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Endocrinology Vol. 144, No. 2 534-543
Copyright © 2003 by The Endocrine Society

ARTICLE

12-Lipoxygenase Pathway Increases Aldosterone Production, 3',5'-Cyclic Adenosine Monophosphate Response Element-Binding Protein Phosphorylation, and p38 Mitogen-Activated Protein Kinase Activation in H295R Human Adrenocortical Cells

Jiali Gu, Yeshao Wen, Angeles Mison and Jerry L. Nadler

Division of Endocrinology, Department of Internal Medicine, University of Virginia, Charlottesville, Virginia 22908

Address all correspondence and requests for reprints to: Jerry L. Nadler, Division of Endocrinology, Department of Internal Medicine, University of Virginia, P.O. Box 801405, Charlottesville, Virginia 22908. E-mail: jln2n{at}virginia.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Evidence suggests that the 12-lipoxygenase (LO) pathway mediates angiotensin II (Ang II)-induced aldosterone synthesis in adrenal glomerulosa cells. To study the mechanisms of 12-LO pathway on aldosterone synthesis, the human adrenocortical cell line, H295R, was transiently transfected with a mouse leukocyte type of 12-LO. Overexpression of 12-LO stimulated aldosterone production 2.7-fold as well as the reporter gene activity of CYP11B2 gene-encoding human aldosterone synthase by 5-fold over that in mock-transfected cells. Ang II further enhanced aldosterone production, which could be blocked by a 12-LO inhibitor, baicalein, in mock cells and cells overexpressing 12-LO. Ang II stimulated cAMP response element-binding protein (CREB) phosphorylation in a dose- and time-dependent fashion in parent H295R cells. Overexpression of 12-LO increased phosphorylation of CREB/activating transcription factor (ATF)-1 1.5-fold over that in mock cells under basal conditions. Ang II led to a further 5.2- and 7.5-fold increase in mock cells and 12-LO cells, respectively. Overexpression of 12-LO induced p38 MAPK activation. The 12-LO product, 12-hydroxyeicosatetraenoic acid, increased phosphorylation of CREB/ATF-1 3.6-fold and phosphorylation of p38 MAPK 8-fold over basal. The p38 MAPK inhibitor SB203580 inhibited Ang II- and 12-LO pathway-induced phosphorylated CREB/ATF-1, suggesting a role of p38 MAPK in Ang II and 12-LO pathway signaling. These results suggest that 12-LO stimulation leads to aldosterone production in H295R cells in part through activation of CREB/ATF-1 and p38 MAPK pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ALDOSTERONE SYNTHASE converts deoxycorticosterone to aldosterone and mediates the final step(s) in aldosterone synthesis (1, 2, 3, 4, 5). The regulation of aldosterone synthase is a key factor in aldosterone production (6). The gene-encoding human aldosterone synthase, CYP11B2, is found in human adrenal glomerulosa cells and expressed within the zona glomerulosa of the adrenal cortex (4). The expression level of aldosterone synthase is controlled at the level of gene transcription. The 5'-flanking region of CYP11B2 has been analyzed in detail. A consensus cAMP response element (CRE) at position -74/-64 plays a critical role in transcriptional regulation (7) of CYP11B2.

Angiotensin II (Ang II) is the major regulator of aldosterone synthesis in the adrenal (6, 8). Ang II can stimulate increase aldosterone synthase mRNA in human primary adrenal zona glomerulosa cells (4). Human adrenocortical H295R cells express the Ang II type 1 receptor (9). Ang II stimulation of these cells results in an increase in aldosterone production (10) and aldosterone synthase mRNA levels (11). The regulatory effect of Ang II on aldosterone production has been shown to be via the activation of CYP11B2 transcription through common cis-elements and maximal induction requires CRE site in the CYP11B2 promoter (7).

Three major isoforms of 12-lipoxygenase (LO), platelet, leukocyte, and epidermal types, have been described in mammals (12, 13). The leukocyte-type enzyme is expressed in a variety of cells (14, 15, 16). Our previous results show that the leukocyte-type 12-LO is expressed in cultured human glomerulosa cells and is up-regulated by Ang II stimulation (17). Evidence also suggests that the 12-LO pathway in part mediates Ang II-stimulated aldosterone production in human and rat adrenal glomerulosa cells (18, 19). Despite these studies, the molecular mechanism and function of 12-LO in the adrenal has not yet been well characterized. The objective of this investigation was to elucidate the mechanism of how 12-LO can regulate aldosterone production. We first examined whether 12-LO stimulates aldosterone production and transcriptional activation of the CYP11B2 promoter. We next assessed whether Ang II and 12-LO stimulates the phosphorylation of the nuclear transcription factor, CRE-binding protein (CREB), and whether MAPK activation plays a role in this response. The results demonstrate that 12-LO overexpression stimulates aldosterone syntheses as well as CYP11B2 transcriptional activity, whereas LO inhibition blocks these actions. Ang II concentration and time dependently increases CREB serine 133 phosphorylation and further enhances 12-LO induced CREB phosphorylation. These effects are associated with p38 MAPK activation. These findings support the role of 12-LO in regulating aldosterone synthesis in part via CREB-dependent CYP11B2 gene expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids and purification
The 12-LO/pcDNAI vector containing the full-length (2.0 kb) mouse leukocyte 12-LO cDNA under the control of the cytomegalovirus promoter was a gift from Dr. Colin Funk (University of Pennsylvania, Philadelphia, PA). The empty vector pcDNAI was purchased from Invitrogen (Carlsbad, CA) and used as mock transfection of 12-LO cDNA. Luciferase reporter gene construct pB2–1521 plasmid containing -1521 to +2 bp of the human CYP11B2 promoter sequence was cloned into the pGL3-basic luciferase reporter plasmid (Promega Corp., Madison, WI) in Dr. Rainey’s lab. The pRSV-LacZ plasmid expressing ß-galactosidase as internal reference plasmid was purchased from Stratagene (La Jolla, CA). All plasmids for transfection were purified by EndoFree plasmid mega kit (QIAGEN Inc., Valencia, CA) following the manufacturer’s protocol.

Cell culture and transient transfection
Human adrenocortical (H295R) cells provided by Dr. Rainey’s lab were cultured in DMEM/Ham’s F12, 1:1 medium (Life Technologies, Inc., Grand Island, NY) supplemented with 2% Ultroser G (BioSepra SA, Villeneuve la Garenne, France), 1% insulin-transferrin-sodium selenite Plus (BD Biosciences, Bedford, MA) and antibiotics as previously described (7). Transient transfection was carried out using DOSPER liposomal transfection reagent (Roche, Indianapolis, IN) following the manufacturer’s instruction. Cells were plated to 30–40% confluency and used 48 h later. Cells were preincubated for 1 h with Opti-MEMI reduced serum medium (Life Technologies, Inc.), and then the transfection mixture containing 80 µg DOSPER and 20 µg plasmid DNA for 100-mm plates was added and incubated for 6 h. The volumes and amount of transfection mixture were adjusted proportionally for using 6- or 12-well plates. In cotransfection experiments, pB2-1521 plasmid or its control plasmid pGL3 as well as 12-LO/pcDNAI or its empty pcDNAI plasmids were added. The pRSV-lacZ vector was also cotransfected to normalize for varying transfection efficiencies. The amount of DNA per transfection was kept constant.

After transfection, cells were incubated in growth medium for overnight to allow recovery. Cells were serum starved for 24 h in medium containing 0.1% BSA and preincubated for 1 h in fresh serum-free medium. Cells were then treated with Ang II or other agents for the times indicated. In some experiments the cells were pretreated with inhibitors before Ang II treatment. Stock solutions of pharmacological inhibitors such as baicalein or SB203580 (Biomol, Plymouth Meeting, PA) were prepared in dimethylsulfoxide (DMSO) at a concentration of 1000-fold so that when they were added to the culture medium, the concentration of DMSO was less than 0.1%.

Measurement of 12-hydroxyeicosatetraenoic acid (HETE)
Transient transfectants were preincubated with fresh media for 1 h and treated with Ang II for 4–24 h. Cells were rinsed twice with PBS, and cell pellets were collected and lysed. Lysates were extracted and deacelylated as described before. The 12-HETE levels were quantitated by a specific RIA as described earlier (18).

Measurement of aldosterone
Transient transfectants were treated with Ang II for 24 h; in some experiments they were preincubated with inhibitor for 2 h before Ang II treatment. At the end of incubation, a duplicate 0.2-ml aliquot of supernatant was removed for the measurement of aldosterone using a Coat-A-Count aldosterone kit (Diagnostic Products, Los Angeles, CA), a solid-phase 125I RIA.

Amplification of reverse transcribed RNA using RT-PCR and blot hybridization
The method was used as described previously (4) with some modifications. The sequences of oligonucleotides for PCR primers or probes were the same as described before (4, 17). Total RNA from cells was extracted using Trizol and treated with deoxyribonuclease 1. A 1-µg aliquot of each RNA sample was reverse transcribed in a 20-µl reaction using 2 U SuperScript RNaseH- reverse transcriptase along with 0.25 µM oligo(deoxythymidine) primer and 10 mM of each deoxynucleotide triphosphate. These reagents were purchased from Invitrogen. A 1-µl aliquot of each sample was mixed with PCR buffer containing the primers and 2.5 U AmpliTaq gold polymerase (Roche, Indianapolis, IN). Samples were amplified for 29 cycles. The conditions used in PCR were a denaturation step at 94 C for 30 sec, annealing at 60 C for 30 sec, and extension at 72 C for 30 sec. The PCR products were analyzed on 1.8% agarose gels and, after staining with ethidium bromide and photography, transferred to nylon membranes (Schleicher & Schuell, Keene, NH). The oligonucleotides used as probes were labeled with 32P using T4 polynucleotide kinase (Invitrogen) and hybridized with the membrane. Blots were washed at 60 C for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe and 65 C for CYP11B2 probe. Negatives of ethidium bromide-stained gels and autoradiograms were scanned.

Luciferase activity and ß-galactosidase enzyme activity assays
Transient transfectants were treated with Ang II for 6 h. At the end of incubation, the cells were rinsed twice with PBS and lysed with lysis buffer (Promega Corp.). Luciferase activity of the cell lysates was measured by using the Luciferase assay system (Promega Corp.) following the protocol provided by manufacturer. The luciferase reaction was measured for 15 sec at room temperature using TD-20/20 luminometer (Turner Design, Sunnyvale, CA). Luciferase values were corrected for transfection efficiency with the ß-galactosidase activity assayed by ß- galactosidase enzyme assay kit (Promega Corp.) in a 96-well plate and the absorbance of samples was read at 420 nm in a plate reader. Luciferase activities were expressed as a fold increase of the basal activity obtained for mock (without 12-LO cDNA) transfection.

Preparation of whole cell lysates and immunoblotting assay
After treatments cells were immediately kept on ice, washed twice with cold PBS, and lysed with 250 µl lysis buffer containing 100 mM Tris-HCl (pH 6.8), 10% sodium dodecyl sulfate, and 100 mM dithiothreitol. The lysate was heated at 95–100 C for 5 min, cooled down on ice, and then sonicated for 2 sec followed by microcentrifugation for 5 min. Immunoblotting was performed per the manufacturer’s instructions (Cell Signaling Technology, Inc., Beverly, MA) with some modification. The blot was incubated with primary antibodies against phosphorylated form of CREB or MAPK. The same blot was stripped and reprobed with an antibody to the corresponding nonphosphorylated forms of CREB or kinase proteins. ERK and c-Jun N-terminal kinase (JNK) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). CREB and p38 MAPK antibodies were purchased from Cell Signaling Technology, Inc. 12-LO polyclonal antibody against porcine leukocyte 12-LO was described as before (17). To detect the first antibody of the blot, the antirabbit IgG-horseradish peroxidase and the enhanced chemiluminescence system was used (Amersham Pharmacia Biotech, Piscataway, NJ). Quantitation was performed by densitometry using an imaging analyzer personal densitometer SI (Molecular Dynamics, Inc., Sunnyvale, CA).

Nuclear extractions and CREB transcription factor assay
Alternatively, phosphorylated-CREB (pCREB) was quantitated using the pCREB/CREB transcription factor assay kit developed by Active Motif Co. (Carlsbad, CA; Ref. 20). This is an ELISA-based assay. In this assay an oligonucleotide containing the CREB consensus-binding site (CRE) was immobilized to a 96-well plate. The active form of CREB in the cell nuclear extract could specifically bind to this CRE oligonucleotide and be detected by incubation with the pCREB/CREB antibody and the anti-IgG-horseradish peroxidase conjugate. A developing solution was then added for a colorimetric readout. In this study cellular nuclear extractions were prepared following the manufacturer’s instruction, and 10–20 µg nuclear protein was used and quantified according to the manufacturer’s protocol. To monitor the specificity, the assay of samples was performed in presence of an excess of the consensus wild-type or mutated CRE oligonucleotides in reactions as a competitor for CREB binding.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Overexpression of 12-LO in H295R cells
The human adrenocortical H295R cells were transiently transfected with 12-LO cDNA-pcDNA1 vector or empty pcDNA1 vector. 12-LO expression was examined by immunoblot analysis. A high level of expression of 12-LO was seen in cells transfected with 12-LO cDNA vector (12-LO cells) (Fig. 1Go, upper panel). In contrast, the expression of 12-LO was undetectable under this condition in cells transfected with empty vector (mock cells), indicating a low level of 12-LO expression in wild-type H295R cells. The 12-LO enzymatic product 12-HETE was also examined. In mock cells, there was 514 ± 63 pg/107 cells 12-HETE formation under basal conditions and a 1.4-, 1.8-, and 2.5-fold increase after 0.1 µM Ang II treatment for 4, 8, and 24 h over that under the basal condition (P < 0.001, respectively, lower panel). The formation of 12-HETE in cells overexpressing 12-LO under basal conditions was 17-fold (8851 ± 946 pg/107 cells) greater than that in mock cells. And there was a 14.4-, 13.9-, and 10.6-fold increase after 0.1 µM Ang II treatment for 4, 8, and 24 h in cells overexpressing 12-LO, compared with that in mock cells, respectively (P = 0.001). The results demonstrate that 12-LO overexpression dramatically increased 12-HETE formation in H295R cells, compared with mock cells, and Ang II stimulated the enzymatic activity in both mock and 12-LO cells.



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  		Figure 1. Overexpression of 12-LO in H295R cells. H295R cells seeded in 100-mm plates were transiently transfected with empty vector pcDNA1 (mock cells) or vector containing 12-LO cDNA-pcDNA1 (12-LO cells) as described in Materials and Methods. After recovery, cells were serum depleted for 24 h and preincubated for 1 h with fresh serum-depleted media before being treated with 0.1 µM Ang II for 4, 8, or 24 h. Cells were harvested and 12-LO protein expression levels and cell-associated 12-HETE formation was evaluated in mock and 12-LO-overexpressing cells using Western blot (upper panel) and RIA (lower panel), respectively. Values of bars represent the mean ± SE of triplicate determination in three similar experiments. *, P = 0.001, compared with basal in mock cells. **, P = 0.001, compared with that of in mock cells, respectively.

 
Overexpression of 12-LO increased aldosterone production
Next, the effect of 12-LO overexpression on the aldosterone production was examined. The 12-LO overexpression was confirmed by immunoblot analysis (Fig. 2Go, upper panel). Aldosterone production was compared between cells overexpressing 12-LO and mock cells under basal condition as well as after treatment with Ang II for 24 h (Fig. 2Go, lower panel). Aldosterone production was increased 2.7-fold in cells overexpressing 12-LO, compared with that seen in mock cells (P < 0.001). Ang II further increased aldosterone production 1.62-fold in 12-LO-overexpressing cells (P < 0.01). These results provide additional support that 12-LO plays an important role in aldosterone synthesis. The effect of baicalein, an inhibitor of 12-LO, on aldosterone production was next examined in mock cells and 12-LO-overexpressing cells. Figure 3Go shows that Ang II-induced aldosterone production was inhibited by baicalein in a concentration-dependent way in mock cells. Baicalein (1 µM and 10 µM) resulted in a 20% and 35% decrease in Ang II-induced aldosterone production, respectively (P = 0.03). Baicalein did not alter basal aldosterone production in mock cells (data not shown). Similar results were observed in cells overexpressing 12-LO in which 10 µM baicalein resulted in a 42% decrease in aldosterone production (P = 0.05).



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  		Figure 2. Effect of 12-LO overexpression on aldosterone production. The H295R cells seeded in two 6-well plates were transiently transfected with pcDNA1 (mock cells) or 12-LO cDNA-pcDNA1 (12-LO cells) as described in Materials and Methods. After recovery, cells were serum depleted for 24 h and then treated with 0.1 µM Ang II in 0.5 ml media for 24 h in triplicate wells for a group. Supernatant was collected from each well and processed for aldosterone measurement. Cells were lysed from each well for total protein measurement and Western blotting analysis. Upper panel shows an example for the expression of 12-LO protein in each wells of mock and 12-LO cells from one experiment. The bars in lower panel show the amounts of secreted aldosterone in each well normalized to the amount of cellular protein (means ± SE) from four experiments with triplicate in a group in each experiment. *, P < 0.001, compared with the basal in mock cells; **, P < 0.01, compared with the basal in 12-LO cells.

 


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  		Figure 3. Effect of baicalein on Ang II-induced aldosterone production in mock and 12-LO cells. Experiments were performed as described in Fig. 2Go except that cells were treated for 24 h with 1 or 10 µM baicalein alone (12-LO cells) or along with 1 nM Ang II (in mock cells). Controls received vehicle, DMSO solvent. The value for the untreated in mock cells was taken as 1. The results are means ± SE from three independent experiments each performed in triplicate. *, P < 0.002, compared with the basal in mock cells; **, P < 0.05, compared with the "a" in mock cells and "b" in 12-LO cells, respectively.

 
To validate the effect of baicalein on aldosterone production in H295R cells, the aldosterone synthase CYP11B2 mRNA levels were examined. Because of the high homology between CYP11B1 and CYP11B2 genes, we used specific CYP11B2 cDNA oligonucleotide probe in RT-PCR assay as we described previously (4). The expression of GAPDH taken as a control housekeeping gene was also determined in the same experiments. As shown in Fig. 4Go, the predicted size of PCR product fragment was amplified, 332 bp for CYP11B2 and 284 bp for GAPDH. Ang II strongly induced CYP11B2 mRNA expression. This is consistent with the observation from the other group (11). Treatment with baicalein resulted in a 50% inhibition of Ang II-induced CYP11B2 mRNA level.



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  		Figure 4. Analysis of CYP11B2 mRNA levels under baicalein treatment using RT-PCR. The H295R cells were treated for 24 h with 0.1 µM Ang II alone or combination of 10 µM baicalein. Total RNA was extracted from these cells and amplified with primers specific for the CYP11B2 or GAPDH gene transcripts. Upper panels are ethidium bromide-stained agarose gels, whereas the lower panels are autoradiograms of blots hybridized with 32P-labeled CYP11B2 or GAPDH probes for the indicated gene. Molecular weight marker was a 100-bp DNA ladder. Quantitation of the bands was done by scanning densitometry.

 
Overexpression of 12-LO increased CYP11B2-luciferase activity
To explore whether 12-LO induces aldosterone production by affecting aldosterone synthase expression, the CYP11B2 reporter gene activity in 12-LO-overexpressing cells and mock cells was measured. The CYP11B2-luciferase reporter construct containing 1521 bp of promoter sequence of CYP11B2 gene (pB2–1521) was transiently cotransfected with 12-LO cDNA or empty vector into H295R cells. Figure 5Go shows luciferase activity in these transfected cells in the presence or absence of Ang II (0.1 µM) for 6 h. The results show that Ang II increased luciferase activity, compared with the basal condition in mock-transfected cells. Cells overexpressing 12-LO had a similar increase in luciferase activity in the absence of Ang II to that of the Ang II-induced activity in mock-transfected cells. Luciferase activity was increased 5-fold in 12-LO-overexpressing cells (P = 0.005). Ang II treatment further enhanced the luciferase activity in the cells overexpressing 12-LO. No effect of basic plasmid pGL3 on luciferase activity was seen at Ang II absent or present in mock and 12-LO cells. The 12-LO overexpression was demonstrated with immunoblot analysis (Fig. 5Go, upper panel). These results indicate that 12-LO can activate CYP11B2 gene transcription.



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  		Figure 5. Human CYP11B2 promoter activity in mock and 12-LO cells. The 295R cells seeded in 12-well plates were transiently cotransfected with pB2–1521 or pGL3; 12LO-pcDNA1 or pcDNA 1 vector; and pRSV-ß-galactosidase. After recovery, cells were incubated in serum-free medium for 24 h and then treated with 0.1 µM Ang II for 6 h. At the end of the incubation, cell lysates were prepared, and transcription was measured by assaying the luciferase activity. The 12-LO protein expression levels in each well in mock cells (transfected with pcDNA1) and 12-LO cells (transfected with 12-LO cDNA) were measured by Western blotting (upper panel). The lower panel shows luciferase activity and is expressed as fold increase over the basal transcription in mock cells. In these lysates, ß-galactosidase activity was also assayed to normalize for efficiency of transfection. The results are mean ± SE for seven independent experiments, each performed in triplicate. *, P < 0.01.

 
Ang II and overexpression of 12-LO increases CREB/activating transcription factor (ATF)-1 phosphorylation
Previous studies have shown that CREB/ATF-1 sites are important in CYP11B2 promoter activity. We therefore explored the role of 12-LO in this pathway. Dose- and time-dependent phosphorylation of CREB at Ser133 by Ang II in parent H295R cells are shown in Fig. 6Go. The nuclear transcription factor CREB was examined with a CRE oligomer immobilized plate, as described in Materials and Methods. CREB was phosphorylated in a time-dependent manner in response to Ang II. There was a 9 ± 1-fold (P < 0.001) increase in CREB phosphorylation at serine 133 at 15 min when H295R cells were stimulated with 0.01 µM Ang II (Fig. 6AGo). The phosphorylation remained elevated for at least 120 min. The dose-response curve showed a significant increase in CREB phosphorylation at 0.001 µM Ang II (P < 0.01) with highest increase at 0.01 µM concentration at the 15-min time point (Fig. 6BGo). The effect of overexpression of 12-LO on CREB phosphorylation was also examined in the presence or absence of Ang II by immunoblot analysis with a CREB phosphorylationspecific antibody (Fig. 7Go). Overexpression of 12-LO showed a significant 1.5-fold (P = 0.02) increase in CREB phosphorylation. There were 5.2- and 7.5-fold increase in CREB phosphorylation, respectively, when mock and 12-LO cells were stimulated with 0.1 µM Ang II (P = 0.001). Total CREB protein was also examined with anti-CREB antibody. We observed an additional band with the antibody specific for the phosphorylation sequence around serine 133 of CREB. Serine 63 phosphorylation of ATF-1 is known to be detected by the same antibody as serine 133-phosphorylated CREB because they are 100% homologous for this consensus phosphorylation sequence (21). The phosphorylation pattern of ATF-1 was parallel to that of CREB in terms of intensity (Fig. 7Go). These results suggest that 12-LO activates the phosphorylation of CREB/ATF-1 and Ang II further increases CREB/ATF-1 phosphorylation. The data support the hypothesis that CREB plays a role in the regulation of the CYP11B2 gene by Ang II and may mediate, in part, in 12-LO effects in H295R cells.



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  		Figure 6. Dose- and time-dependent CREB phosphorylation mediated by Ang II. The H295R cells were cultured in 100-mm dishes to near confluence and then maintained in serum-free medium for 24 h. They were treated with 0.01 µM Ang II for varying periods of time, from 5 to 120 min (A). In another set of experiments, the depleted cells were exposed to increasing concentrations of Ang II for 15 min (B). The cells in both experiments were washed in ice-cold PBS at the end of the incubation period, and the cell nuclear fraction was prepared. The samples containing equal amounts of 20 µg nuclear protein were applied for analysis of CREB phosphorylation with pCREB/CREB transcription factor kit as described in Materials and Methods. The value for the basal was taken as 1. The results are the means ± SE of three observations.

 


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  		Figure 7. Activation of CREB by Ang II and 12-LO. The H295R cells were transfected and treated as described in Fig. 1Go except that the cells were treated with 0.1 µM Ang II for 2 h. At the end of the incubation period, the cell lysates were prepared, and equal amounts of proteins were electrophoresed and immunoblotted with antibody specific for pCREB at Ser 133. The membranes were then stripped and reprobed with CREB antibody. The results shown in the upper panel are representative of three experiments. Quantitation of the bands was done by scanning densitometry. The ratio of pCREB over the nonphosphorylated form was calculated, and the value for the basal of mock cells was taken as 1. The results are the mean ± SE of three independent experiments. * and **, P < 0.01 vs. basal in mock cells; ***, P < 0.01 vs. basal in 12-LO cells.

 
Effect of overexpression of 12-LO and Ang II on ERK 1/2, JNK/SAPK, and p38 MAPK activity
To evaluate the potential signaling mechanisms of Ang II and 12-LO action in H295R cells, experiments were carried out to quantitate phospho-ERK 1/2, JNK, and p38 MAPKs, the active forms of these enzymes. Immunoblotting with phospho-ERK and phospho-JNK/stress-activated protein kinase (SAPK) antibodies showed that Ang II led to a potent 7-fold (P < 0.001) increase in ERK 1/2 activity at 5 min in mock cells (data not shown). This is consistent with the observation in H295R cells from other groups (Refs. 21A and 21B ). However, overexpression of 12-LO did not further activate Ang II-induced ERK 1/2, compared with that in the mock cells (data not shown). Ang II and 12-LO overexpression had no effect on phosphorylation of JNK/SAPK (data not shown).

To characterize the role of p38 MAPK in Ang II-stimulated wild-type H295R cells, p38 MAPK phosphorylation was measured. As shown in Fig. 8AGo, a 2.7-fold (P < 0.01) increase p38 MAPK phosphorylation was seen at 0.001 µM Ang II at the 5-min time and with a peak at 0.1 µM Ang II. Ang II treatment also resulted in time-dependent activation of p38 MAPK. There was a 3.9-fold (P < 0.001) increase in p38 phosphorylation at 5 min with 0.1 µM Ang II and a slow decline over the remaining 180 min (Fig. 8BGo). Effect of 12-LO on the activity of the p38 MAPK was next examined using the mock cells and the cells overexpressing 12-LO (Fig. 9Go). The 12-LO overexpression activated p38 MAPK phosphorylation 1.3-fold (P < 0.001), compared with mock cells in the absence of Ang II. Total amount of p38 MAPK had no change when the same blot was stripped and reprobed with antibody to nonphospho-p38 MAPK.



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  		Figure 8. Dose- and time-dependent p38 MAPK phosphorylation mediated by Ang II. The H295R cells were cultured in 100-mm dishes to near 90% confluence. Cells were then serum depleted for 24 h and treated with increasing concentrations of Ang II, from 0.001 µM to 1 µM for 5 min (A). In another set of experiments, the depleted cells were exposed to 0.1 µM Ang II for varying periods of time, from 5 to 180 min (B). The cells in both experiments were washed in ice-cold PBS at the end of incubation period, and the cell lysates were prepared and harvested for immunoblotting with antibody specific to phospho-p38 MAPK. The membranes were then stripped and reprobed with the antibody to p38 MAPK. The results shown in upper panel are representative of three experiments. Quantitation of the bands was done by scanning densitometry. The value for basal was taken as 1. The results are the means ± SE of three observations.

 


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  		Figure 9. Activation of p38 MAPK by 12-LO. Cells were transiently transfected with pcDNA1 or 12-LO pcDNA1. Cell lysates were immunoblotted with antibodies to phospho-p38 MAPK. The membranes were then stripped and reprobed with the antibodies to p38 MAPK. Quantitation of the bands was done by scanning densitometry. The ratio of intensity in phospho- to nonphospho-p38 MAPK bands was calculated as the bar graph. Values are means ± SE from three experiments. *, P < 0.001.

 
These results demonstrate that 12-LO and Ang II is a potent inducer of the p38 MAPK activity in H295R cells. 12-LO overexpression also activates the p38 MAPK activity but not ERK 1/2 or JNK in H295R cells in the same conditions.

12(S)-HETE increases CYP11B2 luciferase activity, CREB, and p38 MAPK phosphorylation
We then checked the effect of 12-LO metabolism product, 12(S)-HETE, on CYP11B2 luciferase activity, CREB, and p38 MAPK phosphorylation by adding 12(S)-HETE into incubation media of H295R cells. The results shown in Fig. 10Go indicate that 0.1 µM 12(S)-HETE increases CYP11B2 luciferase activity 2.5-fold. No effect of 15(S)-HETE was seen. Dose- and time-dependent phosphorylation of CREB by 12(S)-HETE was examined with CRE oligomer immobilized plate in Fig. 11Go. The dose-response curve showed a 3.6-fold increase in CREB phosphorylation with 0.1 µM 12(S)-HETE treatment for 30 min. CREB was phosphorylated in a time-dependent manner in response to 12(S)-HETE. The phosphorylation returned to basal at 4 h (data not shown). The time cause for p38 MAPK phosphorylation by 12(S)-HETE was measured in Fig. 12Go. There was an 8-fold increase in p38 phosphorylation with treatment of 0.1 µM 12(S)-HETE for 5 min, and then it declined to basal at 20 min.



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  		Figure 10. Effect of 12-HETE on CYP11B2 promoter gene activity in H295R cells. Experiments for the luciferase activity were performed similar to described in Fig. 5Go except that H295R cells were transiently cotransfected with pB2-1521 and pRSV-ß-galactosidase. The treatments were with vehicle ethanol, 0.1 µM Ang II, or 0.1 µM 12(S)-HETE, or 0.1 µM 15(S)-HETE. The results are mean ± SE from three independent experiments, each performed in triplicate. *, P < 0.05 vs. control.

 


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  		Figure 11. Effect of 12-HETE on dose- and time-dependent CREB phosphorylation. Experiments for the CREB phosphorylation were performed similar to that described in Fig. 6Go with a pCREB/CREB transcription factor kit except that treatments were with 12-HETE (0–0.1 µM) for varying periods of time. The results are the means ± SE of three experiments. *, P < 0.05 vs. control.

 


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  		Figure 12. Effect of 12-HETE on p38 MAPK activity. Experiments were performed similar to that described in Fig. 8Go except that cells were stimulated with 0.1 µM 12-HETE for indicated times. The bar graph below shows quantitation of the bands by scanning densitometry. The results are the means ± SE from two experiments.

 
Role of p38 MAPK pathway to stimulate CREB phosphorylation, CYP11B2 reporter gene activity, and aldosterone synthesis in H295R cells
To explore whether the phospho-p38 MAP kinase-signaling pathway is involved in downstream CREB phosphorylation, the effect of the pharmacological p38 MAPK inhibitor SB 203580 (22, 23) on Ang II and 12-LO pathway-induced CREB/ATF-1 phosphorylation was examined. As shown in Fig. 13AGo, 12-LO increased CREB phosphorylation 2.1-fold over basal in mock cells (P = 0.001). Ang II led to a CREB phosphorylation increase of 6.7- and 8.5-fold over basal in mock cells and 12-LO cells, respectively (P < 0.01). These results are similar to those shown in Fig. 7Go. Furthermore, preincubation of mock and 12-LO cells with the inhibitor SB 203580 (10 µM) followed by incubation with 0.1 µM Ang II resulted in a 50% decrease CREB phosphorylation (P < 0.02). SB203580 alone did not have significant effect in mock cells; however, it significantly reduced CREB phosphorylation in 12-LO-overexpressing cells (P < 0.02).



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  		Figure 13. Effect of p38 MAPK inhibitor, SB203580, on Ang II and 12-LO pathway-induced CREB phosphorylation. A, Experiments were performed as described in Fig. 6Go except that cells were treated without (basal) or with 0.1 µM Ang II, 10 µM SB203580, or combinations. B, Experiments were performed as described in Fig. 12Go except that H295R cells were treated with 12-HETE alone or in combination with SB203580. The value for the basal was taken as 1. The results are the means ± SE from three experiments. *, P < 0.02 vs. a; **, P < 0.02 vs. b; ***, P < 0.02 vs. c; +, P < 0.01 vs. d.

 
In addition, the effect of SB203580 on 12-HETE induced CREB phosphorylation was also examined (Fig. 13BGo). The treatment with 5 and 10 µM SB203580 resulted in a 50% and 80% inhibition of 12-HETE induced CREB phosphorylation in H295R cells.

We then checked the effect of SB203580 on CYP11B2 report gene activity and aldosterone production in 12-LO cells. Figure 14Go shows that treatment with SB203580 resulted in a 80% inhibition of CYP11B2 luciferase activity and a 40% inhibition of aldosterone production in 12-LO cells. The effect of SB203580 on CYP11B2 mRNA levels in 12-LO cells was also examined and is shown in Fig. 15Go. Again, treatment with Ang II strongly induced CYP11B2 mRNA expression; addition of SB203580 resulted in an 80% inhibition of CYP11B2 mRNA expression at basal and Ang II treatment.



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  		Figure 14. Effect of p38 MAPK inhibitor, SB203580, on CYP11B2 luciferase activity and aldosterone production in12-LO cells. Experiments were performed as described in Figs. 2Go and 5Go except that 12-LO cells were treated without or with 10 µM SB203580. The results are the mean ± SE from three to four experiments, each performed in triplicate. * and **, P < 0.05 vs. control.

 


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  		Figure 15. Analysis of CYP11B2 mRNA levels under SB203580 treatment in 12-LO cells using RT-PCR. The 12-LO cells were treated for 24 h with 0.1 µM Ang II, 10 µM SB203580, or combinations. RT-PCR was performed as described in Fig. 4Go.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of the current study provide evidence for several molecular mechanisms of how increased 12-LO activity could increase aldosterone production in human adrenal cells. The data suggest a pathway for nuclear signaling of Ang II and 12-LO in H295R cells that involves the p38 MAPK pathway and CREB/ATF-1 phosphorylation.

The leukocyte-type 12-LO has been shown to be present in particular cell types including pancreatic islets, adrenal gland, and pituitary gland (24, 25, 17), suggesting that the enzyme may play a role in hormonal secretion or synthesis. The 12-LO enzyme inhibitors can inhibit Ang II mediated aldosterone secretion in rat and human adrenal glomerulosa cells. The present study in the H295R adrenal cell provides a means to attempt to study the mechanism of 12-LO in mediating aldosterone synthesis and a comprehensive understanding of the role of this enzyme in zona glomerulosa cells of adrenal cortex.

The H295R cell line was used as our model because the cells produce aldosterone in response to Ang II stimulation and previous studies have clearly shown that Ang II can increase CYP11B2 promoter activity in these cells. The results show for the first time that overexpression of 12-LO stimulates aldosterone production and increases CYP11B2 promoter activity in H295R cells. Ang II also enhanced 12-LO-induced aldosterone production and CYP11B2 transcriptional activity. The 12-LO inhibitor, baicalein, reduced these effects, thus providing further support that increased aldosterone production is specific to 12-LO. The reduced effect of 1 µM baicalein on aldosterone production in 12-LO-overexpressing cells appears to be due to high amount of 12-LO activity in these cells.

CREB is a 43-kDa nuclear transcription factor belonging to the CREB/ATF-1 family. It is constitutively expressed and binds to the specific sequence known as CRE. CREB and ATF-1 as a heterodimer binds to CRE and regulates transcription (26). The CREB family was characterized early in the H295R cell line. These cells express high levels of ATF-2, ATF-1, and CRE modulator, whereas CREB levels vary among the strains of H295R cells (27, 28, 29). However, regulation of CREB has not been thoroughly evaluated in H295R cells. Phosphorylation on serine residue 133 of CREB is required for CREB-mediated transcription (30). The phosphorylation does not alter the binding of CREB to CRE, but it increases its association with adapter proteins such as CREB-binding protein, leading to the activation of the transcriptional machinery. The active forms of CREB and ATF-1 could be detected and separated by immunoblotting in 12-LO-overexpressing cells and mock cells. In the method with the pCREB/CREB assay kit, the mix of pCREB/ATF-1 was detected because the phospho-CREB antibody shows cross-reactivity in the present studies. The data obtained using two independent CREB phosphorylation assay methods provided very similar results in the current study. Ang II has been shown to activate gene promoter activity by the CRE and lead to activation of the CREB transcription factor (31). The CYP11B2 5'-flanking region contains a CRE sequence at position -71/-64 and steroidogenic factor-1 element at -129/-114. Ang II uses the CRE-like cis element to regulate human CYP11B2 expression (7). Recent data show that steroidogenic factor-1 is not critical to increase transcription of the CYP11B2 gene in H295R cells (31A ). Our results demonstrate new information on CREB regulation by Ang II in H295R cells. We now present data that Ang II treatment at physiologically relevant concentrations induces phosphorylation of CREB/ATF-1. In addition, we show for the first time that 12-LO can induce activation of CREB in H295R cells.

Additional studies of the time course mechanisms will be needed to fully evaluate the activation of these transcription factors. CREB was identified as a substrate for protein kinase A and mediator of cAMP-regulated gene expression (30). CREB can be phosphorylated and activated by multiple signaling pathways including ERK, protein kinase C, calcium/calmodulin-dependent protein kinases, and p38 MAPK (32, 33, 34, 35). Evidence also suggests that JNK or SAPK plays a role in transducing signals from the cytosol to the nucleus in response to growth factors or cytokines. We earlier showed the role of JNK in Ang II and HETE action in cardiac fibroblasts overexpressing the Ang II type 1 receptor as well as the particular role of p21 activated kinase and small GTP-binding proteins Rac in Ang II action (36). Watanabe et al. (21A ) demonstrated previously that Ang II increased JNK/SAPK activity in H295R cells using immunoprecipitation kinase assays. However, JNK activation by Ang II could not be detected in this study. This may be due to the different method that was used or culture or strain differences of the cells. Ang II also can induce ERK activation in H295R cells and bovine adrenocortical cells (21A, 38).

ERK and p38 have been shown being involved in Ang II-induced growth effects (39, 40). However, little is known about the role of p38 MAPK pathway in the steroidogenic effects of Ang II and 12-LO. A recent publication (41) has shown p38 MAPK involving Ang II activation on calcium homeostasis in bovine adrenal glomerulosa cells. The observation of activation of ERK is consistent with a previous study in these cells (21A ). However, 12-LO overexpression had no direct effect on ERK activity in H295R cells. In contrast, Ang II markedly increased p38 MAPK activity; the peak time was at 5 min. The results show for the first time that Ang II is a potent inducer of the p38 MAPK activity. The 12-LO pathway also increased p38 MAPK activity in H295R cells. In addition, a p38 inhibitor blocked 50% of Ang II and 12-LO induced CREB/ATF phosphorylation. From current inhibitor studies, it appears that the p38 MAPK pathway is playing a role in CREB regulation induced by Ang II and 12-LO. Additional studies will be needed to evaluate the relative contribution of particular p38 isoforms to the effect in Ang II and the 12-LO pathway. These results suggest that multiple signal pathways are involved in Ang II- and 12-LO-induced CYP11B2 transcription in H295R cells. Further studies will be needed to explore other signaling pathways that may be relevant for CYP11B2 transcriptional activity in response to 12-LO.

The interaction of Ang II with their receptors in glomerulosa cells leads to a calmodulin-dependent transient rise in intracellular Ca2+ and phospholipase C activity, which in turn leads to increase the formation of diacylglycerol (DAG) and phosphatidic acid. DAG and phosphatidic acid can be converted into arachidonic acid (AA), and AA may contribute to downstream responses such as sustained increases in aldosterone (42, 43). DAG formation by Ang II action in the adrenal is potentially an important source of AA for LO product formation (44, 45). The 12-LO pathway oxygenates AA to yield 12-HETE, a major product of the 12-LO pathway (12, 13, 46). We have shown that the 12-LO enzyme overexpressed in H295R cells is enzymatically active. Furthermore, Ang II stimulates 12-LO activity as shown by the formation 12-HETE in H295R cells. Previous studies have demonstrated that exposure to exogenous or stimulated endogenous release of 12-LO products plays a role in mediating Ang II-induced aldosterone synthesis in human and rat adrenal glomerulosa cells (18, 19). The current results suggest that p38 MAPK activation and phosphorylation of CREB are involved in Ang II- and 12-LO-induced aldosterone production, at least in part by increasing transcriptional activation of aldosterone synthase CYP11B2.


   Footnotes
 
This work was supported by NIH Grant DK-39721.

Abbreviations: AA, Arachidonic acid; Ang II, angiotensin II; ATF, activating transcription factor; CRE, cAMP response element; CREB, CRE-binding protein; DAG, diacylglycerol; DMSO, dimethylsulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HETE, hydroxyeicosatetraenoic acid; JNK, c-Jun N-terminal kinase; LO, lipoxygenase; pCREB, phosphorylated-CREB; SAPK, stress-activated protein kinase.

Received June 4, 2002.

Accepted for publication October 22, 2002.


   References
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 Results
 Discussion
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