J Korean Med Sci.  2004 Dec;19(6):805-811. 10.3346/jkms.2004.19.6.805.

Aldosterone Upregulates Connective Tissue Growth Factor Gene Expression via p38 MAPK Pathway and Mineralocorticoid Receptor in Ventricular Myocytes

• Lee YS
• Kim JA
• Kim KL
• Jang HS
• Kim JM
• Lee JY
• Shin IS
• Lee JS
• Suh W
• Choi JH
• Jeon ES
• Byun J
• Kim DK

• KMID: 1778560
• DOI: http://doi.org/10.3346/jkms.2004.19.6.805

Abstract

The effect of aldosterone on connective tissue growth factor (CTGF) was examined in rat embryonic ventricular myocytes. Upon aldosterone treatment, CTGF expression was significantly increased in a dose and time-dependent manner. To explore the molecular mechanism for this upregulation, we examined the role of mineralocorticoid receptor. Pre-treatment of an antagonist (spironolactone) at 5-fold excess of aldosterone blocked the CTGF induction by aldosterone, suggesting that the upregulation was mediated by mineralocorticoid receptor. Aldosterone treatment resulted in activation of ERK1/2, p38 MAPK, and JNK pathways with a more transient pat-tern in p38 MAPK. Blocking studies using pre-treatment of the inhibitor of each path-way revealed that p38 MAPK cascade may be important for aldosterone-mediated CTGF upregulation as evidenced by the blocking of CTGF induction by SB203580 (p38 MAPK inhibitor), but not by PD098059 (ERK1/2 inhibitor) and JNK inhibitor I. Interestingly, JNK inhibitor I and PD098059 decreased the basal level of CTGF expression. On the other hand, pre-treatment of spironolactone abrogated the p38 MAPK activation, indicating that mineralocorticoid receptor mechanism is linked to p38 MAPK pathway. Taken together, our findings suggest that aldosterone induces CTGF expression via both p38 MAPK cascade and mineralocorticoid receptor and that cross-talk exists between the two pathways.

Keyword

Connective Tissue Growth Factor; Aldosterone; Mitogen-activated Protein Kinase p38; Spirono-lactone; Receptor, Mineralocorticoid

MeSH Terms

Aldosterone/*pharmacology
Animals
Cells, Cultured
Dose-Response Relationship, Drug
Gene Expression Regulation/drug effects/physiology
Heart Ventricles/drug effects/embryology/metabolism
Immediate-Early Proteins/*metabolism
Intercellular Signaling Peptides and Proteins/*metabolism
Myocytes, Cardiac/*drug effects/*metabolism
Rats
Receptors, Mineralocorticoid/*metabolism
Research Support, Non-U.S. Gov't
Signal Transduction/drug effects/physiology
Spironolactone/pharmacology
Up-Regulation/drug effects/physiology
p38 Mitogen-Activated Protein Kinases/*metabolism

Figure

• 

  Fig. 1 CTGF expression is increased by aldosterone in a dose- and time-dependent manner. The H9c2 cells were treated with various concentrations of aldosterone for 2 hrs (A) and for the indicated times at 1 M concentration (B). After treatment of aldosterone, 10 µg of total RNA from each sample was subjected to Northern blot analysis. The 18S rRNA was used as an internal control to normalize the level of CTGF mRNA. The bar graph shows the value of each sample relative to that of control that received no aldosterone treatment. Representative blot from three separate experiments is shown. The data are means±SEM (n=3, *p<0.05, †p<0.01, ‡p<0.001 vs. control).

• 

  Fig. 2 Spironolactone, a mineralocorticoid receptor antagonist, inhibits aldosterone-mediated CTGF upregulation. The H9c2 cells were pretreated with spironolactone at various concentrations from 0.1 µM to 2.5 µM for 30 min and then exposed to 0.5 µM aldosterone. Ten µg of total RNA was analyzed for the level of CTGF mRNA. The 18S rRNA was used as an internal control to normalize the data. Each bar denotes the value relative to that of control that received no treatment. One of the results from two separate experiments is shown. The data are means±SEM (n=2, *p<0.01 vs. control [lane 1]).

• 

  Fig. 3 Time course of MAPK activation by aldosterone. The H9c2 cells were exposed to 1 µM aldosterone for the indicated times. The cells were then harvested and 20 µg of the lysates subjected to Western blot analysis. To monitor activation of MAPKs, phospho-specific antibodies that selectively recognize the active forms of ERK1/2, p38 MAPK, and JNK were used. Antibodies that bind to either form of ERK1/2, p38 MAPK, and JNK were used to ensure equal loading of the samples. The representative data from two to three experiments are shown.

• 

  Fig. 4 Involvement of p38 MAPK pathway in aldosterone-mediated CTGF upregulation. CTGF induction by aldosterone is inhibited by p38 MAPK inhibitor (SB203580), but not by ERK and JNK inhibitors (PD098059 or JNK inhibitor I, respectively). The H9c2 cells were pre-treated with SB203580 (10 µM), PD098059 (50 µM), or JNK inhibitor I (20 µM) for 30 min and then exposed to 1 µM aldosterone. After 2 hrs, total RNA (10 µg) was harvested and analyzed for the level of CTGF mRNA. Representative result from three separate experiments is shown. The CTGF induction fold is represented as mean±SEM (n=3, *p<0.05 vs. no inhibitor treatment).

• 

  Fig. 5 Effect of spironolactone on p38 MAPK phosphorylation. (A) Western blot analysis of the H9c2 cell lysates harvested at 10 min after aldosterone treatment. One group of cells were pretreated with spironolactone (2.5 µM) for 30 min and then exposed to 0.5 µM aldosterone for 10 min. The other group was treated with aldosterone only. The blots were probed with anti-phospho-p38 MAPK antibody (top), stripped, and then reprobed with anti-p38 MAPK antibody (bottom). (B) Quantification of p38 MAPK activation. Spironolactone pre-treatment resulted in statistically significant inhibition of aldosterone-induced p38 activation. The intensity of each band was quantified by densitometer. Each bar denotes the ratio of phospho-p38 MAPK/total p38 MAPK expressed as mean±SEM. Solid bar: aldosterone only, Hatched bar: spironolactone pre-treatment plus aldosterone (n=3, *p<0.05).

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