factor (EGF), pituitary adenylate cyclase-activating polypeptide (PACAP), and serum. The KCl response was almost abolished by a nuclear Ca2+ clamp, indicating that a rise in nuclear Ca2+ is required. In contrast, the basal expression was not affected by the nuclear Ca2+ clamp, but it was strongly reduced by nifedipine, a specific antagonist of l-type Ca2+ channels. Spontaneous Ca2+ oscillations, blocked by nifedipine, were observed in the cytosol but did not propagate to the nucleus, suggesting that a rise in cytosolic Ca2+ is sufficient for basal c-fos expression. Inactivation of the c-fos promoter cAMP/Ca2+ response element (CRE) had no effect on basal or stimulated expression, whereas inactivation of the serum response element (SRE) had the same marked inhibitory effect as nifedipine. These experiments suggest that in AtT20 cells spontaneous Ca2+ oscillations maintain a basal c-fos transcription through the serum response element. Further induction of c-fos expression by depolarization requires a nuclear Ca2+ increase. immediate early gene cAMP/Ca2+ response element serum response element pituitary adenylate cyclase-activating polypeptide CRE binding protein mitogen-activated protein serum response factor ternary complex factor fluorescence resonance energy transfer acetoxymethyl esters bis-(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester extracellular signal-regulated kinase How short-lived intracellular signals cause marked alterations in gene expression leading to long-term cellular responses is still a puzzling aspect of cell biology. Immediate early genes (IEGs),1 such as c-fos, function as a relay in this process; the transcription and protein levels of IEGs can be changed rapidly as a direct function of cellular signals. IEGs, which are mostly transcription factors, in turn control the expression of "late" responsive genes, which then modify the cellular functions (1Herschman H.R. Annu. Rev. Biochem. 1991; 60: 281-319Crossref PubMed Scopus (946) Google Scholar). The control by intracellular signals of the transcriptional activation of the c-fos gene has been widely studied (2Sheng M. Greenberg M.E. Neuron. 1990; 4: 477-485Abstract Full Text PDF PubMed Scopus (1984) Google Scholar, 3Gallin W.J. Greenberg M.E. Curr. Opin. Neurobiol. 1995; 5: 367-374Crossref PubMed Scopus (104) Google Scholar, 4Ginty D.D. Neuron. 1997; 18: 183-186Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). The c-fos gene includes in its promoter two major response elements that are targets of phosphorylation cascades, the cAMP/Ca2+response element (CRE) and the serum response element (SRE) (2Sheng M. Greenberg M.E. Neuron. 1990; 4: 477-485Abstract Full Text PDF PubMed Scopus (1984) Google Scholar). These two consensus sequences bind different transcription factors, the activities of which are modulated by Ca2+-dependent (11Ahn S. Riccio A. Ginty D.D. Annu. Rev. Physiol. 2000; 62: 803-823Crossref PubMed Scopus (28) Google Scholar, 12Mellstrom B. Naranjo J.R. Curr. Opin. Neurobiol. 2001; 11: 312-319Crossref PubMed Scopus (114) Google Scholar) as well as Ca2+-independent phosphorylation (13Karin M. Hunter T. Curr. Biol. 1995; 5: 747-757Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar). The CRE, which is located ∼60 nucleotides upstream of the transcription initiation site of the c-fos gene, is bound by a leucine zipper transcription factor, CREB (CRE binding protein) (14Shaywitz A.J. Greenberg M.E. Annu. Rev. Biochem. 1999; 68: 821-861Crossref PubMed Scopus (1797) Google Scholar). Following stimulation, CREB is phosphorylated on a critical residue, the serine 133. This phosphorylation allows CREB to recruit the transcriptional adapter CBP (CREB binding protein) and activates transcription of c-fos. Activation of various signaling pathways results in CREB phosphorylation, most notably the cAMP/PKA cascade, Ca2+ signaling acting via Ca2+-calmodulin kinases (CaMkinases), as well as the mitogen-activated protein (MAP) kinases. A second Ca2+ responsive element in the c-fospromoter is the serum response element, SRE, which is located ∼310 nucleotides upstream of the transcriptional initiation site of the c-fos gene (15Treisman R. EMBO J. 1995; 14: 4905-4913Crossref PubMed Scopus (347) Google Scholar). SRE binds the serum response factor (SRF) and its accessory factor TCF, the ternary complex factor. TCFs are encoded by a family of Ets proteins that includes Elk-1, SAP-1a, and SAP-2. Phosphorylation of SRF on serine residue 103 is crucial for it's transcriptional activity. This residue can be phosphorylatedin vitro by MAP kinases and CaMkinases. Therefore, as for CREB, the phosphorylation of SRF may implicate various signaling pathways including Ca2+ signaling cascades mediated by CaMkinase (16Misra R.P. Bonni A. Miranti C.K. Rivera V.M. Sheng M. Greenberg M.E. J. Biol. Chem. 1994; 269: 25483-25493Abstract Full Text PDF PubMed Google Scholar, 17Johnson C.M. Hill C.S. Chawla S. Treisman R. Bading H. J. Neurosci. 1997; 17: 6189-6202Crossref PubMed Google Scholar). Physiological activation of transcription via the SRE is thought to occur predominantly following growth factor stimulation of MAP kinase cascades. Control of gene expression by changes in intracellular calcium (Ca2+) concentration nearly always involves changes in protein phosphorylation. Ca2+ signals can cause such changes directly via Ca2+-calmodulin-activated protein kinases, CaMkinases, or calcineurin, a Ca2+-dependent protein phosphatase (6Finkbeiner S. Greenberg M.E. J. Neurobiol. 1998; 37: 171-189Crossref PubMed Scopus (181) Google Scholar, 7Crabtree G.R. J. Biol. Chem. 2001; 276: 2313-2316Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). A study suggests that finely tuned Ca2+ signals triggered in restricted cellular domains may be able to activate processes that are selectively affecting c-fos gene transcription (5Hardingham G.E. Chawla S. Johnson C.M. Bading H. Nature. 1997; 385: 260-265Crossref PubMed Scopus (643) Google Scholar). An increase of intracellular Ca2+ concentration can occur inside or outside of the nucleus. A cytosolic Ca2+ increase can activate CaMkinase II and/or calcineurin, which then translocate from their site of activation to the nucleus (8Heist E.K. Srinivasan M. Schulman H. J. Biol. Chem. 1998; 273: 19763-19771Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 7Crabtree G.R. J. Biol. Chem. 2001; 276: 2313-2316Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). Transcriptional activation by nuclear Ca2+ can occur by a direct binding on DREAM (downstream regulatory element antagonist modulator), a Ca2+ binding transcriptional repressor (9Carrion A.M. Link W.A. Ledo F. Mellstrom B. Naranjo J.R. Nature. 1999; 398: 80-84Crossref PubMed Scopus (492) Google Scholar), or by the translocation of the Ca2+/calmodulin complex from the cytosol to the nucleus. This translocated complex will then activate a nuclear CaMkinase, such as CaMkinase IV (10Deisseroth K. Heist E.K. Tsien R.W. Nature. 1998; 392: 198-202Crossref PubMed Scopus (546) Google Scholar). Based on the stimulus-secretion coupling concept proposed by W. W. Douglas, pioneering studies in the 1960s demonstrated the electrical excitability of endocrine cells of the pituitary, the pancreas, and the adrenal medulla, now termed neuroendocrine cells (18Douglas W.W. Rubin R.P. J. Physiol. 1961; 159: 40-57Crossref PubMed Scopus (525) Google Scholar, 19Douglas W.W. Nature. 1963; 197: 81-82Crossref PubMed Scopus (45) Google Scholar, 20Douglas W.W. Br. J. Pharmacol. 1968; 34: 453-474Crossref Scopus (796) Google Scholar). These early studies showed that voltage-gated Ca2+ channels were instrumental in the generation of action potentials in a neuroendocrine cell; depolarizing currents were indeed sodium (Na+) and Ca2+ influxes, whereas repolarization was achieved by potassium (K+). Later we demonstrated in neuroendocrine cells that a single action potential may cause a well defined intracellular Ca2+ signal, that those action potentials occur spontaneously and that they are controlled by somatostatin (21Schlegel W. Winiger B.P. Mollard P. Vacher P. Wuarin F. Zahnd G.R. Wollheim C.B. Dufy B. Nature. 1987; 329: 719-721Crossref PubMed Scopus (252) Google Scholar). A wealth of subsequent literature illustrates that the modulation of Ca2+ action potentials in neuroendocrine cells is a versatile signaling option utilized by most releasing factors and releasing modulators, but also by growth factors and other extracellular signals (34Mollard P., and Schlegel W. TEM 7, 361–365.Google Scholar). Activation or inhibition of neuroendocrine cell activity nearly always involves such modulation, which results in alterations of action potential frequency and/or rhythm. In addition, amplitude, duration, and intracellular propagation of action potential-linked Ca2+ signals are modulated as well. In summary, Ca2+ action potentials and the resulting intracellular Ca2+ transients are fundamental signaling units in neuroendocrine cells. Transcriptional control by Ca2+ has been investigated within the context of Ca2+ oscillations generated by intracellular mechanisms originally described in hepatocytes (22Woods N.M. Cuthbertson K.S. Cobbold P.H. Nature. 1986; 319: 600-602Crossref PubMed Scopus (559) Google Scholar). Such Ca2+ oscillations are produced following receptor-mediated phospholipase C (PLC) activation and the generation of the second messenger inositol 1,4,5-trisphosphate (IP3). IP3 binds to the IP3 receptor at the endoplasmic reticulum (ER) and induces the release of Ca2+from the ER. This Ca2+ release from intracellular stores occur in many cell types including pituitary cells (23Stojilkovic S.S. Kukuljan M. Tomic M. Rojas E. Catt K.J. J. Biol. Chem. 1993; 268: 7713-7720Abstract Full Text PDF PubMed Google Scholar). Ca2+ oscillations may activate gene transcription in a manner dependent on their pattern, as demonstrated for the transcription factor nuclear factor AT (NF-AT), which is activated by a mechanism involving the Ca2+-dependent phosphatase, calcineurin (24Dolmetsch R.E., Xu, K. Lewis R.S. Nature. 1998; 392: 933-936Crossref PubMed Scopus (1683) Google Scholar,25Li W. Llopis J. Whitney M. Zlokarnik G. Tsien R.Y. Nature. 1998; 392: 936-941Crossref PubMed Scopus (777) Google Scholar). Action potential-linked Ca2+ transients most likely function also for the activation of gene transcription by mechanisms sensitive to frequency and rhythm of oscillatory Ca2+changes. To test this hypothesis, we used a single cell reporter gene approach (26Zlokarnik G. Negulescu P.A. Knapp T.E. Mere L. Burres N. Feng L. Whitney M. Roemer K. Tsien R.Y. Science. 1998; 279: 84-88Crossref PubMed Scopus (596) Google Scholar) in order to link the observations on Ca2+transients in individual cells to gene transcription. Furthermore, at the single cell level Ca2+ signals can be selectively manipulated by microinjection of high molecular weight Ca2+chelators into specific compartments such as the nucleus. Here we show that Ca2+ transients driven by spontaneous action potentials in the pituitary cell line AtT20 sustain a basal transcriptional activity of the IEG c-fos through a mechanism that involves the SRE and does not require changes in nuclear Ca2+ concentration. The plasmid pBlack-b and the fluorescent substrate CCF-2 AM (CCF-2 acetoxymethyl esters) were purchased from Aurora Biosciences Corp. (San Diego, United States). Fluorescent calcium probes, Fura-2 AM (Fura-2 acetoxymethyl esters) and Fura-2 D70 (Fura-2 Dextran 70 kDa); calcium chelators, Bapta AM (Bapta acetoxymethyl esters), Bapta-D70 (Bapta Dextran 70 kDa), and Texas-Red D70 (TR-D70) were supplied by Molecular Probes (Lucerne, Switzerland). Nifedipine was purchased from Sigma (Buchs, Switzerland). We used the bacterial enzyme β-lactamase as a reporter gene. We already dispose of luciferase reporter constructs containing the c-fos promoter (27Susini S. Van Haasteren G., Li, S. Prentki M. Schlegel W. FASEB J. 2000; 14: 128-136Crossref PubMed Scopus (29) Google Scholar, 28van Haasteren G., Li, S. Ryser S. Schlegel W. Neuroendocrinology. 2000; 72: 368-378Crossref PubMed Scopus (19) Google Scholar). The luciferase coding sequence from our reporter constructs was then excised by NocI and XbaI digestion and replaced by the β-lactamase coding sequence. For our experiments four different constructs were used. The first contains c-fos proto-oncogene for the region of the mouse, extending from −379 to +1073. This construct is called c-fos-βL and corresponds to the wild type c-fospromoter plus part of the gene (first exon and first intron). The second and third constructions contained one each a mutation inactivating the CRE (c-fos-ΔCRE-βL) or the SRE (c-fos-ΔSRE-βL) response elements. The last construction contained two mutations inactivating both the SRE and CRE response elements (c-fos-ΔSRE/CRE-βL). All these constructions are schematically presented in Fig. 5. Mutations were performed by site-directed mutagenesis using the Quick-Change mutagenesis kit from Stratagene (Basel, Switzerland). The strategy of mutagenesis has already been described (5Hardingham G.E. Chawla S. Johnson C.M. Bading H. Nature. 1997; 385: 260-265Crossref PubMed Scopus (643) Google Scholar, 27Susini S. Van Haasteren G., Li, S. Prentki M. Schlegel W. FASEB J. 2000; 14: 128-136Crossref PubMed Scopus (29) Google Scholar, 28van Haasteren G., Li, S. Ryser S. Schlegel W. Neuroendocrinology. 2000; 72: 368-378Crossref PubMed Scopus (19) Google Scholar). Rat corticotrope AtT20 cells were cultured in a Dulbecco's modified Eagle's medium-F12 supplemented with 10% fetal calf serum (FCS) at 37 °C in a humidified atmosphere of 95% air and 5% CO2. Stably transfected AtT20 clones (see below) were selectively maintained with 100 μg/ml G418 (an antibiotic analog to neomycin) added to the culture medium. For all measurements cells were plated on 25 mm coverslips, and serum was removed from the culture medium 24 h before experiments. Five stable AtT20 cell lines were established, four cell lines containing a reporter construct plasmid (c-fos-βL, c-fos-ΔCRE-βL, c-fos-ΔSRE-βL, c-fos-ΔSRE/CRE-βL) and one cell line with viral promoter SV40 (SV40-βL). A pcDNA3 vector containing the neomycin resistance gene co-transfected in a mass ratio of 1:10 was used for selection. The DOSPER liposomal transfection reagent (Roche) was used following the manufacturer's recommendations. The cotransfection was performed as follows. AtT20 cells were grown to ∼60% confluence in 35 mm diameter Petri dishes. After removal of the culture medium and one wash with HBS (Hepes buffer saline, pH 7.4), cells were incubated with 2 ml of HBS containing the plasmids (0.5 μg of pcDNA3 and 5 μg of reporter plasmid) dissolved previously in 40 μl of DOSPER. After 6 h, cells were washed with HBS, and culture was continued in Dulbecco's modified Eagle's medium-F12 (10% fetal calf serum) containing 400 μg/ml G418. After 3 weeks of culture in G418 medium, several neomycin-resistant clones were selected and tested for their ability to induce fluorescence changes in CCF-2 in response to stimulation by 20 mm KCl and 3 μm cpt-cAMP (chlorophenylthio-cyclic AMP). AtT20 cells were injected using an Eppendorf transjector 5246 mounted on a Zeiss Axiovert S100TV microscope. For the measurements of nuclear Ca2+, the injection solution was 50 μm Fura 2-D70, half-strength phosphate-buffered saline, 1 mm MgCl2, pH 7.2. For the nuclear-Ca2+ clamp, 2.5 mm Bapta-D70 and 1 mm Ca2+ were added (free Ca2+concentration was calculated to be 83 nm). To measure the β-lactamase activity, Fura 2-D70 was replaced by 5% TR-D70 as injection marker. After injection, cells were kept in the incubator at 37 °C for 4 to 5 h before experiments. Cytosolic calcium concentration variations were measured using the calcium probe Fura-2. Cells were loaded for 30 min at room temperature with the membrane permeant Fura-2 AM in a medium containing NaCl 140 mm, KCl 5 mm, Ca2+ 1.2 mm, MgCl21 mm, glucose 10 mm, Hepes 20 mm,pH 7.4. Changes of nuclear calcium concentration were measured by nuclear injection of the 70-kDa Dextran conjugate Fura-2 (Fura-2-D70; see "Microinjection" under "Experimental Procedures"). Fura-2 fluorescence (excitation 340/380 nm, emission 510 nm) was monitored with an imaging system. Loaded cells plated on coverslips were mounted on an inverted Zeiss Axiovert S100TV microscope coupled to a Princeton Instruments cooled back-illuminated frame-transfer charge-coupled device camera. The light source for illumination came from a Xenon XBO 75-W lamp. Excitation wavelengths were selected with a PTI (Photon Technology International) monochromator. Images were acquired with the Metamorph 4.1 software (Universal Imaging Corp.). Measurements were performed at 37 °C in an open perfusion micro-incubator (Harward Apparatus). The cytosolic and nuclear calcium (Ca2+) concentrations were determined as described (29Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). AtT20 cells cultured on 25 mm coverslips were loaded with the fluorescent substrate CCF-2AM as described (26Zlokarnik G. Negulescu P.A. Knapp T.E. Mere L. Burres N. Feng L. Whitney M. Roemer K. Tsien R.Y. Science. 1998; 279: 84-88Crossref PubMed Scopus (596) Google Scholar). Briefly, cells were incubated for 1 h at room temperature in culture medium with 1 μm CCF-2AM and 1% pluronic acid. Cells were then washed in the perfusion chamber of the imaging system (as described before for Ca2+measurements). Measurements were made at 37 °C. CCF-2 fluorescence (excitation 405 nm) was imaged alternatively at emission wavelengths 450/530 nm, and data were stored at 3-min intervals. From these raw data in the form of image series at the two wavelengths, the fluorescence emission ratios F(450 nm)/F(530 nm) integrated over the cell area ratio were calculated (Fig. 1). The maximum and minimum CCF-2 ratios were determined by measuring the fluorescence in non-transfected AtT20 cells and in the SV40-βL cell line. The minimum ratio for the CCF-2 fluorescence was 0.48 ± 0.02 (n = 84 non-transfected cells). The maximal CCF-2 ratio was 6.5 ± 0.1 (n = 61 SV40-βL cells). These values are indicated in Fig. 1 (minimum and maximum) and Fig. 5 (minimum only) as dotted lines. To visualize the activation of gene transcription at the single cell level, we used β-lactamase as a reporter gene (26Zlokarnik G. Negulescu P.A. Knapp T.E. Mere L. Burres N. Feng L. Whitney M. Roemer K. Tsien R.Y. Science. 1998; 279: 84-88Crossref PubMed Scopus (596) Google Scholar). Expression of this bacterial enzyme can easily be detected by monitoring the fluorescence of a cell-permeant substrate, CCF-2, composed of two fluorophores linked by a β-lactame ring. The uncleaved CCF-2 emits green light (530 nm) because of fluorescence resonance energy transfer (FRET) between the two fluorophores. Upon β-lactamase expression, the substrate is cleaved and emits blue light (450 nm) because of the loss of FRET. β-lactamase activity is then measured as the change in green to blue CCF-2 fluorescence. To assess c-fos transcription, the β-lactamase coding sequence was placed under control of the promoter and first intron of the c-fos gene, yielding the reporter construct c-fos-βL that is drawn schematically in Fig. 5. Our earlier studies had shown that the first intron of the c-fos gene contains important regulatory elements without which transcription control by Ca2+ is incomplete (27Susini S. Van Haasteren G., Li, S. Prentki M. Schlegel W. FASEB J. 2000; 14: 128-136Crossref PubMed Scopus (29) Google Scholar,28van Haasteren G., Li, S. Ryser S. Schlegel W. Neuroendocrinology. 2000; 72: 368-378Crossref PubMed Scopus (19) Google Scholar). We generated AtT20 cell clones stably expressing c-fos-βL, which were loaded with CCF-2 AM. As seen in Fig. 1 (left panels), stimulation of the CCF-2-loaded c-fos-βL cells with agonists induced a change in the color of CCF-2 fluorescence from green to blue within 15–20 min. The degree of β-lactamase expression was quantified as the ratio of blue (450 nm) to green (530 nm) fluorescence intensities, an increase in CCF-2 ratio (F450/F530) thus reporting c-fos-βL activation. Following cell stimulation, CCF-2 fluorescence images were acquired every 3 min, and CCF-2 ratios were calculated to obtain a time course of c-fos-βL transcriptional activation (Fig. 1, right panels). In the absence of any stimulation, no change in CCF-2 ratio could be detected (top right panel), indicating that the degree of basal c-fos-βL transcriptional activation was stable in AtT20 cells over the 2-h measurement period. In contrast, c-fos-βL reporter expression was markedly enhanced by stimuli known to activate transcription of the c-fos gene in AtT20 cells (Fig. 1, middle and lower panels), such as EGF (20 nm), PACAP (20 nm), and FCS (10%). All stimuli induced a strong change in the CCF-2 ratio, from a mean basal value of 1.8 ± 0.06 (± S.E.) to maximal values after 2 h of stimulation. Other values were 5.4 ± 0.8 for EGF, 4.13 ± 0.1 for PACAP and 4.6 ± 0.1 for serum. These results validate the use of c-fos-βL as a reporter gene, which has the advantage of resolving the kinetics of transcriptional activation of the c-fos gene at the single cell level. To study the effect of Ca2+ on c-fostranscription, cells were depolarized with 20 mm KCl in order to open voltage-gated Ca2+ channels (VOCs) and trigger Ca2+ influx. As shown in Fig.2, KCl depolarization also powerfully activated c-fos-βL transcription, causing a near doubling in the CCF-2 ratio after 2 h of stimulation. This increase was almost completely abolished by preloading the cells with the Ca2+-chelator BAPTA-AM (Fig. 2, bottom panel), confirming that the KCl response was mediated by an increase in cellular Ca2+. Thus, in AtT20 cells a sustained increase in intracellular Ca2+ strongly activates the transcription of the c-fos gene. Although the basal CCF-2 ratio remained stable for up to 2 h in the absence of exogenous stimulus (Fig. 1), there was considerable cell-to-cell heterogeneity among non-stimulated cells (cf. the time 0′ images in Figs. 1 and 2). This suggested that c-fos-βL might already be activated in a fraction of the resting AtT20 cells. Accordingly, the histogram distribution of the CCF-2 ratios could be separated into three subpopulations centered at ratio values of 0.7, 1.8, and 3.5, and comprising 29%, 59%, and 12% of resting c-fos-βL AtT20 cells, respectively (Fig.3 A). This suggested that in the absence of stimulation transcription was strongly repressed in only approximately one-third of the cell population, whereas c-fos-βL was transcribed at intermediate levels in 59% of cells and at high levels in 12% of cells. Interestingly, the ratio value of the "active" population (3.5) was similar to the average CCF-2 ratio measured upon KCl stimulation (Fig. 3 C), suggesting that c-fos transcription was fully activated in 12% of unstimulated cells. To assess whether this heterogeneity could reflect spontaneous Ca2+ transients caused by the basal electrical activity of AtT20 cells, we used thel-type Ca2+ channel inhibitor, nifedipine. Exposure of cells to 1 μm nifedipine for 4 h, a concentration that completely blocks l-type Ca2+ channels, shifted the CCF-2 ratio to lower values, leaving nearly no transcriptionally fully active cells (Fig.3 B). Most cells (61%) now displayed a CCF-2 ratio of 0.8, and the proportion of cells with intermediate c-fos-βL activity (1.6) was reduced to 34%. An opposite effect was observed with a 2-h KCl stimulation (Fig. 3 C); the proportion of cells with fully activated c-fos-βL increased from 12 to 69% (ratio 3.64), whereas intermediate c-fos-βL activity was observed in the remaining 31% of cells (ratio 2.26). Thus, in AtT20 cells, basal c-fos transcription is relatively high as a result of electrical activity. Ca2+ channel blockers strongly down-regulate this endogenous activity, whereas a long-lasting depolarization powerfully activates c-fos transcription. Spontaneous action potentials in pituitary cells generate characteristic Ca2+ transients (21Schlegel W. Winiger B.P. Mollard P. Vacher P. Wuarin F. Zahnd G.R. Wollheim C.B. Dufy B. Nature. 1987; 329: 719-721Crossref PubMed Scopus (252) Google Scholar, 30Guerineau N. Corcuff J.B. Tabarin A. Mollard P. Endocrinology. 1991; 129: 409-420Crossref PubMed Scopus (89) Google Scholar, 31Fiekers J.F. Konopka L.M. Cell Calcium. 1996; 19: 327-336Crossref PubMed Scopus (14) Google Scholar). These Ca2+ elevations could be readily measured in c-fos-βL AtT20 cells using the ratiometric Ca2+ indicator fura-2 (Fig.4). As observed for basal c-fos-βL activity, the Ca2+ activity was heterogenous. 87% of cells (55/63) exhibited Ca2+transients with mean amplitude and frequency of 118 ± 87 nm and 0.1 ± 0.04 Hz, respectively (mean ± S.D., n = 1622, Fig. 4 A), whereas 13% of cells (8/63) lacked electrical activity (not shown). The Ca2+ transients ranged from 30 nm to 400 nm, resembling the distribution of basal c-fos-βL activity (Fig. 4 C). As expected, the spontaneous Ca2+ activity was completely abolished by the addition of 1 μm nifedipine (n = 19, Fig.4 C). Stimulation by 20 mm KCl elicited a transient increase in the cytosolic Ca2+, to 630 ± 200 nm (mean ± S.D., n = 7, Fig.4 D), followed by a sustained plateau (200 ± 70 nm). Note the disappearance of the spontaneous Ca2+ oscillations during the KCl stimulation. The convergent effect of nifedipine on basal Ca2+ transients and c-fos-βL activity suggested an important role for the spontaneous Ca2+ transients in the regulation of gene expression. To assess which element within the c-fos promoter mediated this Ca2+ response, we separately mutated two Ca2+ response elements within the c-fos-βL promoter (SRE and CRE, Fig. 5 A). The CRE and SRE elements were inactivated by site-directed mutagenesis (see Experimental Procedures), and AtT20 clones stably expressing the mutated constructs were established. These two clones (c-fos-ΔCRE-βL and c-fos-ΔSRE-βL) also displayed spontaneous Ca2+ transients, with amplitude and frequency similar to those observed in c-fos-βL or wild type AtT20 cells (data not shown). As shown in Fig. 5 B(hatched bars), deletion of the CRE element only marginally increased the basal CCF-2 ratio and did not affect the ability of cells to respond to nifedipine, but precluded their activation by KCl (2.69 ± 0.37 versus 2.15 ± 0.24,p = 0.07, unpaired t test). In contrast, deletion of the SRE element had a profound effect on endogenous c-fos-βL activity, decreasing the basal CCF-2 ratio from 1.80 ± 0.06 to 0.83 ± 0.03 (filled bars,p < 10−8, unpaired t test). Basal c-fos-ΔSRE-βL transcription could not be further reduced by nifedipine (0.80 ± 0.04), but the cells were still able to respond to a depolarizing stimulus. The ratio increased from 0.83 ± 0.03 to 2.07 ± 0.1 in the presence of KCl. The inactivation of both SRE and SRE elements (c-fos-ΔSRE/CRE-βL, Fig. 5 A) strongly reduced the expression of β-lactamase (dark bars), to values only slightly higher than the minimal values measured in non-transfected cells (dotted line). These cells were insensitive to nifedipine and to KCl stimulation. These data suggest that the CRE element is not required for the basal activation of c-fos. Instead, the SRE element appears to mediate the basal c-fostranscription driven by spontaneous action potentials. In contrast, the SRE element is less essential for the Ca2+-mediated transcriptional activation induced by a large and long-lasting depolarization. The differential regulation of the spontaneous and KCl-induced c-fos transcription suggested that the two types of Ca2+ signals might propagate differently to the nucleus. In particula
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