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Aldosterone Stimulates Proliferation of Mesangial Cells by Activating Mitogen-Activated Protein Kinase 1/2, Cyclin D1, and Cyclin A

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Aldosterone Stimulates Proliferation of Mesangial Cells by Activating Mitogen-Activated Protein Kinase 1/2, Cyclin D1, and Cyclin A Aldosterone Stimulates Proliferation of Mesangial Cells by Activating Mitogen-Activated Protein Kinase 1/2, Cyclin D1, and Cyclin A Yoshio Terada, Takahiko Kobayashi, Hitoshi Kuwana, Hiroyuki Tanaka, Seiji Inoshita, Michio Kuwahara, and Sei Sasaki Department of Nephrology, Tokyo Medical and Dental University, Tokyo, Japan Recently, attention has been focused on the role of aldosterone in the pathophysiology of hypertension and cardiovascular disease. Several clinical and experimental data support the hypothesis that aldosterone contributes to the progression of renal injury. However, the molecular mechanisms of the effects of aldosterone in signal transduction and the cell-cycle progression of mesangial cells are not well known. For determining the signaling pathway of aldosterone in cultured mesangial cells, the effects of aldosterone on the mitogen-activated protein kinase 1/2 (MAPK1/2) pathway and the promoter activities of cyclin D1, cyclin A, and cyclin E were investigated. First, it was shown that the mineralocorticoid receptor (MR) was expressed in rat mesangial cells and glomeruli and that aldosterone stimulated the proliferation of mesangial cells via the MR and MAPK1/2 pathway. Next, it was demonstrated that aldosterone stimulated Ki-RasA, c-Raf kinase, MEK1/2, and MAPK1/2 in rat mesangial cells. Aldosterone induced cyclin D1 and cyclin A promoter activities and protein expressions, as well as the increments of CDK2 and CDK4 kinase activities. The presence of CYP11B2 and 11␤-HSD2 mRNA in rat mesangial cells also was shown. In conclusion, aldosterone seems to exert mainly MR-induced effects that stimulate c-Raf, MEK1/2, MAPK1/2, the activities of CDK2 and CDK4, and the cell-cycle progression in mesangial cells. MR antagonists may serve as a potential therapeutic approach to mesangial proliferative disease. J Am Soc Nephrol 16: 2296–2305, 2005. doi: 10.1681/ASN.2005020129 n recent years, evidence has accumulated that angiotensin- Mesangial cell proliferation is an essential component of converting enzyme (ACE) inhibition or angiotensin II recep- glomerulonephritis. Many cytokines have been shown either to I tor blockage attenuates the decline in renal function and promote or to suppress the cell cycle of mesangial cells in recent structural damage in various kidney diseases (1–5). These benefi- studies (11–13). Because the regulational mechanisms of the cial effects of ACE inhibition and angiotensin II receptor blockage mesangial cell cycle by aldosterone are not well known, learn- are most likely due to the suppression of intrarenal angiotensin II ing more about them would be of great help in developing a concentrations and consequential effects (6). Recent clinical and curative treatment for mesangial proliferative glomerulone- experimental studies have shown that elevated plasma aldoste- phritis. rone levels may also contribute to the progression of cardiac (7) The full complement of hormones and the mechanisms by and renal disease (8–10). In a remnant kidney model, designed which they influence mesangial cell proliferation are not fully using rats that were treated with enalapril and losartan, Greene et appreciated. As in most other cell types, mitogen-activated al. (9) showed a significant suppression of hyperaldosteronism, as protein kinase 1/2 (MAPK1/2; also known as extracellular well as a marked attenuation of proteinuria, hypertension, and signal-regulated kinases 1/2 and p42/p44-MAPK) signaling glomerulosclerosis. In a similar study, Rocha et al. (10) showed mediates the proliferation of mesangial cells through the acti- renoprotective effects of eplerenone and spironolactone in aldo- vation of a number of tyrosine kinase–associated receptors and sterone-stimulated rat models. Chrysostomou and Becker (8) re- ported that the addition of spironolactone to ACE inhibitors mark- G protein–coupled receptors (14,15). Aldosterone was recently edly reduced the urinary excretion rate of protein in chronic renal reported to stimulate MAPK1/2 in cardiac fibroblast (16), epi- failure patients without exerting hemodynamic effects. These thelial cells (17), and renal cortex (18). However, the effects of studies strongly suggested that aldosterone was involved in the aldosterone on mesangial cell signaling, including MAPK1/2, pathogenesis of renal injury. are not known. The growth of mesangial cells and other eukaryotic cells is tightly regulated through a precious balance of positive and Received February 2, 2005. Accepted May 10, 2005. negative regulatory components that confer their effects during the first gap phase (G1) of the cell cycle (19,20). The most critical Published online ahead of print. Publication date available at www.jasn.org. positive-acting components are G1 cyclins (cyclin D, cyclin E, Address correspondence to: Dr. Yoshio Terada, Department of Nephrology, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. and cyclin A) (20,21). These cyclins assemble with cyclin- Phone: 81-3-5803-5214; Fax: 81-3-5803-5215; E-mail: [email protected] dependent kinase (CDK) and phosphorylate the key physio- Copyright © 2005 by the American Society of Nephrology ISSN: 1046-6673/1608-2296 J Am Soc Nephrol 16: 2296–2305, 2005 Aldosterone and Mesangial Cells 2297 logic substrate retinoblastoma protein (22). However, no re- product was predicted to be 382 bp in length. CYP11B2 primer 1 ports have described the effects of aldosterone on the cyclins. (antisense) was 5Ј-GATATCTTCAAAAGAGAGG-3Ј, and primer 2 was Ј Ј The purpose of this study was to investigate the mechanisms 5 -TACTGTTCAGCTAATCACG-3 . The predominant cDNA amplifi- ␤ of aldosterone-induced cell-cycle progression in mesangial cation product was predicted to be 269 bp in length. 11 -HSD2 primer 1 (antisense) was 5Ј-GACTAATGTGAACCTCTGGGAG-3Ј, and primer cells. We attempted to determine the mechanisms of the mes- 2 was 5Ј-TCAGTGCTCGGGGTAGAAGGTG-3Ј. The predominant angial cell proliferation of aldosterone by investigating the cDNA amplification product was predicted to be 269 bp in length. activities of the MAPK cascades, the activities of CDK2 and Glyceraldehyde-3-phosphate-dehydrogenase served as a positive con- CDK4, and cell-cycle analysis in rat mesangial cells. We dem- trol. Primer 1 (antisense) was 5Ј-AGATCCACAACGGATACATT-3Ј, onstrated that aldosterone stimulates MAPK1/2, MEK1/2, and primer 2 was 5Ј-TCCCTCAAGATTGTCAGCAA-3Ј. The predom- c-Raf, Ki-RasA, the activities of CDK2 and CDK4, and the inant cDNA amplification product was predicted to be 309 bp in length. cell-cycle progression in mesangial cells mainly via the mineralocor- The PCR products were size-fractionated by 2% agarose gel electro- ticoid receptor (MR). MR antagonists may serve as a potential ther- phoresis. After electrophoresis and ethidium bromide staining, DNA apeutic approach to mesangial proliferative disease. bands were visualized with an ultraviolet transilluminator (Funakoshi, Tokyo, Japan). The PCR products were sequenced to confirm that these Materials and Methods bands were actually the predicted cDNA. The PCR products were subcloned into a pGEM-TM vector (Promega, Biotec, Madison, WI) and Mesangial Cell Culture and Histologic Examination sequenced as described previously (28). Mesangial cell strains from male Sprague-Dawley rats were isolated and characterized as previously reported (23). Cells were cultured in an RPMI 1640 medium that contained 20% FBS, 100 units/ml penicillin, Reporter Constructs 100 ␮g/ml streptomycin, 5 ␮g/ml insulin, 5 ␮g/ml transferrin, and 5 The cyclin D1 reporter construct used for luciferase assays contained Ϫ ng/ml selenite at 37°C in a 5% CO2 incubator. The cells were seeded in a human cyclin D1 promoter from residues 944 to 139 cloned up- 10-cm dishes for all experiments except the experiment on [3H]thymi- stream of the luciferase gene (gift from Dr. M. Eilers) (29). The cyclin A dine incorporation, which used 24-well dishes. reporter construct contained a human cyclin A promoter from residues Mesangial cells were fixed with 70% ethanol for 10 min for the Ϫ924 to 245 (gift from Dr. J. Sobczak-Thepot) (30), and the cyclin E immunohistologic examination of the MR. The primary antibody was reporter construct contained a human cyclin E promoter from residues anti–human MR purchased from Santa Cruz Biotechnology (Santa Ϫ1195 to 79 cloned upstream of the luciferase gene (gift from Dr. K. Cruz, CA). The secondary antibody was an anti-rabbit IgG FITC-con- Ohtani) (31). Wild-type MEK1 and dominant-negative MEK1 S222A jugated antibody (Sigma, St. Louis, MO). Once fixed, the mesangial were gifts from Dr. E.G. Krebs (32). cells were examined under a confocal laser microscope (Carl Zeiss Japan, Tokyo, Japan) as described previously (24). Transient Transfection and Luciferase Assay Mesangial cells were transfected by the electroporation method with Renal Glomerular Isolation 4 ␮gofthe␤-galactosidase construct and 20 ␮g of the cyclin D1, A, or Male Sprague-Dawley rats that weighed 100 to 150 g were used for E promoter construct. After transfection, the cells were cultured in a these studies. Renal glomerular isolation was performed using previ- medium that contained 20% FCS for 12 h and then changed to either an ously described techniques (25). After incubation in the collagenase FCS(Ϫ) medium that contained an indicated dose of aldosterone or an solution described above, glomeruli were isolated from the cortex using FCS(Ϫ) medium that contained aldosterone for indicated times. Lucif- a grading sieving technique (25). One hundred
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