HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 46, 43599-43607, November 15, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/46/43599    most recent M204787200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Schneider, G. Articles by Schmid, R. M. Search for Related Content PubMed PubMed Citation Articles by Schneider, G. Articles by Schmid, R. M. Social Bookmarking                      What's this?

Cyclosporine Inhibits Growth through the Activating Transcription Factor/cAMP-responsive Element-binding Protein Binding Site in the Cyclin D1 Promoter^*

Günter Schneider, Franz Oswald, Christian Wahl^§, Florian R. Greten, Guido Adler, and Roland M. Schmid^¶

From the  Department of Internal Medicine I and ^§ Department of Medical Microbiology and Hygiene, University of Ulm, 89081 Ulm, Germany

Received for publication, May 16, 2002, and in revised form, September 4, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The immunosuppressive agent cyclosporine affects proliferation depending on the cellular system used. In an attempt to study the inhibitory effect of cyclosporine on proliferation of pancreatic acinar cells, we used AR42J cells as a model system. Here we demonstrate that cyclosporine inhibits growth of these cells by inducing G₁ cell cycle arrest. This effect is mediated by the 5' regulatory region of the cyclin D1 gene and leads to a reduction of cyclin D1 mRNA expression and protein abundance. We show that in AR42J cells the proximal cyclin D1 promoter contains a cis-regulated element, which is important for the maintenance of basal transcriptional activity. This element overlaps the described cAMP-responsive element (CRE) and confers cyclosporine sensitivity to the cyclin D1 promoter. Furthermore, the DNA binding activity of the CRE-binding protein (CREB) decreases through cyclosporine treatment and this is mediated by cyclosporine-induced reduction of CREB steady-state levels. These results demonstrate that cyclosporine can inhibit proliferation of acinar cells by targeting the cyclin D1 promoter at the proximal CRE via a reduction of CREB protein abundance.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Mammalian cell proliferation is regulated in mid- to late G₁ phase of the cell cycle. The D-type cyclins and the associated cyclin-dependent kinases (CDKs)¹ are thought to be the key regulatory components for progression through G₁ phase and for the commitment of cells to enter S phase. Cyclin D1 and its catalytic partners CDK4 and CDK6 phosphorylate the retinoblastoma tumor suppressor gene product (RB), resulting in the release of E2F transcription factors, which are required for the activation of S phase-specific genes (1, 2). Inhibition of cyclin D1 expression prevents transition of cells from G₁ to S phase, whereas ectopic expression of cyclin D1 shortens the G₁ interval in many cell types, suggesting that cyclin D1 may be rate-limiting for G₁ progression (2, 3). The abundance of cyclin D1 is regulated transcriptionally and through the ubiquitin-proteasome pathway via protein degradation (4). The promoter region of the cyclin D1 gene contains multiple cis-acting elements, including binding sites for activator protein-1 (AP-1), for nuclear factor B (NF-B), for signal transducers and activators of transcription, for SP1, and for activating transcription factor (ATF)/cAMP-responsive element-binding protein (CREB). The ATF/CREB-binding site is implicated in the activation of the cyclin D1 gene by pp60^v-src (5). Furthermore, the estrogen-induced activation of the cyclin D1 promoter depends on the ATF/CREB site in which ATF-2 and c-Jun form heterodimers (6). Moreover, the integrin-linked kinase-dependent induction of the cyclin D1 promoter requires an intact ATF/CREB site, which is important for the maintenance of the basal transcriptional activity, but not for the induction of the cyclin D1 promoter activity in vascular endothelial cells (7, 8).

The basic leucine zipper transcription factor CREB is ubiquitously expressed and activated by phosphorylation at serine 133 in response to elevated levels of cAMP, an increase in intracellular calcium concentration, and growth factors (9). In electrically stimulated cultured hippocampal neurons, calcineurin seems to be implicated in the negative regulation of CREB-mediated transcription (10). In contrast, the immunosuppressive drugs cyclosporine and FK506 inhibit CRE-mediated transcription stimulated by membrane depolarization and Ca²⁺ influx in pancreatic islet cells (11). This study provides evidence for a direct involvement of the serine-threonine phosphatase calcineurin in CRE-directed transcription. The effect of calcineurin on CREB-mediated transcription seems to be cell type-specific and modulated through the promoter context. In contrast, the natural CRE-dependent promoters of the rat glucagon, rat somatostatin, and human c-Fos genes negatively respond to cyclosporine treatment in HIT cells; the rat insulin gene promoter is not affected (12, 13). The cyclic peptide cyclosporine binds to cyclophilin and inhibits the serinethreonine phosphatase calcineurin (14-16). This property may account for its different biological actions such as immunosuppression, vasoconstriction, stimulation of TGF- expression, and induction or prevention of apoptosis (17-20).

Moreover, cyclosporine has been shown to alter cell proliferation in different systems. For hematopoietic stem and progenitor cells, smooth muscle cells, and hepatocytes, an induction of cell proliferation or a tumor promoting effect has been shown (21-25). On the other hand, growth of interleukin-3-dependent mast cells, human lung-cancer cells, tubular kidney cells, cultured pituitary cells, T-cells, and keratinocytes seems to be inhibited by cyclosporine (16, 26-30).

In vivo, cyclosporine interferes with the reparative process activated after experimental pancreatitis, at least in part, by inhibiting acinar cell proliferation (31). Because of the short lifetime of dispersed nonimmortalized pancreatic acinar cells, we used AR42J cells, an established model system, to investigate the biology of isolated pancreatic acinar cells (32). Our data show that cyclosporine treatment of the pancreatic acinar cell line AR42J leads to the inhibition of cell growth by inhibiting cell cycle progression in the G₁ phase, which is mediated through transcriptional inhibition of cyclin D1 expression. We mapped a cyclosporine-responsive cis regulatory element in the proximal cyclin D1 promoter, which overlaps the described CREB/ATF-2 binding site. Furthermore, we show that the cyclosporine sensitivity of the cyclin D1 promoter is mediated by a reduction of CREB steady-state levels.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Culture and Transfection-- AR42J cells were cultivated in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 1% (w/v) penicillin/streptomycin. Transfections were performed using FuGENE6 (Roche Molecular Biochemicals) according to the protocol of the manufacturer. To increase transfection efficiency, AR42J cells were treated with 25 µM chloroquine (Sigma) for 2 h prior to transfection. 24 h after transfection cells were treated with cyclosporine (Sigma) or left untreated. Cells were used between passages 10 and 25 for experiments. After another 24 h cells were incubated in lysis buffer (Promega) for 15 min, harvested, and cleared by centrifugation for 15 min. Lysates were normalized for protein content, and luciferase activity of at least three independent transfections was determined in a LB 9501 luminometer (Berthold, Bad Wildbad, Germany) using a luciferase assay system (Promega). Transfections were performed in duplicate.

Plasmids-- The cyclin D1 promoter-containing construct pD1luc, harboring the 1094 to +135 5' regulatory region, was a kind gift of Dr. Claus Scheidereit (33). The cyclin D1 promoter construct 673/+135 was created by partial digestion of pD1luc with BstEII and BamHI following religation. The 267/+135 construct was generated by digesting pD1luc with PstI following religation. The constructs 267/+10 and 64/+10 were created by PCR using the following primers: D164 (5'-CCCCTCGAGCAACAGTAACGTCACACGG-3'), D1267 (5'-CCCCTCGAGGGCTGCAGCGGGGCGATTTGC-3'), and D1+10 (5'-CCCAAGCTTGACAGCCCTCTGGAGGCTCC-3'). The PCR products were cloned into the XhoI - Hind III sites of pGL3basic (Promega). The 267/+10 CRE_mut D1 promoter construct was created using QuikChange^TM site-directed mutagenesis kit (Stratagene) according to the protocol of the manufacturer using the following primers: CRE_{mut sense} (5'-GCTTAACAACAGTAAGTAAGATCTCACGGACTACAGGGGAG-3'), and CRE_{mut antisense} (5'-CTCCCCTGTAGTCCGTGAGATCTTACTGTTGTTAAGC-3'). Sequence confirmation of the respective mutations was performed on both strands. pGL3basic (Promega) and ptk-luc served as control plasmids (34).

Preparation of Total Cell Lysates and Nuclear Extracts-- Whole-cell lysates were prepared by incubating cell pellets for 30 min at 4 °C in immunoprecipitation buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 10% glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 5 mM NaF). Insoluble material was removed by centrifugation, and lysates were aliquoted and stored at 80 °C. Nuclear extracts were prepared, with modifications, according to the method of Schreiber et al. (35). Briefly, cells were collected, washed in phosphate-buffered saline (PBS), and pelleted by centrifugation for 5 min. The cell pellet was resuspended in 400 µl of cold buffer A (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride) and allowed to swell on ice for 15 min. Cells were disrupted by passage through a 26-gauge needle, and nuclei were centrifuged at 500 × g. To avoid cytoplasmatic contamination, the nuclear pellet was washed two times in buffer A. Finally the nuclear pellet was resuspended in 50 µl cold buffer C (20 mM HEPES, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 5 mM NaF) and rocked at 4 °C on a shaking platform for 30 min. The lysates were cleared by centrifugation, aliquoted, and stored at 80 °C.

Electrophoretic Mobility Shift Assay (EMSA) and Oligonucleotides-- EMSAs were performed using 5 µg of nuclear extract in a 20-µl binding reaction containing 4% glycerol, 10 mM Tris, 100 mM NaCl, and 100 ng/µl poly(dI-dC). After a 20-min incubation, 50,000 cpm of a -³²P-labeled double-stranded oligonucleotide was added and incubated for 30 min on ice. For competition a 100-fold molar excess of unlabeled oligonucleotide was used. Complexes were resolved on a 5% polyacrylamide gel in 0.5× TBE at 250 V at room temperature. The gels were dried and exposed on film. Nucleotide sequences of the sense strand of the double-stranded oligonucleotides were as follows: D1/CRE (5'-CTAGTCAACAGTAACGTCACACGGACT-3'), D1/CRE_mut (5'-CTAGTCAACAGTAAGATCTCACGGACT-3'), c-Fos/CRE (5'-TGGTTGAGCCCGTGACGTTTACACTCAT-3'), Oct (5'-CTAGTGTCGAATGCAAATCACTAGAAT-3'), AP-1 (5'-AGCTTACTCAGTACTAGTACG-3'). For supershifts assays antibodies against c-Jun, c-Fos, ATF-2, or CREB (all Santa Cruz Biotechnology) were added to the binding reaction.

Western Blot Analysis-- Extracts were normalized for protein and heated at 95 °C for 5 min in Laemmli buffer. Proteins were resolved on 10% SDS-polyacrylamide gels and electrophoretically transferred to polyvinylidene difluoride (Millipore) membranes in a semidry blotting system. Membranes were blocked in PBS supplemented with 5% skim milk and 0.1% Tween 20, and incubated with antibody for 1 h at room temperature. The membranes were incubated with antibodies against p15^INK4b, p16^INK4a, p21^CIP1/WAF1, p27^KIP1, Cdk4, ATF-2, or CREB (all Santa Cruz Biotechnology) or cyclin D1 and RB (BD PharMingen). Proteins recognized by the antibodies were detected by enhanced chemiluminescence (Amersham Biosciences) using horseradish peroxidase-coupled secondary antibody.

Cell Proliferation Assay-- Cells were plated at a density of 20,000 cells/well in a 24-well plate in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. After seeding the cells were treated with cyclosporine or left untreated. After 18 h [³H]thymidine (1 µCi/well) was added, and incubation was continued for additional 6 h. Afterward the cells were washed in PBS, fixed in methanol, and treated in trichloroacetic acid for 20 min. The precipitated material was resolved in 1 M NaOH and subjected to scintillation counting.

Quantitative RNA Analysis-- Total RNA was isolated and mRNA expression levels were quantified using real-time PCR analysis (TaqMan, PE Applied Biosystems, Norwalk CT) as previously described (36). cDNA synthesis was performed using 2 µg of total RNA by reverse transcription. PCR reaction (denaturation at 95 °C for 2 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 1min; Sybr Green PCR Core Reagents, PerkinElmer Applied Biosystems, Norwalk, CT) was performed using primer combinations listed below. Endogenous cyclophilin mRNA levels were used as internal controls, and cyclin D1 mRNA levels were normalized to cyclophilin mRNA levels. The following primers were used: cyclophilin-FP (5'-ATGGTCAACCCCACCGTGT-3'), cyclophilin-RP (5'-TTCTTGCTGTCTTTGGAACTTTGTC-3'), cyclinD1-FP (5'-TACCCTGACACCAATCTCCTCA-3'), and cyclinD1-RP (5'- TTTCCGCATGGATGGCAC-3').

Cell Cycle Analysis and Annexin V Staining-- For cell cycle analysis cells were washed twice in PBS and redissolved in propidium iodide (PI) staining buffer containing 0.1% sodium citrate, 0.1% Triton X-100, and 50 µg/ml PI. After 1 h of incubation at 4 °C, flow cytometry was performed using a BD Biosciences FACScan. The distribution of cells in different cell cycle stages (G₁/S/G₂+M) was determined according to their DNA content. Thus, cells possessing 2n (diploid) DNA were assigned to be in G₁ and those having a DNA content between 2n and 4n (tetraploid) were defined as S phase cells. Finally, cells having ~4n DNA content were assumed to be in G₂+M. The sum of all cells in FACS analysis was set to 100% and the distribution into G₁, S, and G₂+M phases was determined by counting the cells in each window according to the definitions above.

For staining of apoptotic cells, annexin V was used in conjunction with the vital dye PI to distinguish apoptotic (annexin V-positive, PI-negative) from necrotic cells (annexin V-positive, PI-positive). Unfixed AR42J cells were washed twice with PBS and resuspended in a binding buffer containing 10 mM HEPES/NaOH (pH 7.5), 140 mM NaCl, 2.5 mM CaCl₂. Cells were incubated for 15 min with annexin V-FITC (BD PharMingen) and PI and subsequently analyzed by flow cytometry with a fluorescence-activated cell sorter (FACStar, BD Biosciences).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cyclosporine Inhibits Cell Growth of AR42J Cells-- To test the effect of cyclosporine on the growth of pancreatic acinar cells, we treated AR42J cells, a cell culture model system for pancreatic acinar cells, with cyclosporine (32). Untreated AR42J cells were 80% confluent after 48 h, whereas the growth of cyclosporine-treated cells was strongly diminished without overt morphological alterations (Fig. 1A). To confirm the clear difference between untreated and cyclosporine-treated cells, we monitored cell number over time. Treatment of AR42J cells with cyclosporine led to a dose-dependent decrease in the growth rate of the cells over a time period of 3 days (Fig. 1B). The inhibition of AR42J cell proliferation by cyclosporine was not caused by cytotoxicity because the cells remained viable, as determined by trypan blue exclusion (Fig. 1C). The inhibitory effect of cyclosporine on cell proliferation was further supported by the [³H]thymidine incorporation assay. Treatment of AR42J cells with cyclosporine induced a dose-dependent decrease in [³H]thymidine incorporation (Fig. 1D). This suggests that cyclosporine negatively influences the growth of pancreatic acinar cells. This effect seems to be specific for pancreatic acinar cells, because the cyclosporine-induced growth inhibition was not observed in HeLa cells, pancreatic carcinoma cell lines, like MiaPaCa2 or Panc1, neuroendocrine Bon cells, or neuronal cell lines, like SK-N-SH (data not shown).

View larger version (55K): [in this window] [in a new window]   Fig. 1.   Cyclosporine inhibits growth of AR42J cells. A, photomicrographs (original magnification, ×16) of AR42J cells grown without (left) or with 1.2 µg/ml cyclosporine (right) for 48 h. Note the lack of morphological alterations of cyclosporine-treated cells. B, AR42J cells were plated at a density of 0.72 × 105 cells/10-mm dish, treated with the indicated concentrations of cyclosporine, or left untreated. Numbers of cells was determined daily. C, AR42J cells were cultivated in the presence of 1.2 µg/ml cyclosporine and viability was determined daily using trypan blue exclusion. D, cells were plated and treated with cyclosporine at the indicated concentrations or left untreated. After 18 h cells were pulsed with [3H]thymidine for an additional 6 h. Afterward the [3H]thymidine incorporation was determined. Data were obtained from three independent experiments done in duplicate and are presented as mean and standard error of the mean (S.E.).

Cyclosporine Does Not Induce Apoptosis in AR42J Cells-- The finely tuned balance between mitosis and apoptosis determines the net growth of cells. Exposure of certain cell lines to cyclosporine led to the induction of apoptosis (19, 20, 37). To exclude that the effect of cyclosporine on AR42J cells is caused by the induction of apoptosis, we investigated markers for apoptosis (Fig. 2). The exposure of phosphatidylserine on the outer leaflet of the cytoplasmic membranes is an early apoptotic process. Annexin V is an anticoagulant that preferentially binds to negatively charged phospholipids and can be used to identify apoptotic cells. As shown in Fig. 2, the treatment of AR42J cells with cyclosporine over a time period of 48 h did not lead to an increase of annexin V-positive cells. In contrast, UV light augmented the annexin V-positive fraction to 71% 8 h after irradiation. Moreover, we could not detect apoptosis in cyclosporine-treated AR42J cells compared with untreated cells assayed by 4,6-diamidino-2-phenylindole staining or by Western analysis of the caspase substrate p116 poly(ADP-ribose) polymerase (data not shown). Therefore, these data suggest that the observed growth defect of cyclosporine-treated AR42J cells is not a result of the induction of apoptosis.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Cyclosporine does not induce apoptosis in AR42J cells. Flow cytometric analysis of apoptotic cells using annexin V-FITC. AR42J cells were treated with 1.2 µg/ml cyclosporine for 24 and 48 h or left untreated. As a positive control, AR42J cells were exposed to UV light for 5 min and annexin V binding was assayed after irradiation for 8 h. Cells were incubated with annexin V-FITC in a buffer containing PI and analyzed by flow cytometry. PI-negative cells were gated and shown as histograms.

Exposure of AR42J Cells to Cyclosporine Induces a G₁ Arrest Independent of Up-regulation of Cyclin-dependent Kinase Inhibitors (CKIs)-- Next we investigated cell cycle distribution of untreated and cyclosporine-treated AR42J cells using PI staining and FACS analysis. Cyclosporine treatment increased the fraction of cells in G₁ phase from 62 to nearly 75% after 24 h. As shown in Fig. 3A, the G₁ fraction of cyclosporine-treated cells was further increased to 83% after 48 h, whereas the cells in the S and the G₂/M phase decreased to 11 and 6% compared with the untreated control. Because a growth arrest in the G₁ phase of the cell cycle could be the result of enforced expression of CKIs, we examined the protein abundance of p15^INK4b, p16^INK4a, p21^CIP1/WAF1, and p27^KIP1. We could not demonstrate an increase in the expression level of these proteins induced by cyclosporine for the time period of 48 h for all four CKIs investigated (Fig. 3B). Moreover, the protein levels of the G₁ phase CDK4 were not altered by cyclosporine treatment (Fig. 3C). Taken together these results argue for a cyclosporine-induced G₁ phase arrest in AR42J cells independent of the induction of the CKIs p15^INK4b, p16^INK4a, p21^CIP1/WAF1, and p27^KIP1.

View larger version (40K): [in this window] [in a new window]   Fig. 3.   Cyclosporine causes a G1 phase arrest independent of up-regulation of CKIs. A, FACS analysis of cell cycle distribution. AR42J cells were treated with 1.2 µg/ml cyclosporine for 24 h (middle histogram) and 48 h (right histogram) or left untreated (left histogram). After collecting, the cells were stained with PI and FACS analyses were performed. Fractions of cells in G1, S, and G2/M phase are indicated. One representative experiment out of four is presented. B, Western blot analysis of CKI expression. AR42J cells were treated for 24 h (lanes 2 and 5) and 48 h (lanes 3 and 6) with 1.2 µg/ml cyclosporine or left untreated (lanes 1 and 4). Whole cell extracts were prepared and probed with antibodies against p15INK4b, p16INK4a, p21CIP1/WAF1, and p27KIP1. Coomassie-stained membranes show equal protein loading. C, Western blot analysis of CDK4 expression. AR42J cells were treated for 24 h (lane 2) and 48 h (lane 3) with 1.2 µg/ml cyclosporine or left untreated (lane 1). Whole cell extracts were prepared and probed with an antibody against CDK4. A nonspecific band (n.s.) indicates equal protein loading.

Cyclosporine Leads to the Transcriptional Down-regulation of Cyclin D1 and to RB Hypophosphorylation-- The progression of the G₁ phase is largely regulated by D-type cyclins (2, 38). This prompted us to investigate whether cyclin D1 is regulated by cyclosporine in AR42J cells. The protein abundance of cyclin D1 is decreased in cyclosporine-treated AR42J cells compared with the untreated control (Fig. 4A). The cyclin D1 levels were slightly reduced after 24 h and almost completely suppressed after 72 h. A nonspecific band indicated equal protein loading. To test whether the observed decrease in cyclin D1 abundance is reflected by alterations in the phosphorylation status of RB, we performed Western blot analyses. In untreated, randomly cycling AR42J cells, RB was mainly hyperphosphorylated (Fig. 4B, lane 1). Treatment with cyclosporine led to the sustained accumulation of hypophosphorylated RB over a time period of 72 h (Fig. 4B, lanes 2-4). These data further confirm that cyclosporine induces a G₁ phase arrest in AR42J cells because RB is predominantly hypophosphorylated in the early G₁ phase (1). Cyclin D1 expression depends highly on transcriptional regulation (4). Consistent with protein levels, quantitative RNA analysis using real-time PCR revealed a reduction of cyclin D1 mRNA levels in AR42J cells 24-48 h after cyclosporine treatment (Fig. 4C). To verify whether decreased cyclin D1 mRNA expression is mediated by the 5' regulatory region of the cyclin D1 gene, we carried out transient transfection experiments. As shown in Fig. 4D, basal cyclin D1 promoter activity is strongly and dose-dependently reduced by cyclosporine in AR42J cells. This supports the conclusion that a transcriptional mechanism is responsible for the cyclosporine-induced decrease of cyclin D1 protein and RNA levels.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Cyclosporine leads to transcriptional down-regulation of cyclin D1 and RB hypophosphorylation. A, Western blot analysis of cyclin D1 expression. AR42J cells were treated with 1.2 µg/ml cyclosporine for 24 h (lane 2), 48 h (lane 3), and 72 h (lane 4) or left untreated (lane 1). Whole cell extracts were probed with an anti-cyclin D1 antibody. A nonspecific band confirms equal protein loading. B, Western blot analysis of RB expression. AR42J cells were treated with 1.2 µg/ml cyclosporine for 24 h (lane 2), 48 h (lane 3), and 72 h (lane 4) or left untreated (lane 1). Whole cell extracts were probed with an anti-RB antibody. To confirm equal protein loading, the Coomassie-stained membranes are shown. C, quantitative cyclin D1 mRNA expression analysis. Total RNA was prepared after 24 and 48 h of treatment with 1.2 µg/ml cyclosporine or from untreated AR42J cells. mRNA expression levels were quantified using real-time PCR analysis and normalized to cyclophilin mRNA levels. D, transient transfection of cyclin D1 promoter constructs in AR42J cells. 5 µg of the cyclin D1 luciferase reporter construct were transfected into AR42J cells. After 24 h the cells were treated with 1.2 µg/ml cyclosporine or left untreated. After an additional 24 h, cells were harvested and luciferase activity was determined. Data were obtained from three independent experiments done in duplicate and are presented as mean and standard error of the mean (S.E.).

The Proximal Cyclin D1 Promoter Contains a Cyclosporine-responsive Element-- To determine the region of the cyclin D1 promoter required for cyclosporine sensitivity, deletion constructs of the cyclin D1 5' promoter were transfected into AR42J cells following stimulation with cyclosporine (Fig. 5). Deletions from 1094 to 64 reduced the basal promoter activity to one third. This indicates that a cis regulatory element that contributes to the maintenance of the basal transcriptional activity in AR42J cells is located in the proximal 64 base pairs of the cyclin D1 promoter. It has been shown that the promoter region between 883 and 831 functions as a silencer element, explaining the 2-fold elevation of the basal transcriptional activity of the 673/+135 cyclin D1 promoter construct (39, 40). To exclude that a region downstream of the initiation site is involved in the cyclosporine sensitivity of the cyclin D1 promoter, we compared the 267/+135 construct with a 267/+10 cyclin D1 promoter construct. As shown in Fig. 5, both constructs show the same characteristics in AR42J cells, harboring 70% of the basal transcriptional activity, as compared with the 1094/+135 cyclin D1 promoter construct. In addition, both constructs responded similarly to cyclosporine treatment. Interestingly the basal activity of all cyclin D1 promoter 5' deletion constructs is reduced to approximately one fifth to one third following cyclosporine treatment, whereas both control vectors showed no cyclosporine sensitivity. These results suggest that the first 64 base pairs upstream of the initiation site in the cyclin D1 promoter are involved in the maintenance of basal transcriptional activity as well as cyclosporine sensitivity.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   The proximal D1 promoter contains a cyclosporine-responsive element. 5 µg of the indicated 5' deletion cyclin D1 promoter luciferase constructs or the control vectors (pGL3basic and ptk-luc) were transiently transfected into AR42J cells. 24 h after transfection, cells were treated with 1.2 µg/ml cyclosporine or left untreated. After an additional 24 h, cells were harvested and luciferase activity was determined. Data were obtained from three independent experiments done in duplicate and are presented as mean and standard error of the mean (S.E.).

CREB Confers Cyclosporine Sensitivity to the Proximal Cyclin D1 Promoter-- The nucleotide sequence of the proximal cyclin D1 promoter is depicted in Fig. 6A. This promoter region contains cis regulatory elements for NF-B and ATF/CREB (33, 41-43). We were not able to detect NF-B activity in nuclear extracts from AR42J cells to an oligonucleotide probe corresponding to the B site of the cyclin D1 promoter. Moreover, cyclin D1 promoter constructs with mutations in the proximal NF-B binding site were equally sensitive to cyclosporine as the wild type constructs in transient transfection assays (data not shown). Therefore, we assessed the role of the ATF/CRE binding site. EMSAs were performed with nuclear extracts of unstimulated AR42J cells using a -³²P-labeled cyclin D1 CRE as probe. As demonstrated in Fig. 6B, three complexes could be visualized termed A, B, and C (Fig. 6B, lane 1). Treatment of the cells with cyclosporine for 24 and 48 h led to the disappearance of all three complexes, suggesting that cyclosporine-sensitive factors bind to the cyclin D1 CRE site (Fig. 6B, lanes 2 and 3). The same extracts were probed with an Oct consensus-containing oligonucleotide as control. Nuclear extracts of untreated AR42J cells show a typical Oct-containing complex (Fig. 6B, lane 4). The Oct binding activity was not significantly affected by cyclosporine over a time period of 24 h (Fig. 6B, lane 5) and 48 h (Fig. 6B, lane 6) compared with three complexes formed at the cyclin D1 CRE. For further characterization of the complexes formed at the cyclin D1 CRE, we performed competition analyses. All three complexes were abolished after preincubation with a 100-fold molar excess of unlabeled wild type probe (Fig. 6C, lane 2) but not with a mutant probe (Fig. 6C, lane 3). Addition of a 100-fold molar excess of the CRE derived from the c-Fos promoter also completely prevented complex formation, indicating the specificity of these complexes (Fig. 6C, lane 4). It has been shown that cyclin D1 CRE binds the AP-1 transcription factors c-Fos and c-Jun in mouse embryonic fibroblasts. Moreover, c-Jun was shown to be implicated in the estrogen-induced activation of the cyclin D1 gene via the ATF/CREB binding site (6, 43). Addition of a 100-fold molar excess of a unlabeled AP-1 oligonucleotide did not disturb the formation of the three complexes detected in nuclear extracts of AR42J cells (Fig. 6C, lane 5), which suggests that AP-1 is not involved in complex formation at the cyclin D1 CRE in AR42J cells. In an attempt to determine the composition of these complexes, we performed supershift assays with antibodies against CREB, ATF-2, c-Fos, and c-Jun. The CREB antibody greatly reduces the intensity of all three complexes, leading to a supershifted band (Fig. 6D, lane 2). The antibodies against ATF-2, c-Fos, and c-Jun did not influence the formation of complexes A, B, and C. This further confirms that AP-1 factors are not part of the complexes formed at the cyclin D1 CRE in AR42J cells. In some experiments a low mobility cyclosporine-sensitive complex is detectable. This complex is supershifted with the ATF-2 and CREB antibodies; therefore, we cannot exclude ATF-2 binding to the cyclin D1 CRE in AR42J cells (data not shown). These data identify CREB as a cyclosporine-sensitive factor that binds to the cyclin D1 CRE.

View larger version (63K): [in this window] [in a new window]   Fig. 6.   CREB confers cyclosporine sensitivity to the cyclin D1 CRE. A, nucleotide sequence of proximal cyclin D1 promoter. The NF-B and the CRE binding sites are indicated. In addition, the mutated CRE binding site is shown. B, EMSA of nuclear extracts from AR42J cells treated for 24 h (lanes 2 and 5) or 48 h (lanes 3 and 6) with 1.2 µg/ml cyclosporine or untreated cells (lanes 1 and 3). In lanes 1-3 the -32P-labeled cyclin D1 CRE was used as probe, whereas in lanes 4-6 a -32P-labeled Oct consensus oligonucleotide was used. C, competition analysis of the complexes formed at the cyclin D1 CRE. Nuclear extracts of untreated AR42J cells were incubated with the -32P-labeled CRE of the cyclin D1 promoter (lane 1) or competed with a 100-fold molar excess of the unlabeled wild type oligonucleotide (lane 2), or unlabeled mutated cyclin D1 CRE (lane 3), the c-Fos CRE (lane 4), or an AP-1 consensus oligonucleotide (lane 5). D, supershift analysis of the complexes formed at the cyclin D1 CRE. Nuclear extracts of untreated AR42J cells were preincubated with no antibody (lane 1), a CREB antibody (lane 2), an ATF-2 antibody (lane 3), a c-Fos antibody (lane 4), or a c-Jun antibody (lane 5). The supershift obtained with the CREB antibody is indicated with ss.

Cyclosporine Induces a Reduction of CREB Steady-state Levels in AR42J Cells-- To test whether the observed loss of CREB binding to the cyclin D1 CRE is a result of a reduction of the binding affinity or a reduction of CREB expression, we performed Western blot analysis using whole cell extracts of untreated and cyclosporine-treated AR42J cells. Compared with the untreated control (Fig. 7, lane 1), a strong decrease in the CREB protein abundance was detected following cyclosporine treatment for 24 h (Fig. 7, lane 2) and 48 h (Fig. 7, lane 3). Reprobing the membrane with an ATF-2 antibody tested the specificity of this effect on CREB expression. As shown in Fig. 7, protein levels of ATF-2 were not altered over a time period of 48 h. These results demonstrate that the loss of CREB binding to the cyclin D1 CRE is caused by a cyclosporine-induced decrease of CREB protein abundance.

View larger version (34K): [in this window] [in a new window]   Fig. 7.   Western blot analysis of CREB and ATF-2 expression in AR42J cells. AR42J cells were treated for 24 h (lane 2) and 48 h (lane 3) with 1.2 µg/ml cyclosporine or left as an untreated control (lane 1). Whole cell extracts were first probed with an antibody against CREB. After stripping membranes were reprobed with an ATF-2 antibody.

Mutation of the ATF/CREB Cis Regulatory Element in the Context of the Proximal Cyclin D1 Promoter Leads to the Reduction of the Basal Activity and Loss of Cyclosporine Sensitivity-- To further study the influence of the ATF/CREB cis regulatory element regarding maintenance of the basal cyclin D1 promoter activity and cyclosporine sensitivity, we carried out transient transfection experiments in AR42J cells with a cyclin D1 reporter construct harboring a CRE mutation. In these experiments the activity of the cyclin D1 267/+10 wild type reporter was reduced to nearly one third upon cyclosporine treatment (Fig. 8). In contrast the CRE-mutated cyclin D1 construct, D1 267/+10 CRE_mut does not respond to cyclosporine (Fig. 8). Moreover, the basal activity in AR42J cells was only 40% compared with the basal activity of the wild type construct. These results further underline the importance of the ATF/CREB cis regulatory element for the maintenance of the basal cyclin D1 promoter activity and cyclosporine sensitivity.

View larger version (13K): [in this window] [in a new window]   Fig. 8.   Transient transfection of CRE mutated cyclin D1 reporter constructs. 5 µg of the cyclin D1 267/+10 wild type reporter construct or the 267/+10 cyclin D1 reporter gene construct containing a mutated CRE binding site (D1 267/+10 CREmut) were transiently transfected in AR42J cells. 24 h after transfection, cells were treated with 1.2 µg/ml cyclosporine or left untreated. After an additional 24 h, cells were harvested and luciferase activity was determined. Data were obtained from three independent experiments done in duplicate and are presented as mean and standard error of the mean (S.E.).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Here we show that cyclosporine inhibits proliferation of AR42J cells, a pancreatic acinar cell line, by inducing a G₁ arrest through reduction of cyclin D1 levels. A similar effect has been reported for psoriatic skin lesions, where a reduced cell turnover and a reduced expression of cyclin D1 after cyclosporine treatment were shown in vivo. In vitro, FK506 and cyclosporine induced a G₁ phase arrest in growth factor-stimulated cultured human keratinocytes (44, 45). The G₁ phase arrest induced by cyclosporine was also observed in the tubular kidney cell line LLC-PK₁. Cyclosporine treatment of LLC-PK1 cells increased the fraction of cells in the G₁ phase from 62 to 78% (46). Cyclosporine has been shown to stimulate TGF- expression, which is known to exert an inhibitory effect on proliferation of acinar cells (47, 48). However, the effect of cyclosporine on cell proliferation of AR42J cells most likely is independent of TGF-, because protein levels of the cyclin-dependent kinase inhibitors p15^INK4b and p21^CIP1/WAF1, known targets of TGF-, are not altered in cyclosporine-treated AR42J cells (49, 50). This conclusion is further supported by the unaltered expression of CDK4, which is drastically reduced upon TGF- treatment of TGF--responsive cells (51).

In AR42J cells cyclin D1 is down-regulated at the transcriptional level by cyclosporine. Previous reports have shown that the proximal cyclin D1 promoter is controlled by a CREB/ATF/AP-1 and an NF-B binding site (33, 41, 42). Although binding of the AP-1 transcription factors c-Jun and c-Fos was demonstrated in mouse embryonic fibroblasts and MCF-7 cells, we did not detect AP-1 binding to the cyclin D1 CRE in AR42J cells (6, 43). We provide evidence that members of the CREB/ATF protein family, mainly CREB, bind the CRE site within the cyclin D1 promoter. First, in EMSA competition analysis, the formed complexes are completely competed with the cyclin D1 CRE and the c-Fos CRE, whereas the mutated cyclin D1 CRE had no effect on complex formation. Second, all three complexes were supershifted by an antibody, which recognizes CREB, ATF-1, and CREM. Third, Western blot analysis revealed that CREB has the highest abundance among the three different proteins recognized by the antibody (results not shown).

In line with our data in AR42J cells, CREB has been found to be important for the maintenance of the basal cyclin D1 promoter activity in vascular endothelial cells (8). Phosphorylation of CREB at serine 133 is crucial for its ability to trans-activate target genes and its ability to bind to CBP (9). A recent report showed that CREB is constitutively phosphorylated at serine 133 in AR42J cells. This would explain the observed CREB- dependent basal cyclin D1 promoter activity (52). In addition to maintenance of basal cyclin D1 promoter activity, CREB also confers cyclosporine sensitivity to the proximal promoter part. Transcriptional activation of CREB by depolarization was shown to be inhibited by cyclosporine and FK506 in non-immune cells (13, 53). The mechanisms leading to loss of CREB transcriptional activity are largely unknown and seem to be independent of CREB phosphorylation at serine 119 (in CREB-327, corresponding to serine 133 in CREB-341) (13). In AR42J cells, treatment with cyclosporine leads to a nearly complete loss of CREB protein expression, which is different from the results reported by Schwaninger et al. (53). Reduction of CREB expression is a specific effect because protein abundance of ATF2 is unaltered. Recently, Taylor et al. (54) have demonstrated phosphorylation-dependent targeting of CREB to the ubiquitin proteasome pathway. In epithelial cells hypoxia led to hyperphosphorylation, ubiquitination and subsequent proteasome-mediated degradation of CREB. This has been correlated with the degradation of the serine-threonine phosphatase PP1, a phosphatase involved in CREB dephosphorylation and regulation of transcriptional activity (54, 55). A similar regulation might be relevant in our cellular system because the molecular target of cyclosporine calcineurin is known to stimulate PP1 activity (56).

The cyclosporine-induced targeting of CREB to the ubiquitin proteasome pathway is an attractive model, but we cannot exclude a transcriptional mechanism responsible for the diminished CREB protein abundance. Loss of the transcriptional activity of CREB, as observed by Schwaninger et al. (53), could lead to down-regulation of the CREB promoter, which is known to be autoregulated (57). Furthermore, we cannot exclude destabilization of the CREB mRNA by cyclosporine, as demonstrated for cytokines like interleukin-3 (26). This regulation will be subject to further experiments.

CREB is a potential candidate for mediating the observed effects of cyclosporine on cell cycle regulation. CRE transcription factor decoy oligonucleotides are able to block growth of a broad spectrum of cancer cells (58). Park et al. (59) recently demonstrated an inhibition of cyclin D1 expression in MCF-7 cells upon treatment with a CRE decoy oligonucleotide. They found transcriptional down-regulation of cyclin D1 with consecutive decreased cyclin D/CDK4 activity and RB hypophosphorylation. These data suggest an important role for CREB in controlling cyclin D1 levels.

In summary, here we identify an important role for the CRE for cyclin D1 promoter activity in AR42J cells. CREB mediates basal promoter activity and confers cyclosporine sensitivity to the cyclin D1 promoter. These data might explain the proliferative defect of acinar cells induced by cyclosporine in the regenerative phase after acute pancreatitis. Furthermore, this pathway could be an attractive target to inhibit growth of certain kinds of cancer.

	ACKNOWLEDGEMENTS

We are indebted to Janet Köhler, Ester Rüber, und Rosi Rittelmann for excellent technical assistance. We thank Drs. Leopold Ludwig and Hana Algül for critically reading the manuscript and helpful discussion.

	FOOTNOTES

^* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB518 (to R. M. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Internal Medicine I, University of Ulm, Robert-Koch-Str. 8, D-89081 Ulm, Germany. E-mail: roland.schmid@medizin.uni-ulm.de.

Published, JBC Papers in Press, September 4, 2002, DOI 10.1074/jbc.M204787200

	ABBREVIATIONS

The abbreviations used are: CDK, cyclin-dependent kinase; CRE, cAMP-responsive element; CREB, cAMP-responsive element-binding protein; ATF, activating transcription factor; RB, retinoblastoma protein; FACS, fluorescence-activated cell sorting; AP-1, activator protein-1; NF-B, nuclear factor B; PI, propidium iodide; FITC, fluorescein isothiocyanate; TGF-, transforming growth factor ; CKI, cyclin-dependent kinase inhibitor; EMSA, electrophoretic mobility shift assay; PBS, phosphate-buffered saline.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				S. Periyasamy, M. Warrier, M. P. M. Tillekeratne, W. Shou, and E. R. Sanchez The Immunophilin Ligands Cyclosporin A and FK506 Suppress Prostate Cancer Cell Growth by Androgen Receptor-Dependent and -Independent Mechanisms Endocrinology, October 1, 2007; 148(10): 4716 - 4726. [Abstract] [Full Text] [PDF]

				M. Reichert, D. Saur, R. Hamacher, R. M. Schmid, and G. Schneider Phosphoinositide-3-Kinase Signaling Controls S-Phase Kinase-Associated Protein 2 Transcription via E2F1 in Pancreatic Ductal Adenocarcinoma Cells Cancer Res., May 1, 2007; 67(9): 4149 - 4156. [Abstract] [Full Text] [PDF]

				S. Semba and K. Huebner Protein Expression Profiling Identifies Cyclophilin A as a Molecular Target in Fhit-Mediated Tumor Suppression Mol. Cancer Res., August 1, 2006; 4(8): 529 - 538. [Abstract] [Full Text] [PDF]

				U. Haussler, G. von Wichert, R. M. Schmid, F. Keller, and G. Schneider Epidermal growth factor activates nuclear factor-{kappa}B in human proximal tubule cells Am J Physiol Renal Physiol, October 1, 2005; 289(4): F808 - F815. [Abstract] [Full Text] [PDF]

				V. Waters, S. Sokol, B. Reddy, G. Soong, J. Chun, and A. Prince The Effect of Cyclosporin A on Airway Cell Proinflammatory Signaling and Pneumonia Am. J. Respir. Cell Mol. Biol., August 1, 2005; 33(2): 138 - 144. [Abstract] [Full Text] [PDF]

				J. Crespo, J. Martinez-Gonzalez, J. Rius, and L. Badimon Simvastatin inhibits NOR-1 expression induced by hyperlipemia by interfering with CREB activation Cardiovasc Res, August 1, 2005; 67(2): 333 - 341. [Abstract] [Full Text] [PDF]

				J. M. Giger, F. Haddad, A. X. Qin, and K. M. Baldwin Effect of cyclosporin A treatment on the in vivo regulation of type I MHC gene expression J Appl Physiol, August 1, 2004; 97(2): 475 - 483. [Abstract] [Full Text] [PDF]

				J. Rius, J. Martinez-Gonzalez, J. Crespo, and L. Badimon Involvement of Neuron-Derived Orphan Receptor-1 (NOR-1) in LDL-Induced Mitogenic Stimulus in Vascular Smooth Muscle Cells: Role of CREB Arterioscler. Thromb. Vasc. Biol., April 1, 2004; 24(4): 697 - 702. [Abstract] [Full Text] [PDF]

				C. R. Kahl and A. R. Means Calcineurin Regulates Cyclin D1 Accumulation in Growth-stimulated Fibroblasts Mol. Biol. Cell, April 1, 2004; 15(4): 1833 - 1842. [Abstract] [Full Text] [PDF]

				J. M. Colomer, M. Terasawa, and A. R. Means Targeted Expression of Calmodulin Increases Ventricular Cardiomyocyte Proliferation and Deoxyribonucleic Acid Synthesis during Mouse Development Endocrinology, March 1, 2004; 145(3): 1356 - 1366. [Abstract] [Full Text] [PDF]

				C. R. Kahl and A. R. Means Regulation of Cell Cycle Progression by Calcium/Calmodulin-Dependent Pathways Endocr. Rev., December 1, 2003; 24(6): 719 - 736. [Abstract] [Full Text] [PDF]

				Y. Li, X. Li, and F. H. Sarkar Gene Expression Profiles of I3C- and DIM-Treated PC3 Human Prostate Cancer Cells Determined by cDNA Microarray Analysis J. Nutr., April 1, 2003; 133(4): 1011 - 1019. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 277/46/43599    most recent M204787200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Schneider, G. Articles by Schmid, R. M. Search for Related Content PubMed PubMed Citation Articles by Schneider, G. Articles by Schmid, R. M. Social Bookmarking                      What's this?