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

Home , MAPK1

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) reported 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 glomeruli were used for erase and ␤-galactosidase activities were measured according to the the reverse transcription–PCR (RT-PCR) procedure, and 300 glomeruli protocols of the manufacturer (Promega) (33). Luciferase enzyme units were used for the Western blot analysis, as described previously (26). were normalized to ␤-galactosidase.

Antibodies 3 Antibodies against anti-mouse cyclin D1, anti-rabbit cyclin A, anti- [ H]Thymidine Incorporation rabbit cyclin E, anti-mouse CDK2, anti-mouse CDK4, and anti-mouse Mesangial cells were plated in 24-well plates and incubated in an Ϫ Ki-RasA were purchased from Santa Cruz Biotechnology. Antibodies FCS( ) medium that contained aldosterone (1 nM to approximately 100 ␮ 3 against anti-mouse c-Raf, anti-mouse phospho-c-Raf, anti-mouse nM) for 24 h (34). For the last 4 h, 1 Ci [ H]thymidine (Amersham) was MEK1/2, anti-mouse phospho-MEK1/2, anti-mouse MAPK1/2, and added to the medium. The cells were washed three times in 4°C PBS. anti-mouse phospho-MAPK1/2 were purchased from Cell Signaling After cold 10% TCA was added to precipitate the protein and DNA, the Technology (Beverly, MA). mixture was redissolved in 0.5 M NaOH. Aquasol-2 scintillation cock- tails (NEN Research Products, Boston, MA) were counted in a scintillation counter. RT-PCR The mRNA was extracted from the mesangial cells, glomeruli, rat renal cortex, and rat adrenal gland using TRI-REAGENT (Life Tech- Cell-Cycle Analysis by Flow Cytometry nologies, Gaithersburg, MD) (27). RT was performed using a cDNA Mesangial cells were cultured in 10-cm dishes and then incubated for 24 h synthesis kit (Boehringer-Mannheim, GmbH) according to the manu- in either an FCS(Ϫ) medium or 20% FCS medium, each with indicated doses facturer’s instructions. Reaction tubes were incubated at 42°C for 60 of aldosterone. The samples were washed twice with PBS and then resus- min in the Programmed Tempcontrol System (Astec, Tokyo, Japan). pended in 70% ethanol for1hat4°C. Fixed and permeated cells were PCR was performed using the GeneAmp DNA Amplification Reagent collected by centrifugation, washed with PBS, treated with RNase, and stained Kit (Perkin-Elmer Cetus, Norwalk, CT). MR primer 1 (antisense) was with propidium iodide. The cell counts in the G1, S, and G2/M phases were 5Ј-AGAAGATGCATCAGTCTGCC-3Ј, and primer 2 was 5Ј-GTGAT- analyzed by flow cytometry using a FACS Calibur (Becton Dickinson, San GATCTCCACCAGCAT-3Ј. The predominant cDNA amplification Jose, CA), as described previously (34). 2298 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 2296–2305, 2005

Western Blot Analysis Whole-cell lysates that were extracted from the cultured mesangial cells, rat isolated glomeruli, and rat renal cortex were lysed in SDS sample buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 1 ␮g/ml aprotinin, 1 ␮g/ml leupeptin, 1 mM PMSF, and 0.1 mM sodium orthovanadate) at 4°C. Twenty micrograms of protein extracts was analyzed by Western blotting with anti–cyclin E, anti–cyclin D1, anti–cyclin A, anti–Ki- RasA, or anti-MR. Twenty micrograms of protein extracts was analyzed by Western blotting with anti–phospho-MAPK1/2, anti-MAPK1/2, anti–phospho-MEK1/2, anti-MEK1/2, anti–phospho-c-Raf, or anti–c- Raf. The proteins were separated in SDS-polyacrylamide gels and transferred to an Immobilon P membrane (Daiichikagaku, Tokyo, Ja- pan). The blots were incubated in 5% nonfat dry milk (in 1ϫ PBS and 0.1% Tween 20) for1hatroom temperature and then incubated with the indicated antibodies (1:1000 dilution in 5% nonfat dry milk and 0.1% Tween 20 in 1ϫ PBS) for 2 h. After three washes in 0.1% Tween 20 in 1ϫ PBS, the blots were incubated with horseradish peroxidase– conjugated secondary antibodies (1:2500 dilution) for 1 h. The mem- branes were visualized by the Amersham ECL system (Amersham Figure 1. The presence of the mineralocorticoid receptor (MR), Corp., Arlington Heights, IL) after three more washes using the previ- CYP11B2, and 11␤-HSD2 in cultured mesangial cells and iso- ously described methods (35). lated glomeruli. (A) Reverse transcription–PCR (RT-PCR) analysis of the MR was performed from RNA that was extracted CDK4 and CDK2 Kinase Assays from isolated glomeruli (Glm), mesangial cells (MG), the renal cortex (Cort), and H O (negative control). Ethidium bromide– Immune complex kinase assays were performed using essentially the 2 stained agarose gels for the MR (top) and glyceraldehyde-3- same methods as those described previously (36). Rat mesangial cells were phosphate-dehydrogenase (GAPDH; bottom). (B) Immunoblot- pretreated with an FCS(Ϫ) medium for 48 h. A medium that contained 100 ting of the MR (top) and actin (bottom) was performed from nM aldosterone was added for the last 12 h, and the cells were lysed at the protein that was extracted from isolated Glm, MG, and Cort. end of the incubation period. After the cell lysates were incubated for 2 h (C) RT-PCR analysis of CYP11B2 was performed from RNA at 4°C with 10 ␮l of the anti-mouse CDK4 antibody or anti-mouse CDK2 that was extracted from isolated Glm, MG, the adrenal gland antibody (Santa Cruz), 20 ␮l of protein G-plus agarose (Oncogene Science, (Adr), and H O. Ethidium bromide–stained agarose gels for Uniondale, NY) was recovered together with the immune complexes by 2 CYP11B2 (top) and GAPDH (bottom). (D) RT-PCR analysis of centrifugation. Immunoprecipitated proteins were suspended in 30 ␮lofa 11␤-HSD2 was performed from RNA that was extracted from kinase buffer (50 mM HEPES [pH 7.5], 10 mM MgCl , and 1 mM dithio- 2 isolated Glm, MG, Adr, and H O. Ethidium bromide–stained threitol) that contained a substrate (0.2 ␮g of soluble glutathione S-trans- 2 agarose gels for 11␤-HSD2 (top) and GAPDH (bottom). Similar ferase–retinoblastoma protein fusion protein for the CDK4 kinase assay; results were obtained from five repeated experiments. Histione H1 [Boehringer Mannheim] for the CDK2 assay; and 2.5 mM EGTA, 10 mM ␤-glycerophosphate, 1 mM NaF, 20 ␮M ATP, and 10 ␮Ci of [␥-32P]ATP [NEN, Dupont, Boston, MA] 6000 Ci/mmol). After incubation for 30 min at 30°C with occasional mixing, the samples were boiled in a tivity for a protein of the appropriate size (approximately 120 polyacrylamide gel sample buffer that contained SDS and separated by kD) was observed in both isolated glomeruli and mesangial electrophoresis. Phosphorylated proteins were visualized by autoradiog- cells. A protein extract of the rat renal cortex was used as a raphy. positive control. Our next step was to examine the presence of CYP11B2 and 11␤-HSD2 from both isolated glomeruli and cul- Statistical Analyses tured rat mesangial cells, using adrenal RNA as a positive All values are expressed as means Ϯ SEM. The differences were control. As shown in Figure 1, C and D, mRNA of CYP11B2 and Ͻ tested using ANOVA. P 0.05 was considered significant. 11␤-HSD2 could be detected clearly from both isolated glomeruli and cultured rat mesangial cells. Immunohistologic exami- Results nation confirmed the presence of the MR in the cytosolic lesions Presence of MR, CYP11B2, and 11␤-HSD2 in Cultured of rat mesangial cells in the absence of aldosterone (Figure 2A). Mesangial Cells and Isolated Glomeruli The staining of the MR was shifted to the nucleus after 60 min We first examined the presence of the MR, CYP11B2, and of incubation with aldosterone (10 nM; Figure 2B). These results ␤ 11 -HSD2 in rat cultured mesangial cells and rat isolated glo- confirmed the presence of the MR in rat mesangial cells. meruli. The presence of the MR was examined by RT-PCR and Western blot analysis. As shown in Figure 1A, mRNA of the Aldosterone Increases [3H]Thymidine Incorporation and Cell MR was clearly detected from both isolated glomeruli and Counts of Cultured Mesangial Cells in S and G2/M Phases cultured rat mesangial cells. We used rat renal cortex as a We next examined the effects of aldosterone on mesangial positive control for MR expression. Western blot analysis also proliferation by measuring [3H]thymidine uptake and under- revealed the presence of the MR in isolated glomeruli and taking a cell-cycle analysis. Mesangial cells were treated with cultured mesangial cells. As shown in Figure 1B, positive reac- aldosterone (1 nM to approximately 100 nM) for 24 h with J Am Soc Nephrol 16: 2296–2305, 2005 Aldosterone and Mesangial Cells 2299

levels of phospho-MEK1/2 were 441 Ϯ 57 and 393 Ϯ 45% of the control levels at 3 and 6 h, respectively. Similarly, aldosterone significantly increased phospho-MAPK1/2 levels 612 Ϯ 58% by 3h(n ϭ 6). The total MEK1/2 levels and MAPK1/2 levels remained unchanged at the same 3-h time points (n ϭ 5). In our examination of Ki-RasA expression in mesangial cells, aldosterone substantially increased the amount of Ki-RasA expression at 1 h (315 Ϯ 35%; n ϭ 6) and 3 h (287 Ϯ 32%; n ϭ 6).

Figure 2. Immunohistochemistry of the MR in the presence and absence of aldosterone in cultured rat mesangial cells. Mesan- Aldosterone Stimulates MAPK1/2 Signaling Cascades in gial cells were incubated in FCS(Ϫ) RPMI 1640 in the absence Rat Mesangial Cells through MR (A) or presence (B) of aldosterone (10 nM) for 60 min. Rat The experiments shown in Figure 5 tested the possibility that mesangial cells were fixed with 70% ethanol and stained with aldosterone activates Ki-RasA and MAPK1/2 signaling cas- an MR antibody. Magnification, ϫ400. cades via the MR in mesangial cells. The representative Western blots demonstrate the dose-dependent effects of aldosterone in the absence and presence of MR (spironolactone) and glucocor- [3H]thymidine pulsing for the last 4 h. Figure 3A shows the ticoid receptor (RU-486) antagonists on the levels of Ki-RasA, dose-dependent induction of [3H]thymidine uptake by aldoste- phospho-c-Raf, total c-Raf, phospho-MEK1/2, total MEK1/2, rone compared with the control. These data indicate that aldo- phospho-MAPK1/2, and total MAPK1/2 (Figure 5A). The mes- sterone stimulates mesangial cell proliferation. To examine the angial cells in these experiments were treated for 3 h with effects of endogenous aldosterone on mesangial cell prolifera- aldosterone and both with and without the antagonists (0.1 ␮ tion, we added spironolactone (0.1 ␮M) to the medium in the M). As shown in the summary graph of Figure 5B, aldosterone absence of exogenous aldosterone in mesangial cells. As shown at 10 nM significantly increased the relative levels of phospho- in Figure 3A (right), the [3H]thymidine uptake did not change c-Raf, phospho-MEK1/2, phospho-MAPK1/2, and Ki-RasA, significantly in response to the spironolactone treatment. This whereas the total c-Raf, MEK1/2, and MAPK1/2 levels were finding indicated that the endogenous aldosterone did not not affected. Spironolactone markedly attenuated the actions of impart significant effects on mesangial cell proliferation, at least aldosterone (10 nM) on phospho-c-Raf, phospho-MEK1/2, in our experimental conditions. To verify the stimulatory ef- phospho-MAPK1/2, and Ki-RasA, whereas RU-486 had no fects of mesangial cell proliferation by exogenous aldosterone, such inhibitory effect. we performed a cell-cycle analysis by FACS under two conditions: A serum-free condition and a normal cell-cycle condition Aldosterone Increases Protein Expressions of Cyclin D1 and (20% FCS). Mesangial cells were incubated in an FCS(Ϫ) me- Cyclin A in Mesangial Cells dium with aldosterone for 24 h (Figure 3B). Aldosterone (10 We next examined the protein expressions of G1/S phase nM) increased the cell numbers of the S and G2/M phases, as cyclins (cyclin E, cyclin D1, and cyclin A) by Western blot shown in Figure 3C. Spironolactone inhibited the aldosterone- analysis. Aldosterone increased the protein levels of cyclin D1 induced increments of the cell counts in the S and G2/M phases and cyclin A significantly and dose dependently at 12 h (Figure in serum-free condition. Mesangial cells were incubated in 20% 6, A and B). FCS medium with aldosterone for 24 h (Figure 3D). The aldo- We further examined the time course of the protein ex- sterone (10 nM) increased the cell numbers of the S and G2/M pressions of cyclin D1, cyclin A, and cyclin E. As shown in phases, as shown in Figure 3E. Spironolactone inhibited the Figure 6, C and D, the protein expression of cyclin D1 was aldosterone-induced increments of the cell counts in the S and increased from 6 h and sustained up to 24 h. The protein G2/M phases under the normal cell-cycle condition. These data expression of cyclin A was increased from 12 h and upregu- suggest that aldosterone stimulates the cell-cycle progression lated up to 24 h. The protein expression of cyclin E was via the MR in rat mesangial cells. slightly increased at 12 h and peaked at 18 h. The data are summarized in Figure 6D. These stimulatory effects of cyclin Aldosterone Stimulates Ki-RasA and MAPK1/2 Cascade in D1 and cyclin A were inhibited by spironolactone (0.1 ␮M), Cultured Rat Mesangial Cells PD-98059 (10 ␮M), and transfection of the dominant-negative Many proliferative signals stimulate MAPK1/2 cascades in MEK1S222A (Figure 7). mesangial cells. In this phase of the study, we tested whether aldosterone stimulates MAPK1/2 signaling in rat-cultured Aldosterone Augments in Promoter Activities of Cyclin D1 mesangial cells. As shown in Figure 4A (and summarized in and Cyclin A Figure 4B), aldosterone was found to increase the activated To investigate the role of aldosterone in the transcriptional (phosphorylated) protein levels of several members of the regulation of cyclin D1, cyclin A, and cyclin E, we measured MAPK1/2 cascade in rat mesangial cells. Phospho(Ser259)- promoter activity using plasmids that contain cyclin D1, A, and c-Raf levels were significantly increased after3hofaldosterone E promoter regions and luciferase reporter genes in mesangial treatment (455 Ϯ 49%; n ϭ 6). In contrast, aldosterone left the cells (Figure 8A). Aldosterone (100 nM) increased cyclin D1 total cellular c-Raf expression unchanged (n ϭ 6). The relative promoter activity and cyclin A promoter activity to 795 Ϯ 114 2300 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 2296–2305, 2005

Figure 4. Effect of aldosterone on MAPK1/2, MEK1/2, c-Raf-phosphorylation, and Ki-RasA levels in rat mesangial cells. (A) Aldoste- rone (10 nM) was added to the medium, and total cellular protein was extracted at indicated times. Twenty micrograms of protein was loaded for Western blot analysis for phospho-c-Raf, total-c-Raf, phospho-MEK1/2, total-MEK1/2, phospho-MAPK1/2, total-MAPK1/2, and Ki-RasA. Similar results were obtained from six repeated experiments. (B) Quantitative analyses were performed for phospho-c-Raf, phospho-MEK1/2, phospho-MAPK1/2, and Ki-RasA using a densitometer. Each bar represents the mean Ϯ SEM (n ϭ 6); *P Ͻ 0.05 versus control (0 h) by the ANOVA test.

and 1290 Ϯ 187% of the control levels, respectively. These data suggest that the induction of the cyclin D1 and cyclin A promoter activities and the protein expression that upregulates the G1/S transition may play roles in the aldosterone-induced

Mesangial cells were treated with indicated doses of aldosterone for 24 h, with [3H]thymidine pulsed in the medium for the last4hofincubation (n ϭ 6, mean Ϯ SEM, *P Ͻ 0.05 versus control by the ANOVA test; left). Mesangial cells were treated with spironolactone (0.1 ␮M) for 24 h, with [3H]thymidine pulsed in the medium for the last4hofincubation (n ϭ 6, mean Ϯ SEM, NS versus control by the ANOVA test; right). (B) Cell-cycle analysis was performed in 10 nM aldosterone (Aldo)- stimulated rat mesangial cells in the presence or absence of spironolactone (Spiro; 0.1 ␮M) in serum-free condition. (C) Percentages of G0/G1-, S-, and G2/M-phase cells that were treated with or without aldosterone (Ald; 10 nM) and spironolactone (AϩS; 0.1 ␮M)) in serum-free condition. Each bar represents the mean Ϯ SEM (n ϭ 5); *P Ͻ 0.05 by the ANOVA test. (D) Cell-cycle analysis was performed in 10 nM Aldo-stimulated rat mesangial cells in the presence or absence of Spiro (0.1 ␮M) in a normal cell-cycle condition (20% FCS). (E) Percentages Figure 3. Aldosterone increases [3H]thymidine incorporation of G0/G1, S, and G2/M-phase cells that were treated with or and the cell counts of cultured mesangial cell in the S and without Ald (10 nM) and AϩS (0.1 ␮M) in a normal cell-cycle G2/M phases. (A) Effects of aldosterone on [3H]thymidine condition (20% FCS). Each bar represents the mean Ϯ SEM (n ϭ incorporation and cell-cycle progression of rat mesangial cells. 5); *P Ͻ 0.05 by the ANOVA test. J Am Soc Nephrol 16: 2296–2305, 2005 Aldosterone and Mesangial Cells 2301

Aldosterone Stimulates CDK2 and CDK4 Activities in Rat Mesangial Cells We examined whether the induction of cyclin D1 and cyclin A by aldosterone contributes to the increased kinase activities of CDK4 and CDK2, respectively. Twelve hours of aldosterone treatment increased CDK4 kinase activities and CDK2 activities to 7.5 times and 12.7 times the control levels, respectively (Figure 9). These stimulatory effects of aldosterone on CDK4 and CDK2 were significantly inhibited by spironolactone (0.1 ␮M). These data suggest that the effect of aldosterone in inducing the CDK4 and CDK2 activities that are responsible for the upregulation of the G1/S transition may play roles in the aldosterone-induced stimulation of mesangial cell proliferation. These effects are mediated by the MR.

Aldosterone Promotes Rat Mesangial Cell Proliferation through MR and MAPK1/2 Signaling MAPK1/2 signaling is known to stimulate cell proliferation. In view of the confirmed effects of aldosterone in promoting mesangial cell proliferation and activating MAPK1/2 signaling cascades, we questioned whether aldosterone stimulates mesangial cell proliferation via the MAPK1/2 cascade and the MR. As shown in Figure 10, spironolactone (0.1 ␮M), PD-98059 (10 ␮M), and transfection of the dominant-negative MEK1S222A significantly attenuated the aldosterone-induced mesangial cell proliferation measured by [3H]thymidine incorporation.

Discussion Figure 5. Effects of MR (spironolactone) and glucocorticoid In this study, we demonstrate that the MR is expressed in rat receptor (RU-486) antagonists on aldosterone-stimulated mesangial cells and glomeruli and that aldosterone stimulates MAPK1/2, MEK1/2, c-Raf-phosphorylation, and Ki-RasA lev- the proliferation of mesangial cells via the MR and MAPK1/2 els in rat mesangial cells. Rat mesangial cells were treated for pathway. We also confirm the effects of aldosterone in inducing 3 h with aldosterone both with and without the antagonist (0.1 the promoter activities and protein expressions of cyclin D1 and ␮ M). (A) Twenty micrograms of protein was loaded for West- cyclin A, the increments of CDK2 and CDK4 activities, and the ern blot analysis for phospho-c-Raf, total-c-Raf, phospho- increment of Ki-RasA in rat mesangial cells. Last, we show the MEK1/2, total-MEK1/2, phospho-MAPK1/2, total-MAPK1/2, presence of CYP11B2 and 11␤-HSD2 mRNA in rat mesangial and Ki-RasA. (B) Graph summaries of A using a densitometer. cells. Aldosterone at 1 and 10 nM significantly increased relative levels Recent attention has been focused on the role of aldosterone of phospho-c-Raf, phospho-MEK1/2, phospho-MAPK1/2, and Ki-RasA. Spironolactone markedly attenuated the actions of aldo- in the pathophysiology of hypertension and cardiovascular sterone (10 nM) on phospho-c-Raf, phospho-MEK1/2, phospho- disease. Several clinical and experimental data support the MAPK1/2, and Ki-RasA, whereas RU-486 exerted no such effect. hypothesis that aldosterone contributes to the progression of Each bar represents the mean Ϯ SEM (n ϭ 6); *P Ͻ 0.05 versus renal injury (8–10). The administration of spironolactone did aldosterone (10 nM) by the ANOVA test. not alter BP but markedly ameliorated renal injury in stroke- prone spontaneously hypertensive rats (37). However, the exact mechanisms of aldosterone that lead to mesangial cell proliferation have not been clarified. This study is the first to demon- stimulation of mesangial cell proliferation. Spironolactone and strate that aldosterone stimulates mesangial cell proliferation PD-98059 inhibited these aldosterone-induced stimulatory ef- via the MR, c-Raf, MEK1/2, MAPK1/2, cyclin D1, and cyclin A fects of cyclin D1 and cyclin A promoter activities (data not pathways. These in vitro results are consistent with both the in shown). vitro (38) and in vivo (8,18) works of others using other types of We further examined the time course of the promoter activ- cells and experiments. The data presented here along with ities of cyclin D1, cyclin A, and cyclin E. As shown in Figure 8B, those of previous studies suggest that the aldosterone-depen- the promoter activity of cyclin D1 was increased from6hand dent activation of the MAPK1/2 cascade in mesangial cells sustained up to 24 h. The promoter activity of cyclin A was could play a role in the recently documented pathologic actions increased from 12 h and upregulated up to 24 h. The promoter of this steroid hormone on the kidney. activity of cyclin E was slightly increased at 12 h and reached its Recent studies demonstrated that aldosterone increases Ki- peak at 18 h. The data are summarized in Figure 8B. RasA levels in epithelia (17,39,40) and cardiac fibroblasts (16). 2302 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 2296–2305, 2005

Figure 7. Aldosterone increases the protein expressions of cyclin D1 and cyclin A via the MEK1-MAP1/2 pathway in mesangial cells. Rat mesangial cells were treated for 12 h with aldosterone both with and without the antagonists. Twenty micrograms of protein was loaded for Western blot analysis for cyclin D1 and cyclin A. Spironolactone, PD98059, and transfection of MEK1S222A markedly attenuated the actions of aldosterone (10 nM) on cyclin D1 and cyclin A. Similar results were obtained from five repeated experiments.

This action is dependent on corticosteroid-mediated changes in the transcription of the Ki-rasA gene at identified cis-elements (41,42). To the best of our knowledge, the effects of aldosterone on Ki-RasA levels in mesangial cells have not been investigated. Aldosterone increased Ki-RasA protein levels in rat mesangial cells in our experiments. This effect was marked after only 1 h and significantly by as early as 3 h. The MR antagonist spironolactone attenuated the induction of Ki-RasA in mesangial cells by aldosterone, suggesting that this effect was mediated by the classical MR. These data are the first to link directly aldosterone action in rat mesangial cells to the induction of Figure 6. Aldosterone increases the protein expressions of cyclin Ki-RasA, a signaling factor that is capable of promoting cellular D1 and cyclin A in mesangial cells. (A) Aldosterone (1 nM to proliferation. approximately 100 nM) was added to the medium, and total Our results also establish that aldosterone in mesangial cells cellular protein was extracted at 12 h. Twenty micrograms of activates downstream signaling effectors of Ki-RasA in the protein was loaded for Western blot analysis for cyclin D1, MAPK1/2 cascade, including phosphorylated forms of c-Raf, cyclin A, cyclin E, and actin. (B) Graph summaries of A using a MEK1/2, and MAPK1/2. The time course of the activation of densitometer. Aldosterone significantly increased the relative the MAPK1/2 cascade constituents, which seems maximal at levels of cyclin D1 and cyclin A. Each bar represents the 3 h, is consistent with that of Ki-RasA. Thus, it seems that this Ϯ ϭ Ͻ mean SEM (n 5); *P 0.05 versus control by the ANOVA process drives MAPK1/2 signaling in mesangial cells. Nish- test. (C) Aldosterone (100 nM) was added to the medium, and iyama et al. (18) reported that MAPK is activated via reactive total cellular protein was extracted at indicated times. Twenty oxygen species in aldosterone/salt-induced renal injury in vivo. micrograms of protein was loaded for Western blot analysis for cyclin D1, cyclin A, cyclin E, and actin. (D) Graph summaries of However, they did not examine the upstream signals of C using a densitometer. Aldosterone significantly increased the MAPK1/2, such as Ki-RasA, c-Raf, and MEK1/2. The Ras relative levels of cyclin D1, cyclin A, and cyclin E. Each bar MAPK1/2 cascade is a well-known mitogenic pathway in represents the mean Ϯ SEM (n ϭ 5); *P Ͻ 0.05 versus control by many diverse cell types and is activated in some instances the ANOVA test. during proliferation of mesangial cells (43–45). With this in J Am Soc Nephrol 16: 2296–2305, 2005 Aldosterone and Mesangial Cells 2303

Figure 9. Effect of aldosterone on CDK2 and CDK4 activities in rat mesangial cells in the presence or absence of the MR antagonist. Rat mesangial cells were pretreated with an FCS(Ϫ) medium for 48 h. A medium that contained 100 nM aldosterone either with or without 0.1 ␮M spironolactone was added for the last 12 h, and the cells were lysed at the end of the incubation period. Cell lysates were incubated for2hat4°Cwith 10 ␮lof the anti-rat CDK4 antibody or the anti-rat CDK2 antibody, then a kinase assay was performed as described in Materials and Methods. Similar results were obtained from five repeated experiments. Figure 8. Involvement of aldosterone in cyclin E, D1, and A promoter activities in rat mesangial cells. (A) The cells were strated the presence of CYP11B2 in many types of cells or transiently transfected with cyclin E, D1, and A promoter plas- organs such as heart and vascular smooth muscle cells, includ- mids that contained the luciferase reporter gene by the electro- ing the adrenal gland (46). The existence of a locally synthe- poration method. Cells were incubated in an FCS(Ϫ) medium with indicated doses of aldosterone for 12 h. The cells were sized aldosterone system may play a role in the pathologic collected and assayed for luciferase activities. Each bar repre- actions of aldosterone in mesangial proliferative glomerular sents the mean Ϯ SEM (n ϭ 6); *P Ͻ 0.05 versus control by the diseases. In this study, however, we examined only the effects ANOVA test. (B) The cells were transiently transfected with of spironolactone on the [3H]thymidine uptake in the absence cyclin E, D1, and A promoter plasmids that contained the of added aldosterone. As shown in Figure 3A, spironolactone luciferase reporter gene by the electroporation method. Cells did not have significant effects on the [3H]thymidine uptake in Ϫ were incubated in an FCS( ) medium with aldosterone (100 the absence of added aldosterone. We thus confirmed that the nM) for indicated times. The cells were collected and assayed locally synthesized aldosterone did not have a significant effect Ϯ for luciferase activities. Each bar represents the mean SEM on mesangial cell proliferation under our experimental condi- (n ϭ 6); *P Ͻ 0.05 versus control by the ANOVA test. tion. mind, we tested whether the aldosterone-induced activation of the MAPK1/2 cascade promoted the proliferation of rat mesangial cells. Inhibition of the MAPK1/2 cascade by PD-98059 and transfection of MEK1S222A abrogated the effect of aldosterone in stimulating mesangial cell proliferation in this study. This is the first finding to link directly aldosterone-induced MAPK1/2 signaling to the stimulation of rat mesangial cell proliferation. The complete absence of serum in the experiments for these proliferation studies suggests that aldosterone alone is a potent mitogenic signal for rat mesangial cells. This study also demonstrated that the aldosterone-induced MAPK1/2 activation triggers the induction of cyclin D1 and cyclin A and activation of CDK2 and CDK4. Thus, the activa- Figure 10. Involvement of the MR and the MEK1-MAPK1/2 tion of the MAPK1/2 cascade and induction of the cyclin-CDK pathway in the aldosterone-induced mesangial cell proliferation. The mesangial cells were transfected with MEK1 wild- pathway may represent a point of signaling convergence that type or dominant negative MEK1 S222A. The mesangial cells stimulates rat mesangial cell proliferation and contributes to were incubated with 10 nM aldosterone in the presence or the pathologic actions of aldosterone in mesangial proliferative absence of PD-98059 (10 ␮M) and spironolactone (0.1 ␮M). glomerular diseases. Mesangial cells were treated with indicated doses of aldoste- Our experiments are also the first to demonstrate the pres- rone for a 24-h incubation period, with [3H]thymidine pulsing ence of CYP11B2 and 11␤-HSD2 mRNA in rat mesangial cells in the medium for the last4h(n ϭ 6, mean Ϯ SEM); *P Ͻ 0.05 and isolated glomeruli. Several recent reports have demon- versus aldosterone (10 nM) by the ANOVA test. 2304 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 2296–2305, 2005

Xio et al. (46,47) reported that aldosterone potentiates angio- ical investigation: Studies on the renin-angiotensin system. tensin II–stimulated rat vascular smooth muscle cell prolifera- Hypertension 35: 150–154, 2000 tion. Angiotensin II has been known to stimulate mesangial cell 7. Brilla CG, Matsubara LS, Weber KT: Anti-aldosterone proliferation and hypertrophy. It is of interest to investigate the treatment and the prevention of myocardial fibrosis in interaction of aldosterone and angiotensin II on mesangial cell primary and secondary hyperaldosteronism. J Mol Cell proliferation and cell signaling pathways such as MAP1/2. Cardiol 25: 563–575, 1993 8. Chrysostomou A, Becker G: Spironolactone in addition to Further studies of these issues may help us to expand our ACE inhibition to reduce proteinuria in patients with understanding of mesangial proliferative diseases. chronic renal disease. N Engl J Med 345: 925–926, 2001 Although it is often difficult and sometimes inappropriate to 9. Greene EL, Kren S, Hostetter TH: Role of aldosterone in the extend in vitro results to in vivo conditions, our results are remnant kidney model. J Clin Invest 98: 1063–1068, 1996 consistent with the above-mentioned studies in support of the 10. Rocha R, Stier CT Jr, Kifor I, Ochoa-Maya MR, Rennke HG, close link between aldosterone and renal function via the MR. Williams GH, Adler GK: Aldosterone: A mediator of myo- Further investigation will be needed to determine whether the cardial necrosis and renal arteriopathy. Endocrinology 141: actions of aldosterone in the kidney involve other cell types and 3891–3898, 2000 receptors. The current experimental design tested only whether 11. Abboud HE: Growth factors in glomerulonephritis. Kidney aldosterone affects Ki-RasA and MAPK1/2 signaling in rat Int 43: 252–267, 1993 mesangial cells via the MR. We did not directly test the parallel 12. Floege J, Eng E, Young BA, Johnson RJ: Factors involved in hypothesis that this corticosteroid at higher doses is also capa- the regulation of mesangial cell proliferation in vitro and in ble of affecting mesangial cells via glucocorticoid receptors or vivo. Kidney Int 43: S48–S54, 1993 whether aldosterone exerts nongenomic actions on renal tis- 13. Johnson RJ, Floege J, Couser WG, Alpers CE: Role of plate- sues. Further studies will be necessary to develop an under- let-derived growth factor in glomerular disease. JAmSoc Nephrol 4: 119–128, 1993 standing of the pathophysiologic actions of aldosterone in mes- 14. Terada Y, Nakashima O, Inoshita S, Kuwahara M, Sasaki S: angial cells and glomeruli. Our studies may suggest that MR TGF-beta-activating kinase-1 inhibits cell cycle and expres- antagonists may prove useful as a therapeutic approach to sion of cyclin D1 and A in LLC-PK1 cells. Kidney Int 56: mesangial proliferative disease. 1378–1390, 1999 15. Bokemeyer D, Guglielmi KE, McGinty A, Sorokin A, Li- anos EA, Dunn MJ: Activation of extracellular signal-reg- Acknowledgments ulated kinase in proliferative glomerulonephritis in rats. This work was supported by the Uehama Memorial Foundation and J Clin Invest 100: 582–588, 1997 a grant from the Ministry of Education, Science, Culture and Sports of 16. Stockand JD, Meszaros JG: Aldosterone stimulates prolif- Japan, as well as Health and Labor Science Research Grants for Re- eration of cardiac fibroblasts by activating Ki-rasA and search on Specific Diseases from Ministry of Health, Labor, and Wel- MAPK1/2 signaling. Am J Physiol 284: H176–H184, 2003 fare. 17. Tong Q, Booth RF, Worrell RT, Stockand JD: Regulation of We thank Drs. E.G. Krebs, M. Eilers, J. Sobczak-Thepot, and K. Na transport by aldosterone: Signaling convergence and Ohtani for providing plasmids. cross talk between PI3-K and MAPK1/2 cascades. Am J Physiol 286: F1232–F1238, 2004 18. Nishiyama A, Yao L, Nagai Y, Miyata K, Yoshizumi M, References Kagami S, Kondo S, Kiyomoto H, Shokoji T, Kimura S, 1. Anderson S, Meyer TW, Rennke HG, Brenner BM: Control Kohno M, Abe Y: Possible contributions of reactive oxygen of glomerular hypertension limits glomerular injury in rats species and mitogen-activated protein kinase to renal in- with reduced renal mass. J Clin Invest 76: 612–619, 1985 jury in aldosterone/salt-induced hypertensive rats. Hyper- 2. Cattran DC, Greenwood C, Ritchie S: Long-term benefits of tension 24: 841–848, 2004 angiotensin-converting enzyme inhibitor therapy in pa- 19. Hunter T, Pines J: Cyclins and cancer. II: Cyclin D and tients with severe immunoglobulin A nephropathy: A CDK inhibitors come of age. Cell 79: 573–582, 1994 comparison to patients receiving treatment with other an- 20. Sherr CJ: Mammalian G1 cyclins. Cell 73: 1059–1065, 1993 tihypertensive agents and to patients receiving no therapy. 21. Hunter T: Braking the cycle. Cell 75: 839–841, 1993 Am J Kidney Dis 23: 247–254, 1994 22. Weinberg RA: The retinoblastoma protein and cell cycle 3. Hommel E, Parving HH, Matjiesen E, Edsberg B, Nielsen control. Cell 81: 323–330, 1995 MD, Giese J: Effect of captopril on kidney function in 23. Owada A, Tomita K, Terada Y, Sakamoto H, Nonoguchi H, insulin-dependent diabetic patients with nephropathy. Marumo F: Endothelin (ET)-3 stimulates cyclic guanosine BMJ 293: 467–470, 1986 3Ј,5Ј-monophosphate production via ETB receptor by pro- 4. Ishidoya S, Morrissey J, Cracken MC, Reyes A, Klahr S: ducing nitric oxide in isolated rat glomerulus, and in cul- Angiotensin II receptor antagonist ameliorates renal tubu- tured rat mesangial cells. J Clin Invest 93: 556–563, 1994 lo-interstitial fibrosis caused by unilateral renal obstruc- 24. Terada Y, Tanaka H, Okado T, Shimamura H, Inoshita S, tion. Kidney Int 47: 1285–1294, 1995 Kuwahara M, Sasaki S: Wnt4 expression and function of 5. Rosenberg ME, Smith LJ, Correa-Rotter R, Hostetter TH: the developmental gene Wnt-4 during experimental acute The paradox of the renin-angiotensin system in chronic renal failure in rats. J Am Soc Nephrol 14: 1223–1233, 2003 renal disease. Kidney Int 45: 403–410, 1994 25. Terada Y, Tomita K, Yoshiyama N, Shiigai T, Marumo F: 6. Hollenberg NK: Implications of species difference for clin- Leukotriene D4 inhibits atrial natriuretic factor-dependent J Am Soc Nephrol 16: 2296–2305, 2005 Aldosterone and Mesangial Cells 2305

cGMP production in rat glomerulus. Am J Physiol 256: perosmolality in renal cells. J Biol Chem 269: 31296–31301, F95–F99, 1989 1994 26. Terada Y, Yamada T, Takayama M, Nonoguchi H, Sasaki S, 36. Inoshita S, Terada Y, Nakashima O, Kuwahara M, Sasaki S, Tomita K, Marumo F: Presence and regulation of Raf-1-K Marumo F: Roles of E2F1 in mesangial cell cycle in vitro. (Kinase), MAPK-K, MAP-K, and S6-K in rat nephron seg- Kidney Int 56: 2086–2095, 1999 ments. J Am Soc Nephrol 6: 1565–1577, 1995 37. Rocha R, Chander PN, Khanna K, Zuckerman A, Stier CT 27. Terada Y, Moriyama T, Martin BM, Knepper MA, Garcia- Jr: Mineralocorticoid blockade reduces vascular injury in Perez A: RT-PCR micro-localization of mRNA for guanylyl stroke-prone hypertensive rats. Hypertension 31: 451–458, cyclase-coupled ANF receptor in rat kidney. Am J Physiol 1998 261: F1080–F1087, 1991 38. Schorb W, Conrad KM, Singer HA, Dostal DE, Baker KM: 28. Terada Y, Tomita K, Nonoguchi H, Yang T, Marumo F: Angiotensin II is a potent stimulator of MAP-kinase activ- Different localization and regulation of two types of vaso- ity in neonatal rat cardiac fibroblasts. J Mol Cell Cardiol 27: pressin receptor messenger RNA in microdissected rat 1151–1160, 1995 nephron segments using reverse transcription polymerase 39. Stockand JD, Spier BJ, Worrell RT, Yue G, Al-Baldawi N, ϩ chain reaction. J Clin Invest 92: 2339–2345, 1993 Eaton DC: Regulation of Na reabsorption by the aldoste- 29. Solomon DL, Philipp A, Land H, Eilers M: Expression of rone-induced small G protein K-Ras2A. J Biol Chem 274: cyclin D1 mRNA is not upregulated by Myc in rat fibro- 35449–35454, 1999 blasts. Oncogene 11: 1893–1897, 1995 40. Spindler B, Verrey F: Aldosterone action: induction of p21(ras) and fra-2 and transcription-independent decrease 30. Desdouets C, Ory C, Matesic G, Soussi T, Brechot C, Sob- in myc, jun, and fos. Am J Physiol 276: C1154–C1161, 1999 czak TJ: ATF/CREB site mediated transcriptional activa- 41. Pethe V, Shekhar PV: Estrogen inducibility of c-Ha-ras tion and p53 dependent repression of the cyclin A pro- transcription in breast cancer cells. Identification of func- moter. FEBS Lett 385: 34–38, 1996 tional estrogen-responsive transcriptional regulatory ele- 31. Ohtani K, DeGregori J, Nevins JR: Regulation of the cyclin ments in exon 1/intron 1 of the c-Ha-ras gene. J Biol Chem E gene by transcription factor E2F1. Proc Natl Acad Sci 274: 30969–30978, 1999 USA92: 12146–12150, 1995 42. Strawhecker JM, Betz NA, Neades RY, Houser W, Pelling 32. Seger R, Seger D, Reszka AA, Munar ES, Eldar FH, Do- JC: Binding of the 97 kDa glucocorticoid receptor to the 5Ј browolska G, Jensen AM, Campbell JS, Fischer EH, Krebs upstream flanking region of the mouse c-Ha-ras oncogene. EG: Overexpression of mitogen-activated protein kinase Oncogene 4: 1317–1322, 1989 kinase (MAPKK) and its mutants in NIH 3T3 cells. Evi- 43. Bokemeyer D, Sorokin A, Dunn MJ: Multiple intracellular dence that MAPKK involvement in cellular proliferation is MAP kinase signaling cascades. Kidney Int 49: 1187–1195, regulated by phosphorylation of serine residues in its ki- 1996 nase subdomains VII and VIII. J Biol Chem 269: 25699– 44. Shankland SJ: Cell-cycle control and renal disease. Kidney 25709, 1994 Int 52: 294–308, 1997 33. Hanada S, Terada Y, Inoshita S, Lormann SM, Sasaki S, 45. Terada Y, Yamada T, Nakashima O, Sasaki S, Marumo F: Marumo F: Cyclic GMP using adenovirus inhibits cyclin E Overexpression of cell cycle inhibitors (p16INK4 and transcription and mesangial cell cycle. Am J Physiol 280: p21Cip1) and cyclin D1 using adenovirus vectors regulates F851–F859, 2001 proliferation of rat mesangial cells. J Am Soc Nephrol 8: 34. Terada Y, Inoshita S, Nakashima O, Yamada T, Tamamori 51–60, 1997 M, Ito H, Sasaki S, Marumo F: Cyclin D1, p16, and retino- 46. Xiao F, Puddefoot JR, Vinson GP: Aldosterone mediates blastoma gene regulate mitogenic signaling of endothelin angiotensin II-stimulated rat vascular smooth muscle cell in rat mesangial cells. Kidney Int 53: 76–83, 1998 proliferation. J Endocrinol 165: 533–536, 2000 35. Terada Y, Tomita K, Homma MK, Nonoguchi H, Yang T, 47. Xiao F, Puddefoot JR, Barker S, Vinson GP: Mechanism for Yamada T, Yuasa Y, Krebs EG, Marumo F: Sequential aldosterone potentiation of angiotensin II-stimulated rat activation of Raf-1 kinase, mitogen-activated protein vascular smooth muscle cell proliferation. Hypertension 44: (MAP) kinase kinase, MAP kinase, and S6 kinase by hy- 340–345, 2004