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Endocrinology, doi:10.1210/en.2005-1222
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Endocrinology Vol. 147, No. 6 2850-2863
Copyright © 2006 by The Endocrine Society

Identification of a Functional AP1 Element in the Rat Vasopressin Gene Promoter

Masanori Yoshida, Yasumasa Iwasaki, Masato Asai, Shinobu Takayasu, Takafumi Taguchi, Keiichi Itoi, Kozo Hashimoto and Yutaka Oiso

Department of Endocrinology, Toyota Memorial Hospital (M.Y.), Toyota 471-8513, Japan; Department of Endocrinology, Metabolism, and Nephrology, Kochi Medical School, Kochi University (Y.I., T.T., K.H.), Nankoku 783-8505, Japan; Departments of Endocrinology and Diabetes (M.Y., M.A., Y.O.) and Clinical Pathophysiology (S.T.), Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan; and Laboratory of Information Biology, Graduate School of Information Science, Tohoku University (K.I.), Sendai 980-8579, Japan

Address all correspondence and requests for reprints to: Dr. Masanori Yoshida, Department of Endocrinology, Toyota Memorial Hospital, 1-1 Heiwa-cho, Toyota 471-8513, Japan. E-mail: masanori_yoshida{at}mail.toyota.co.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Arginine vasopressin (AVP) is expressed in paraventricular, supraoptic, and suprachiasmatic nuclei of the hypothalamus, where transcription of the AVP gene is activated by various forms of stress, such as hyperosmolality, inflammation, and photic stimulation. In vasopressinergic neurons, the expression of the Fos/Jun family proteins is known to be rapidly induced after these stimuli as well. However, it is still unknown whether these proteins actually mediate AVP gene expression. In this study we examined in vitro the role of Fos/Jun protein in transcriptional regulation of the AVP gene using the BE(2)M17 neuroblastoma cell line. We found that 5'-promoter activity of the rat AVP gene (–803/+26) markedly increased when all combinations of the Fos/Jun family proteins were overexpressed. Coexpression of the cAMP-responsive element-binding protein-binding protein and steroid receptor coactivator-1a further enhanced the Fos/Jun-mediated transcription. Using site-directed mutagenesis and EMSA techniques, we identified an activation protein 1 (AP1)-like element (–134/–128; TGAATCA) in the AVP gene 5'-promoter region, which is the sole responsible site for the Fos/Jun-mediated transcription. We also found that 12-O-tetradecarbonyl phorbol 13-acetate stimulates AVP gene transcription partly via the AP1 site through the activation of ERK signaling. Together, these results suggest that a variety of Fos/Jun family member proteins stimulate transcription of the AVP gene through the AP1 site we identified. Furthermore, this effect may be activated by both protein kinase A and protein kinase C signaling pathways.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A NONAPEPTIDE HORMONE, arginine vasopressin (AVP), also known as an antidiuretic hormone, is distributed mainly in the supraoptic nuclei (SON), paraventricular nuclei (PVN), and suprachiasmatic nuclei (SCN) of the hypothalamus. Expression of the AVP gene in the SON and PVN is regulated by various forms of stress, such as hyperosmolality, inflammation, cytokines, and emotional stress (1). In the SCN, there is much evidence indicating that AVP is associated with circadian regulation (2). However, the precise molecular mechanism(s) responsible for the stress-induced regulation of AVP gene transcription is not entirely clear.

Fos/Jun family proteins are components of the transcription factor, activator protein 1 (AP1), which regulates the expression of multiple genes associated with cell growth, differentiation, and transformation (3, 4). In neuronal cells, the expression of these proteins is known to be induced rapidly in response to diverse extracellular stimuli as an immediate-early gene product. Indeed, c-Fos has been widely used as a marker of functional activity of neurons (5). To date, five Fos family proteins (c-Fos, Fra-1, Fra-2, FosB, and the natural truncated form {Delta}FosB) and three Jun family proteins (c-Jun, JunB, and JunD) have been identified. These proteins have a leucine zipper domain and adjacent basic regions (bZip), which are required for protein-protein interactions or binding to the target DNA sequences (6, 7). They bind to the specific sequence TGAC/GTCA (known as AP1) of the promoter region of the target genes by forming a homodimer (Jun/Jun) or heterodimer (Fos/Jun) at the portion of the Zip (8, 9). These homodimers or heterodimers of Fos/Jun proteins bind to the AP1 site with different affinities and effects (10). In contrast, Jun family members are known to bind to cAMP-responsive element (CRE) by forming homodimers or heterodimers with other bZip proteins, such as activating transcription factor (ATF) family proteins (11).

Previous studies have shown that in response to a variety of extracellular stimuli, Fos/Jun family proteins are rapidly expressed in vasopressinergic neurons in the hypothalamus, synchronizing with the activation of AVP gene expression (12, 13, 14). Indeed, the number of c-Fos-positive AVP neurons is reported to increase in response to stimuli (14). These results strongly suggest that Fos/Jun family proteins may mediate the rapid transcriptional induction of the AVP gene by extracellular stimuli, although a canonical AP1 site is not recognized in the 5'-promoter region of AVP (15).

The precise role of Fos/Jun family proteins in regulation of the AVP gene promoter has not been clarified, partly because of the lack of appropriate cell lines that endogenously express the AVP gene. Using bovine AVP gene promoter and fibroblast-derived CV-1 cells, Pardy et al. (16) showed the binding of c-Jun homodimer to a CRE, which is also known to be a binding site of CRE-binding protein (CREB)/ATF family transcription factors. More recently, Grace et al. (17) reported that in uterine cervix-derived HeLa cells, Fos family proteins bind to a noncanonical AP1 site of the rat AVP gene promoter, as determined by EMSA. The discrepancy may be derived from either species differences or differences in the cell lines used in each study. Furthermore, in the latter study no functional analysis of the putative AP1 site was carried out, and thus, the role of AP1 in transcriptional regulation of the AVP gene remains obscure.

In the present study we attempted to clarify whether Fos/Jun proteins mediate AVP transcription via the putative CRE element or another AP1-responsive element(s) using the BE(2)M17 neuroblastoma cell line (18), which endogenously expresses AVP mRNA. We identified a functional AP1 element in the 5'-promoter region of the rat AVP gene, which is almost solely responsible for c-Fos/c-Jun-mediated transcriptional activation. Our data also suggest the possible involvement of protein kinase C (PKC)-regulated signaling pathways in regulation of the AVP gene.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmid construction
cDNAs of all the mouse Fos/Jun family (c-Fos, Fra-1, Fra-2, c-FosB, {Delta}FosB, c-Jun, JunB, and JunD) expression vectors were cloned by RT-PCR using total RNA extracted from mouse AtT20 corticotroph cells and inserted into a pRc/cytomegalovirus expression vector (BD Clontech, Palo Alto, CA). Specific primers used for PCR were as follows: c-fos: forward, 5'-CCTCCAAGCTTGCCACCATGATGTTCTCGGGTTTCAACGG-3'; reverse, 5'-CTCGAGCGGCCGCTCACAGGGCCAGCAGCGTGGGTG-3'; c-jun: forward, 5'-CCTCCAAGCTTGCCACCATGACTGCAAAGATGGA-AACGAC-3'; reverse, 5'-CTCGAGCGGCCGCTCAAAACGTTTGCAACT-GCTGCG-3'; fra-1: forward, 5'-AAGCTTGCCACCATGTACCGAGACTACGGGGAACCG-3'; reverse, 5'-GCGGCCGCTCACAAAGCCAGGAG-TGTAGGAGAGC-3'; fra-2: forward, 5'-AAGCTTGCCACCATGTACCA-GGATTATCCCGGGAACTTTG-3'; reverse, 5'-GCGGCCGCTTACAGGG-CTAGAAGTGTGGGGGAG-3'; fosB and {Delta}fosB: forward 5'-TTGCGGCCG-CCAGGGAAATGTTTCAAGCTTTTCCC-3'; reverse, 5'-TTTCTAGAG-AGTTTACAGAGCAAGAAGGGA-3'; junB: forward, 5'-TTAAGCTTGCC-GCCCGGATGTGCACGAAAATGGAAC-3'; reverse, 5'-TTGCGGCCG-CGCTCTCAGAAGGCGTGTCCC-3'; and junD: forward, 5'-TTAAGC-TTGGAGGTGGGGATGGAAACGCCCTTCTATG-3'; reverse, 5'-TTGCG-GCCGCGGCTCAGTACGCCGGGACCTG-3'. The cAMP response element-binding protein-binding protein (CBP) expression vector (RSV/CBP) was provided by Dr. Toshihiro Nakashima (University of Tsukuba, Tsukuba, Japan). The steroid receptor coactivator-1a (SRC-1a) expression vector pcDNA3SRC-1a was provided by Prof. Shigeaki Kato (Tokyo University, Tokyo, Japan). A series of deletion mutant constructs of the rat AVP gene promoter (VP803, VP266, and VP36) was described previously (17). Two additional deletion mutants (VP170 and VP74) were constructed using the specific primers as follows: VP170: forward, 5'-TTTGGATC CTCTGCATTGACAGGCCCA-3'; reverse, 5'-TTTGGTACCGGCACTGCGTGCAGCTCT-3'; and VP74: forward, 5'-TATGGATCCGGGGGTTAGCAGCCACGC-3'; reverse, 5'-TTTGGTACCGGCACTGCGTGCAGCTCT-3'. Furthermore, two mutant constructs in which either AP1 or CRE was mutated (VP803/AP1m or VP803/CREm, respectively) were made from the VP803 by a site-directed mutagenesis technique using the primers as follows: VP803/AP1m: forward, 5'-GAGGGAGCTGC TGACAGCTTGGGAC-3'; reverse, 5'-CAGCAGCTCCCTCGGCATCTGGGGA-3'; and VP803/CREm: forward, 5'-CTCTGATGTGGGACCTGTCAGCTGT-3'; reverse, 5'-GTCCCACATCAGAGGCAGTGATTCAGGCAT-3'. c-fos/luciferase (Luc) and c-jun/Luc constructs were made by inserting the 5'-promoter region of mouse c-fos (–733/+77) and c-jun (–1192/+74) into pA3Luc using the following primers: c-fos/Luc: forward, 5'-GGTACCGCAGGAACAGTGCTAGTATTGC-3'; reverse, 5'-AAGCTTCACGGTCACTGCTCGTTCGC-3'; and c-jun/Luc: forward, 5'-GGTACCACACACTTTCGTCCTAAGGG-3'; reverse, 5'-AAGCTTGTGTCTGTCTGT-CTGCCTGAC-3'. The nucleotide sequences of the elements inserted in Cons/AP1/Luc, Cons/CRE/Luc, VP/AP1/Luc, and VP/CRE/Luc are shown in Fig. 5BGo. For making an expression vector for short interfering RNA (siRNA), the hairpin siRNA template oligonuleotide derived from the cDNA sequence of human c-fos was designed as follows: sense 5'-GAT- CCGCGGAGACAGACCAACTAGTTCAAGAGACTAGTTGGTCTGTC- TCCGCTTTTTTGGAAA-3', antisense 5'-AGCTTTTCCAAAAAAGCGG-AGACAGACCAACTAGTCTCTTGAACTAGTTGGTCTGTCTCCGCG-3'. Then the two oligonucleotides were annealed and ligated into pSilencer 2.0-U6 (Ambion, Austin, TX) according to the manufacturer’s protocol. This siRNA template corresponds to nucleotides 499–517 of human c-fos mRNA (+1 indicates the translation start site).


Figure 5
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  		FIG. 5. Analysis of the AP1-like element and CRE in the heterologous promoter. A, Comparison of the nucleotide sequences of the putative AP1-like element and CRE in the 5'-promoter of AVP genes (rat, mouse, bovine, and human). The AP1-like element was completely conserved among species. B, Schematic representation of the plasmids containing the tandem repeat of consensus AP1, consensus CRE, AP1-like element (VP/AP1), or CRE element (VP/CRE). C, Effects of c-Fos/c-Jun overexpression on the transcription activity of the heterologous promoters shown above. A marked stimulatory effect of Fos/Jun coexpression was observed on the promoters containing the consensus AP1 or VP/AP1. Data are expressed as the fold increase over the control group. *, P < 0.05 vs. corresponding control.

 
Cell culture and transfection
BE(2)M17 and Neuro2A cells were maintained in T75 culture flasks using DMEM/Ham’s F-12 culture medium (Sigma-Aldrich Corp., St. Louis, MO) supplemented with 10% (vol/vol) fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 µg/ml) at 37 C with 5% CO2. In each experiment, cells were plated in 3-cm diameter culture dishes, then transient transfection was carried out using FuGene 6 reagent (Roche, Indianapolis, IN; 3 µl FuGene 6/1 µg plasmid for each dish). When different doses of an expression plasmid were used in an experiment, the total amounts of transfected plasmid DNA were kept constant by the addition of empty control vectors. Twenty-four to 48 h after transfection, cells were harvested, and an Luc assay was performed as described previously (19).

Semiquantitative RT-PCR
Total RNA was extracted from BE(2)M17 cells with an acid guanidinium thiocyanate-phenol-chloroform method using Sepasol-RNA I super (Nacalai Tesque, Kyoto, Japan). First-strand cDNA was synthesized using 1 µg RNA each by reverse transcriptase (RevaTra Ace, Osaka, Japan) and then applied for PCR. The PCR protocol we used was as follows: 94 C for 2 min for initial denaturation, 10 cycles of amplification [97 C for 10 sec, 70–60 C (the temperature decreased by 1 C/cycle) for 1 min, 72 C for 1 min], followed by 23–33 cycles (depending on the experiment) of amplification (97 C for 10 sec, 60 C for 1 min, and 72 C for 1 min), and a final extension (72 C for 7 min). Primer sets used are as follows: human AVP: forward, 5'-ATGCCTGACACCATGCTGCC-3'; reverse, 5'-GCAGCGCCTCAGCCGTGCCC-3'; mouse AVP: forward, 5'-ATGCTCAACACTA CGCTCTCC-3'; reverse, 5'-GCGCCTCGGCGGTGCCCAC-3'; human CRH: forward, 5'- AGAAAGGAAGACAACCTCCAG-3'; reverse, 5'-AGCAGCTGCTGCAGCAACAC-3'; human glyceraldehyde-3-phosphate dehydrogenase (GAPDH): forward, 5'-TGATGACATCAAG AAGGTGGTGAAG-3'; reverse, 5'-TCCTTGGAGGCCATGTGGGCCAT-3'; mouse GAPDH: forward, 5'-TGCACCACCAACTGCTTAG-3'; reverse, 5'-GGATGCAGGGATGATG-TTC-3'; and c-fos: forward, 5'-CGCCTGTCAACGCGCAGGAC-3'; reverse, 5'-TCTTCTTCTGGAGATAACTG-3'. The RT-PCR products of AVP mRNA were confirmed by nucleotide sequencing (3730 DNA analyzer, Applied Biosystems, Foster City, CA).

EMSA
BE(2)M17 cells were plated in 6-cm diameter plates and transfected with c-Fos/c-Jun (1.5 µg each) or pRc/cytomegalovirus (3.0 µg), then nuclear cell extracts were obtained using nuclear and cytoplasmic extraction reagents (NE-PER, Pierce Chemical Co., Rockford, IL). Protein concentration was determined using a bicinchoninic acid protein assay kit (Pierce Chemical Co.). The protein amount of each lane was constant in each experiment. Biotin-labeled or unlabeled probes containing the AP1 element of the rat AVP promoter region, altered AP1 element, or consensus AP1 sequence were synthesized as shown below and annealed. A gel mobility shift assay was carried out using a LightShift chemiluminescent EMSA kit (Pierce Chemical Co.). For supershift assay, 8 µl c-Fos antibody (Santa Cruz Biotechnology, Inc., catalog no. sc-52) was added to the nuclear extract 1 h before the binding reaction at 4 C. Binding reactions were loaded onto an 8 x 8 x 0.1-cm 6% polyacrylamide gel in 0.5x Tris-buffered EDTA (TBE) buffer and electrophoresed at 100 V at 4 C for 2 h. Biotin-labeled, double-stranded DNA was transferred to positively charged nylon membrane (Hybond-N; Amersham Pharmacia Biotech, Little Chalfont, UK) using the capillary method. For competition experiments, a 600-fold excess of unlabeled competitor oligonucleotide containing the consensus AP1 sequence was included in the reaction with biotin-labeled probe and incubated simultaneously with the nuclear extracts of BE(2)M17 cells. Biotin-labeled DNA were integrated with streptavidin-horseradish peroxidase conjugate. Finally, chemiluminescence was detected using Light Capture (LAS-3000 Luminoimage Analyzer, FujiFilm, Minnamiashigara, Japan). The probes and competitor were as follows: AP1VP: forward, 5'-AGATGCCTGAATCACTGCTGA-3' (biotin labeled); reverse, 5'-TCAGCAGTGATTCAGGCATCT-3'; AP1cons: forward, 5'-CGCTTGATGACTCAGCCGGAA-3' (biotin labeled and unlabeled); reverse, 5'-TTCCGGCT-GAGTCATCAAGCG-3'; and AP1mVP: forward, 5'-AGATGCCGAGGGAGCTGCTGA-3' (biotin labeled); reverse, 5'-TCAGCAGCTCCCTCGGCATCT-3'.

Statistics
Samples in each group of experiments were made in triplicate. All experiments were performed more than twice to confirm the reproducibility, and representative data are shown in Results. Data are expressed as the mean ± SE. The differences between experimental values were analyzed by Student’s t test. Statistical analysis was performed by one-way ANOVA. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Protein kinase A (PKA)- and PKC-mediated transcription of the AVP gene in homologous BE(2)M17 neuronal cells
In this study we used human neuroblastoma-derived BE(2)M17 cells, which are known to express endogenous CRH mRNA (18). We first confirmed the presence of AVP as well as CRH mRNAs in BE(2)M17 cells by RT-PCR analysis using intron-spanning primer sets (Fig. 1AGo). The expression of both mRNAs was also recognized in the differentiated cells with retinoic acid treatment (10 µM; 72 h). We verified that the nucleotide sequence of the PCR products exactly matched that of human AVP mRNA. Semiquantitative RT-PCR showed that the density of the band correlated with the number of PCR cycles, whereas no amplification was obtained using RNA from another neuroblastoma cell line, Neuro2A (Fig. 1BGo). Together, these data suggest that BE(2)M17 is an appropriate cell line for studying transcriptional regulation of the AVP gene.


Figure 1
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  		FIG. 1. cAMP/PKA- and PKC-dependent transcription of the AVP gene. A, Analysis of endogenous expression of AVP and CRH mRNAs. Their expression was confirmed by RT-PCR in the BE(2)M17 neuroblastoma cell line. In differentiated cells treated with retinoic acid (10 µM; 72 h), their expression was also recognized. B, Semiquantitative RT-PCR in BE(2)M17 and Neuro2A cell lines. The cycle-dependent (37–43 cycles) amplification of AVP mRNA was observed in BE(2)M17 cells, but not in Neuro2A cells. GAPDH mRNA was used as an internal control. C, Effects of PKA/PKC stimulation on AVP gene expression. Fsk (10 µM, 5 h) and TPA (100 nM, 12 h) significantly stimulated the 5'-promoter activity of the AVP gene (VP803). These effects were abolished by the PKA inhibitor, H89 (10 µM), and the PKC inhibitor, RO320432 (1 µM), respectively. BG, Background. D and E, Effects of PKA/PKC stimulation on c-Fos/c-Jun gene expression. Fsk (10 µM; 5 h) and TPA (100 nM; 12 h) also stimulated the 5'-promoter activity of the c-fos (–733/+77), but not the c-jun (–1192/+74), gene. F and G, The time-course effects of Fsk (10 µM) and TPA (100 nM) on endogenous expression of c-fos mRNA in BE(2)M17 cells by RT-PCR. The PCR cycle number was 35 (Fsk) or 33 (TPA). GAPDH mRNA was used as an internal control. Data are expressed as the fold increase over the untreated control group. *, P < 0.05 vs. corresponding control; #, P < 0.05 vs. TPA or Fsk alone.

 
The cAMP/PKA pathway has been shown to regulate AVP gene transcription in the heterologous cell line (19, 20, 21), whereas the involvement of PKC is currently controversial (22, 23). Using BE(2)M17 cells, we first tested whether cAMP/PKA and/or PKC signal transduction pathways are involved in transcriptional regulation of the AVP gene. We found that the 5'-promoter activity of the AVP gene (–803/+26; VP803) was significantly enhanced by treatment with both forskolin (Fsk) and the phorbol ester, 12-O-tetradecarbonyl phorbol 13-acetate (TPA). Induction by the PKA or PKC activator was completely abolished by the PKA inhibitor, H89 (10 µM), or by the classical PKC inhibitor, RO320432 (1 µM), respectively (Fig. 1CGo). These results suggest that PKC-mediated, as well as PKA-mediated, signaling pathways contribute to transcriptional activation of the AVP gene.

Previous studies have shown that TPA is a potent activator of AP1 (24). Fos/Jun family proteins, components of AP1, are known to be induced by various extracellular stimuli. Furthermore, activation of the cAMP/PKA pathway stimulates the expression of Fos/Jun proteins (25). Because a variety of forms of stress activate the Fos/Jun expression in vasopressinergic neurons, we assumed that these proteins might activate the transcriptional activity of the AVP gene via both PKA and PKC signaling pathways, and we tested the transcriptional activity of Fos and Jun genes under the activators of these kinases. The results showed that the transcriptional activity of the c-fos gene (–733/+77) was significantly enhanced by both Fsk and TPA treatments, but that of the c-jun promoter (–1192/+74) was not changed (Fig. 1Go, D and E). We also analyzed by RT-PCR the time-course effect of endogenous c-fos expression in BE(2)M17 cells after Fsk or TPA treatment. As expected, the level of c-fos mRNA was increased by both Fsk and TPA treatments (Fig. 1Go, F and G). Thus, we confirmed that in BE(2)M17 cells, the expression of c-fos was indeed induced by the activation of both cAMP/PKA and PKC signaling pathways.

Fos/Jun potently stimulates AVP gene transcription
We then focused on the role of Fos/Jun family proteins in transcriptional regulation of the AVP gene. We found that coexpression of Fos and/or Jun family proteins markedly enhanced transcriptional activity of the AVP gene (VP803). More precisely, the expression of c-Jun and JunB alone, but not Fos proteins alone, produced a 3- to 4-fold increase (Fig. 2AGo), and combined expression of Fos/Jun showed more robust effects (10- to 70-fold increase; Fig. 2BGo). These results suggest that the immediate-early genes play an important role in transcriptional induction of the AVP gene. Interestingly, AVP responsiveness to the coexpression of Fos/Jun differed among the various patterns of combination. c-Fos, not alone, but with Jun family proteins, induced AVP gene promoter activity more potently than other Fos-related members. Among the Jun family, JunD had the smallest effect. In contrast, {Delta}FosB, a natural truncated form of FosB (26), had a minimal enhancing effect compared with FosB when coexpressed with c-Jun (Fig. 2CGo). In addition, the combined effects of c-Fos and c-Jun proteins were dose dependent: AVP promoter activity gradually increased in proportion to the increasing doses of c-Fos (Fig. 2DGo) or c-Jun (Fig. 2EGo) expression. Together, these results strongly suggest that both c-Fos and c-Jun proteins positively regulate AVP promoter activity in a dose-dependent manner.


Figure 2
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  		FIG. 2. Effects of the expression of Fos/Jun family proteins on AVP gene expression. A, Effects of Fos/Jun proteins alone. The 5'-promoter activity of the AVP gene (VP803) was significantly enhanced by the coexpression of Jun family proteins (c-Jun and JunB), but not by Fos family proteins (c-Fos, Fra-1, Fra-2, and FosB). B, Effects of combined expression of Fos/Jun proteins. The 5'-promoter activity of the AVP gene (VP803) was markedly enhanced by any combination of Fos/Jun family proteins. The most prominent effect (>70-fold) was observed when c-Fos and JunB were coexpressed. C, Effect of coexpression of {Delta}FosB, an endogenous dominant negative form of FosB. The combined effect of {Delta}FosB/c-Jun on the 5'-promoter activity of the AVP gene was much less than that of FosB/c-Jun. D and E, Dose-dependency of the combined effects of Fos/Jun proteins. The 5'-promoter activity of the AVP gene (VP803) was significantly stimulated by increasing the dosage of either c-Fos or c-Jun (the ratio of DNA dosage ranged between 0 and 3.0). The total amounts of the transfected plasmid DNA were kept constant by the addition of empty control vectors. Data are expressed as the fold increase over the control group. *, P < 0.05 vs. corresponding control; #, P < 0.05 vs. FosB/c-Jun-transfected group.

 
Mapping of Fos/Jun-responsive elements in the 5'-promoter region of the AVP gene
To identify the AP1-responsive element in the AVP gene promoter, we analyzed the effect of c-Fos/c-Jun expression using five serial deletion mutant constructs. The basal promoter activity of each deletion mutant is shown in Fig. 3AGo (left panel). In VP803, VP266, or VP170, marked stimulatory effects (40- to 50-fold) were observed with c-Fos/c-Jun coexpression (Fig. 3AGo, right panel). This effect, however, was almost completely lost in the shorter constructs (VP74 and VP36), indicating that the region between –170 and –74 bp of the AVP gene 5'-promoter is important for the c-Fos/c-Jun-mediated effects. In this region, we found one AP1-like element (TGAATCA, –134/–128), in which only one nucleotide (underlined) was different from the canonical AP1 consensus motif (TGAC/GTCA; Fig. 3BGo). A putative CRE and two AP2 sites were also located (15).


Figure 3
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  		FIG. 3. Mapping of the Fos/Jun response element in the 5'-promoter of the AVP gene. A, Deletion analysis using various lengths of AVP 5'-promoter-luciferase constructs. The basal promoter activity of each deletion mutant (left panel) and the stimulatory effects of overexpression of c-Fos/c-Jun (right panel) are shown. The stimulatory effect of the combined expression of c-Fos/c-Jun was markedly blunted in VP74 or the shorter construct in BE(2)M17 cells. B, The nucleotide sequences of the rat AVP gene 5'-promoter (–200/–1). One putative AP1-like element as well as a previously identified CRE are boxed. C, Mutagenesis analysis. Two mutated constructs of the VP803, in which either the putative AP1-like element or CRE was altered (shown in the upper panel), were tested as described in B. The stimulatory effect of the combined expression of c-Fos/c-Jun was almost completely abolished only when the putative AP1-like element was mutated. Similar effects were obtained using another neuroblastoma cell line, Neuro2A. Data are expressed as the fold increase over the control group. *, P < 0.05 vs. corresponding control; #, P < 0.05 vs. VP803- and c-Fos/c-Jun-transfected group.

 
To help clarify the roles of these elements, we used mutated constructs in which the sequences of the AP1-like and CRE-like elements were altered by site-directed mutagenesis (VP803/AP1m and VP803/CREm, respectively; Fig. 3CGo). When the AP1-like element was eliminated (VP803/AP1m), the stimulatory effect by c-Fos/c-Jun coexpression was almost completely lost. In contrast, elimination of CRE (VP803/CREm) did not affect the c-Fos/c-Jun-mediated induction. These effects suggest that the AP1-like sequence (–134/–128), but not CRE (–123/–116), is the functional AP1 element introducing the c-Fos/c-Jun-mediated effect in the 5'-promoter of the AVP gene.

Binding of AP1 protein on the AP1-like element identified within the 5'-promoter region of the AVP gene
To confirm that AP1 proteins indeed bind the AP1-like element, we performed EMSA with oligonucleotides containing the putative AP1-like element or consensus AP1 element, using nuclear extracts obtained from BE(2)M17 cells. When c-Fos/c-Jun proteins were overexpressed, the DNA-protein complex was clearly recognized using probes containing the putative (AP1VP) or consensus (AP1cons) AP1 motif (Fig. 4AGo). The band formation was completely eliminated by the addition of unlabeled consensus AP1 oligonucleotide. Furthermore, we observed supershift of the band with c-Fos antibody (Fig 4BGo), suggesting the specificity of AP1 binding. Quantitative analysis showed that the density of the band corresponding to the DNA-AP1 complex significantly increased (1.7 ± 0.2-fold; n = 3) in c-Fos/c-Jun-overexpressed cells compared with that in untreated cells. A representative image of this is shown in Fig. 4CGo. In addition, the DNA-protein complex was not observed using the biotin-labeled oligonucleotides in which the AP1 element was mutated (Fig. 4CGo).


Figure 4
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  		FIG. 4. EMSA. A, BE(2)M17 cells were transfected with c-Fos/c-Jun expression vectors. Then the nuclear protein was extracted and applied for EMSA using biotin-labeled probes containing the AP1-like element in the AVP gene promoter (AP1VP; lane 1) or the consensus AP1 sequence (AP1cons; lane 2). An excess amount (600-fold) of unlabeled consensus AP1 probe was used as a competitor (lane 3). B, Supershift analysis. In the presence of a specific c-Fos antibody, the AP1 complex was supershifted. C, BE(2)M17 cells were transfected with mock (lane 1) or c-Fos/c-Jun (lanes 2–4) expression vectors. Then the nuclear extracts were applied for EMSA using the biotin-labeled AP1VP (lanes 1–3) or mutated oligonucleotide in which the AP1-like element was altered (AP1mVP; lane 4). An excess amount (600-fold) of unlabeled AP1cons probe was also used as a competitor (lane 3).

 
The nucleotide sequence of the AP1-like element was highly conserved among the species examined (rat, mouse, bovine, and human; Fig. 5AGo). We next tested, using a heterologous promoter (PathDetect cis-reporting system), whether the AP1-like element was responsible for the c-Fos/c-Jun protein-induced effect (Fig. 5BGo). When a reporter plasmid, containing consensus AP1 (7xAP1) or CRE (4xCRE) was cointroduced with the c-Fos/c-Jun expression vector, a marked transcriptional induction was observed only with AP1-containing reporter (Fig. 5CGo, left). We then performed a similar experiment using reporter plasmids containing two tandem repeats of the AP1- or CRE-like elements in the AVP gene and found a response only in the former construct (Fig. 5CGo, right). Similar results were obtained in Neuro2A cells (data not shown). Together, these results indicate that the AP1-like element we identified is functional and mediates c-Fos/c-Jun-induced AVP gene transcription.

CBP and SRC-1 enhance AVP gene transcription through AP1
CREB-binding protein (CBP) is identified as a cofactor for a number of transcriptional factors, including CREB and AP1 (27). To clarify the possible involvement of CBP in AVP gene expression, we examined the effect of CBP overexpression on transcriptional activity of the AVP gene. We found that CBP did not enhance basal transcriptional activity, but significantly enhanced c-Fos/c-Jun-induced transcriptional activity (Fig. 6AGo). This effect was also observed in the heterologous promoter containing only the AP1 element in the AVP promoter (Fig. 6BGo).


Figure 6
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  		FIG. 6. Roles of CBP and SRC-1a in the c-Fos/c-Jun-mediated transcription activity of the AVP gene. A and B, The effect of CBP coexpression. The expression of CBP did not influence basal, but significantly enhanced c-Fos/c-Jun-mediated, transcription of VP803 (A) or VP/AP1(B). C and D, The effect of SRC-1a coexpression. Enhancing effects of SRC-1a similar to those of CBP were observed for VP803 (C) and VP/AP1 (D). Data are expressed as the fold increase vs. the control (vehicle) group. *, P < 0.05 vs. corresponding control; #, P < 0.05 vs. c-Fos/c-Jun-transfected group.

 
We also examined the role of SRC-1 in the AP1-mediated transcription of the AVP gene. When SRC-1a, a major isoform of SRCs in the hypothalamus (28), was overexpressed, an amplifying effect, similar to that of CBP on the whole AVP gene promoter (VP803)- or AP1 element-dependent transcription, was obtained (Fig. 6Go, C and D). Together, these data suggest that CBP and SRC-1a are involved in AVP gene regulation in such a way that they do not influence basal transcription activity, but are acting as coactivators of AP1-induced AVP gene expression.

PKC regulates AVP promoter activity via the AP1 element through the ERK signaling pathway
We also examined whether the PKC signaling pathway is involved in c-Fos/c-Jun-induced transcriptional activation of AVP gene. The 5'-promoter region of the c-fos gene contains the serum response element (SRE), which is known to be an essential element for TPA-induced transcription (29). Previous reports show that activation of the -fos gene by PKC is elicited through the MAPK, in particular, the ERK, signaling pathway (30). It may also be possible that other MAPKs, such as p38 or c-Jun N-terminal protein kinase (JNK), are involved in PKC-induced activation of the c-fos gene promoter (31). We found that TPA-induced transcriptional activity of the AVP gene was abolished completely by the classical PKC inhibitor (RO302432) and also partly blocked by p42/44 MAPK kinase (MEK) inhibitor (PD98059), but not by p38 MAPK inhibitor (SB203580) or JNK inhibitor (SP600125) (Fig. 7AGo). In contrast, Fsk-induced AVP gene transcription was significantly suppressed by H89, but not by other MAPK or PKC inhibitors (Fig. 7BGo).


Figure 7
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  		FIG. 7. Effects of various MAPK inhibitors on TPA-induced AVP gene expression. A, Effects on AVP promoter. TPA (100 nM; 12 h) significantly enhanced the 5'-promoter activity of the AVP gene (VP803), which was abolished by a PKC inhibitor (RO320432; 1 µM) or was significantly diminished by an ERK inhibitor (PD98059; 20 µM). B, Fsk (10 µM; 5 h) significantly enhanced the 5'-promoter activity of the AVP gene (VP803), which was abolished by PKA inhibitor (H89; 1 µM), but not by other inhibitors. C and D, Effects on the AP1 element in AVP promoter or on c-fos gene promoter. Effects similar to those in A were observed on the isolated AP1 element in a heterologous promoter (PD basic) or on a c-fos gene 5'-promoter. In all cases, treatment of the cells with the p38 MAPK inhibitor, SB203580 (10 µM), had enhancing effects, whereas the JNK inhibitor. SP600125 (10 µM). had no effect on the promoter activity of each gene. Data are expressed as the fold increase vs. the control (vehicle) group. *, P < 0.05 vs. control; #, P < 0.05 vs. TPA or Fsk alone.

 
We then examined whether the ERK/AP1 signal transduction cascade is involved in TPA-induced transcriptional activation of AVP gene. The transcriptional activity of the heterologous promoter containing the AP1 element in the AVP gene (VP/AP1/Luc) was also induced by TPA treatment. This induction was markedly eliminated by PD98059, but not by SP600125 (Fig. 7CGo), and was enhanced by SB203580. Similar effects were obtained using c-fos/Luc plasmid (Fig. 7DGo). These data indicate that the classical PKC-induced transcription activity of AVP gene is mediated, at least partially, via the ERK-dependent pathway.

To confirm the role of MAPK, we examined the effect of overexpression of MEK1, an activator of ERK, on AP1-induced AVP gene transcription. Coexpression of MEK1 significantly stimulated the transcriptional activity of the AP1 element containing heterologous promoter (VP/AP1/Luc) as well as a c-fos gene promoter in a dose-dependent manner (Fig. 8AGo). Likewise, MEK1 overexpression stimulated the transcriptional activity of VP803 (Fig. 8BGo). TPA-stimulated transcription activity was significantly (~50%) attenuated by elimination of the AP1 element (VP74) compared with VP170 containing this element (Fig. 8CGo). A similar result was obtained in VP803/AP1m-transfected, compared with VP803-transfected, cells (Fig. 8DGo). Together, our results suggest that PKC plays an important role in the induction of AVP gene expression, at least partly via the AP1 site through the ERK signaling pathway.


Figure 8
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  		FIG. 8. Role of the AP1 element in ERK/TPA-mediated AVP gene expression. A and B, Effects of MEK coexpression. Overexpression of MEK significantly enhanced the transcriptional activity of the reporter plasmids containing the isolated AP1 element in the AVP gene (VP/AP1) or the c-fos gene promoter in a dose-dependent manner (A). A similar effect was observed on the wild-type AVP gene promoter (VP803; B). C and D, Effects of TPA on the promoter activity of the AVP gene with/without the AP1 element. The stimulatory effect of TPA (100 nM) on the transcription activity of the AVP gene without the AP1 element was much less than that on the gene containing the AP1 element (C, VP803 vs. VP74; D, VP803 vs. VP803/AP1m). E, Effect of TPA on the formation of the DNA-AP1 complex. BE(2)M17 cells were treated with or without TPA (100 nM; 12 h). The nuclear extracts were applied for EMSA using biotin-labeled AP1VP (lanes 1–3). An excess amount (600-fold) of unlabeled AP1cons probe was used as a competitor (lane 3). F, Quantification by EMSA of AP1 binding levels after TPA stimulation. G, Effect of c-fos siRNA on the TPA-induced promoter activity of the AVP gene. Overexpression of the c-fos siRNA expression vector significantly suppressed the stimulatory effect of TPA (100 nM; 12 h) on the transcription activity of the AVP gene compared with control plasmid. Data are expressed as the fold increase vs. the control (vehicle) group. *, P < 0.05 vs. corresponding control; #, P < 0.05 vs. TPA-treated or VP170- or VP803-transfected group (C and D, respectively), or the pSilencer 2.0-U6-transfected control group (G).

 
We also examined by EMSA whether TPA-stimulated transcription activity is induced via the AP1 element. We found that after TPA treatment, the density of the bands corresponding to the DNA-AP1 complex was significantly increased. Similar to the manner demonstrated in Fig. 4Go, band formation was completely eliminated by the addition of cold competitor (Fig. 8EGo). Quantitative analysis of the density of the bands showed that AP1 binding after TPA stimulation was about 2-fold greater than that of untreated control cells (Fig. 8FGo). Moreover, introduction of the c-fos siRNA expression vector significantly suppressed the stimulatory effect of TPA on transcriptional activity of the AVP gene compared with the control vector (Fig. 8GGo). Together, these data strongly suggest that PKC activates the endogenous AP1 proteins and subsequently stimulates transcription of the AVP gene through the AP1 site (–134/–128).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we examined the role of the Fos/Jun family transcription factors in the transcriptional regulation of the AVP gene in a homologous BE(2)M17 neuroblastoma cell line. Using deletion analysis and the site-directed mutagenesis technique as well as heterologous promoter constructs, we identified one AP1 element (–134/–128) that is almost comprehensively responsible for the marked induction of AVP gene 5'-promoter activity by Fos/Jun family protein. EMSA verified the direct binding of the c-Fos/c-Jun complex to this AP1 site. Furthermore, the PKC-mediated pathway is also involved in regulation of the AVP gene, at least partly though the AP1 site. Together, our data strongly suggest that the AP1 (–134/–128) motif we identified is one of the major regulatory elements, through which AP1 protein induced by a variety of stress elicits transcriptional activation of the AVP gene.

A differential expression pattern (both spatial and temporal) of Fos/Jun family proteins in the hypothalamus was observed depending on the type of stress (32, 33, 34, 35, 36). Regarding osmotic stress, Luckman et al. (37) extensively studied the expression of Fos/Jun family genes in the SON and PVN after various degrees of acute osmotic stimuli and observed an increase in c-fos, c-jun, and junB mRNAs in vasopressinergic neurons. Sustained expression of Fos family proteins was also reported during chronic hypertonic saline treatment (38). In SCN in which light stimulation is the major regulator of the AVP gene, the expression of c-Fos, Fra-2, and FosB was induced by light pulses, some of which were colocalized with AVP-positive neurons (12). These results indicate that Fos/Jun family proteins play an important role in transcriptional activation of the AVP gene regardless of the type of stress. However, there is no direct evidence indicating that Fos/Jun family proteins can induce the promoter activity of the AVP gene.

In in vitro studies, there are some discrepancies regarding the role of Fos/Jun family proteins in AVP gene expression. Pardy et al. (16) suggested the binding of c-Jun homodimer not to the AP1, but to the CRE, using footprint and chloramphenicol transferase assay in CV-1 cells. In contrast, Grace et al. (17) reported the binding of Fos family proteins using EMSA in HeLa cells to the AP1 element, which corresponds to the element we found in BE(2)M17 cells. However, in that study the binding was influenced by the length of the probe used, and no functional analyses was carried out. Furthermore, nonneuronal cells were used in both reports, and thus, the functional role of the AP1 site in AVP gene transcription in neuronal cells remains unclear. We clarified that the AP1 site (–134/–128) is the sole and an important responsive site for activation of AVP gene transcription by almost all combinations of Fos/Jun or Jun/Jun dimers in BE(2)M17 cells.

We screened a number of neuronal cell lines, including BE(2)M17 cells, which are reported to express CRH mRNA (18). We found that this cell line endogenously expresses AVP as well as CRH. The mRNA level estimated by semiquantitative RT-PCR, however, was much lower than that of GAPDH, possibly due to a lower copy number of AVP mRNA or to low amplification efficiency of PCR because of the extremely high GC-rich content of the AVP gene product. We also failed to determine immunoreactive AVP in culture medium by RIA, probably because of the low expression level or the lack of expression of processing enzymes required for making mature AVP. Nevertheless, BE(2)M17 is the only available cell line to date expressing endogenous AVP, and thus, we assume that it is the most appropriate as host cells for the analysis of AVP gene transcription in the neuronal milieu.

In this study we indeed recognized that overexpression of Fos/Jun family genes potently enhances AVP gene transcription. Interestingly, a different combination of Fos/Jun family proteins causes differing responsiveness of AVP promoter activity, which may reflect the diverse responses of AVP gene expression after a variety of extracellular stimuli in vivo. Among the proteins, those of the Jun family (c-Jun and JunB) showed a significant stimulatory effect (up to 4-fold), whereas the Fos family protein alone had no effect. This is consistent with the previous finding that Jun, but not Fos, family proteins can form a homodimer. More obviously, coexpression of Fos/Jun proteins dramatically enhanced (~70-fold) the transcriptional activity of the AVP gene. Because the Fos/Jun heterodimer and the Jun/Jun homodimer serve as AP1 transcription factors, these results strongly suggest that AP1 is involved in transcriptional activation of the AVP gene.

We identified the AP1-like element in the 5'-promoter region of the AVP gene that is responsible for the c-Fos/c-Jun-mediated gene induction. When the element was isolated and incorporated into a heterologous promoter, remarkable transcriptional induction (>200-fold increase) by c-Fos/c-Jun was observed. Moreover, deletion or mutation of this element caused a dramatic decrease in the c-Fos/c-Jun-induced effect. Thus, the identified AP1 site is almost solely responsible for c-Fos/c-Jun-mediated transcriptional induction. EMSA also supports the idea that the c-Fos/c-Jun complex directly binds to this AP1 element. A similar effect was observed in another neuronal cell line, Neuro2A, indicating that our results are not a cell line-specific event.

In contrast to the above findings, the effect of c-Fos/c-Jun protein expression on the CRE element (–123/–116), located only 5 bp downstream of the AP1 element, was minimal. Although CRE-mediated transcription is usually mediated by CREB, other bZip transcription factors, such as c-Jun/c-Jun homodimer or c-Jun/ATF family heterodimer, are reported to bind and activate the transcription, because of the close similarity of palindromic consensus sequences between CRE (TGACGTCA) and AP1 (TGAC/GTCA). However, our current data suggest that at least in the case of the AVP gene, the CRE does not appear to be the alternative site of AP1 action. Instead, the CRE may be acting as a binding site of CREB, mediating the different signaling inputs through the cAMP/PKA pathway (19).

In this study we found that CBP and SRC-1, two representative cofactors of various transcription factors, act as a coactivator of Fos/Jun-dependent AVP gene transcription as well. CBP is known to be ubiquitously expressed and acts as a general coactivator (27). Another coactivator, SRC-1, has been shown to be highly expressed in the brain, including the hippocampus, piriform cortex, amygdala, anterior pituitary, cerebellar Purkinje cells, and hypothalamus (28), and acts in cooperation with a variety of nuclear receptors along with the recruitment of histone acetyltransferase (39). Moreover, SRC-1 interacts with other transcription factors, such as AP1 (40) or nuclear factor-{kappa}B (41). In the hypothalamus, abundant expression of the SRC-1a mRNA isoform is identified in SON, both magnocellular and parvocellular parts of PVN, and SCN (28), where AVP is expressed. Therefore, we hypothesized that SRC-1 is involved in AVP gene expression through AP1. Our current data clearly show the involvement of these cofactors in AP1-mediated transcription of the AVP gene and thus suggest that they act as an enhancer of stress-induced AVP gene expression.

It is still controversial whether PKC activation stimulates AVP gene promoter activity, although it is generally accepted that transcription of the AVP gene is positively regulated via the cAMP/PKA pathway (19, 20, 21). Several reports showed that activation of PKC increases AVP mRNA in fetal hypothalamic cultures (22, 42, 43), whereas other reports did not observe this (23). In this study, using a homologous cell line, we obtained evidence suggesting that PKC stimulates AVP gene transcription by c-Fos/c-Jun induction through the ERK (p42/44 MAPK) pathway. In these cells, TPA, an activator of classical PKC, potently enhanced c-fos expression and AVP gene expression, both of which are significantly eliminated by ERK inhibitor. The c-fos promoter region includes the CRE, SRE, and the sis-inducible element, conferring the responsiveness to a wide variety of stimuli via several intracellular signaling pathways, including cAMP/PKA, PKC, calcium-dependent, and MAPK-dependent signaling pathways (44, 45, 46). In particular, the SRE element is an important region of PKC-induced transcription of the c-fos gene, mediated through activation of the MEK-ERK signal cascade by TPA treatment (33). Our results are in accordance with the above findings. In addition, we found that p38 MAPK inhibitor enhanced AVP gene promoter activity under TPA-stimulated conditions, suggesting the suppressive role of the signaling pathway. Furthermore, we confirmed by EMSA that AP1-binding activity increased in TPA-treated cells. Conversely, the TPA-stimulated transcription activity was significantly suppressed by the knockdown of c-fos mRNA using siRNA. Our results suggest that the PKC-ERK-c-Fos/c-Jun pathway is another signal transduction pathway, in addition to cAMP/PKA, in the regulation of AVP gene transcription. The presence of this signaling cascade is also supported by the finding that the effect of TPA is significantly diminished after elimination of the AP1 element in the AVP gene promoter. However, there remains a substantial stimulatory effect of TPA in this experimental condition, suggesting the presence of a PKC-dependent, but AP1-independent, pathway(s). In this case, TPA may activate CRE-dependent transcription by ERK-mediated CREB phosphorylation.

In conclusion, our results suggest strongly that the immediate-early gene products, Fos/Jun family proteins, act as potent inducers of AVP gene transcription. In this process, the PKC-ERK-c-Fos/c-Jun signaling pathway may play an important role in AVP gene expression, at least partly via the AP1 site we identified (Fig. 9Go). Thus, we assume that transcriptional regulation of the AVP gene is regulated in a complicated manner, including both cAMP/PKA and PKC/MAPK pathways, probably depending on the type of signaling input from the other area of the brain. Additional studies of an experimental animal model using a paradigm employing a variety of forms stress is absolutely necessary to clarify the precise role of each Fos/Jun family protein in the transcription of AVP mRNA in vivo.


Figure 9
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  		FIG. 9. Proposed model of intracellular signaling cascades regulating AVP gene transcription in neuronal cells. A variety of signaling pathways are involved in transcriptional regulation of the AVP gene. The cAMP/PKA pathway phosphorylates CREB, resulting in up-regulation of c-fos and AVP genes via the CRE(s). The phospholipase C (PLC)/PKC pathway induces the expression of the c-fos gene via the MEK-ERK pathway and subsequently stimulates AVP gene expression through the AP1 element we identified. Other Fos/Jun or Jun/Jun dimers may be involved in the regulation as well. Cofactors, such as CBP and SRC-1a, enhance AP1-mediated AVP gene expression. Overall, we assume that Fos/Jun family proteins play important roles in stress-induced transcriptional regulation of the AVP gene.

 


   Acknowledgments
 
We thank Prof. Shigeaki Kato for providing the SRC-1a expression vector. We also thank Dr. Toshihiro Nakashima (Tsukuba University, Tsukuba, Japan) for providing the CBP expression vector.


   Footnotes
 
All authors (M.Y., Y.I., M.A., S.T., T.T., K.I., K.H., and Y.O.) have nothing to declare.

First Published Online March 16, 2006

Abbreviations: AP1, Activator protein 1; bZip, basic leucine zipper domain; CBP, CREB-binding protein; CRE, cAMP-responsive element; CREB, cAMP-responsive element-binding protein; Fsk, forskolin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; JNK, c-Jun N-terminal protein kinase; Luc, luciferase; mCRE, mutated CRE; MEK, MAPK kinase; PRA, protein kinase A; PKC, protein kinase C; PVN, paraventricular nucleus; SON, supraoptic nucleus; SRC-1a, steroid receptor coactivator-1a; SRE, serum response element; TPA, 12-O-tetradecarbonyl phorbol 13-acetate.

Received September 23, 2005.

Accepted for publication March 8, 2006.


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 Introduction
 Materials and Methods
 Results
 Discussion
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