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. 2011 Sep;164(2):394-404.
doi: 10.1111/j.1476-5381.2011.01375.x.

Angiotensin II type 1 receptor signalling regulates microRNA differentially in cardiac fibroblasts and myocytes

Pia Lindgren Jeppesen  1 , Gitte Lund Christensen, Mikael Schneider, Anne Yaël Nossent, Hasse Brønnum Jensen, Ditte Caroline Andersen, Tilde Eskildsen, Steen Gammeltoft, Jakob Lerche Hansen, Søren Paludan Sheikh
Affiliations

Angiotensin II type 1 receptor signalling regulates microRNA differentially in cardiac fibroblasts and myocytes

Pia Lindgren Jeppesen et al. Br J Pharmacol. 2011 Sep.

Abstract

Background and purpose: The angiotensin II type 1 receptor (AT(1)R) is a key regulator of blood pressure and cardiac contractility and is profoundly involved in development of cardiac disease. Since several microRNAs (miRNAs) have been implicated in cardiac disease, we determined whether miRNAs might be regulated by AT(1)R signals in a Gαq/11-dependent or -independent manner.

Experimental approach: We performed a global miRNA array analysis of angiotensin II (Ang II)-mediated miRNA regulation in HEK293N cells overexpressing the AT(1)R and focused on separating the role of Gαq/11-dependent and -independent pathways. MiRNA regulation was verified with quantitative PCR in both HEK293N cells and primary cardiac myocytes and fibroblasts.

Key results: Our studies revealed five miRNAs (miR-29b, -129-3p, -132, -132* and -212) that were up-regulated by Ang II in HEK293N cells. In contrast, the biased Ang II analogue, [Sar1, Ile4, Ile8] Ang II (SII Ang II), which selectively activates Gαq/11-independent signalling, failed to regulate miRNAs in HEK293N cells. Furthermore, Ang II-induced miRNA regulation was blocked following Gαq/11 and Mek1 inhibition. The observed Ang II regulation of miRNA was confirmed in primary cultures of adult cardiac fibroblasts. Interestingly, Ang II did not regulate miRNA expression in cardiac myocytes, but SII Ang II significantly down-regulated miR-129-3p.

Conclusions and implications: Five miRNAs were regulated by Ang II through mechanisms depending on Gαq/11 and Erk1/2 activation. These miRNAs may be involved in Ang II-mediated cardiac biology and disease, as several of these miRNAs have previously been associated with cardiovascular disease and were found to be regulated in cardiac cells.

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Figures

Figure 1
Figure 1
The AT1R regulates miRNA expression profiles by Gαq-dependent mechanisms. (A) Schematic illustration of AT1R signal transduction. Erk1/2, Jnk and p38 can be activated by both Gαq-dependent and -independent mechanisms. Erk1/2 activated by Gαq-independent signalling is sequestered in the cytosol and it is therefore questionable whether MAP kinases activated by this pathway can regulate miRNA expression. (B) Representative Western blots comparing p-Erk1/2, p-p38 and p-Jnk activation in AT1R-HEK cells treated with angiotensin II (Ang II) or [Sar1, Ile4, Ile8] Ang II (SII Ang II) for 3 min. (C) Heatmap showing differential miRNA expression in AT1R-HEK cells treated with Ang II or SII Ang II for 24 h (n = 3). (D)q-PCR validation of regulated miRNAs. The expression is normalized against the stably expressed miRNAs miR-17 and miR-191. Note the difference in the normalized expression at the y-axis. *P < 0.05, tested with a paired two-tailed t-test.
Figure 2
Figure 2
The effect of angiotensin II (Ang II) on cell number and volume of AT1R-HEK cells. (A,B) Cells were treated with 100 nM Ang II, 18.7 µM [Sar1, Ile4, Ile8] Ang II (SII Ang II), or 5% FBS for 24 h and cell number and volume were measured on a Coulter counter. *P < 0.05 tested with a paired t-test (n = 4). (C)The effect of confluence on miRNA expression in AT1R-HEK cells was investigated by treatment of cells seeded at low (10 000 cm−2), medium (20 000 cm−2) and high densities (60 000 cm−2) and with vehicle or Ang II for 24 h. The expression of miR-7 is significantly regulated by plating densities alone. The expression is normalized against the stably expressed miRNAs miR-17 and miR-191. The effect of confluence was tested for significance by two-way anova analysis (n = 3). Note the difference in expression at the y-axis.
Figure 3
Figure 3
Angiotensin II (Ang II)-mediated expression of miR-29b, miR-129-3p, miR-132, miR-132* and miR-212 in AT1R-HEK cells is dependent on Gαq and Erk1/2. Cells were treated with 100 nM Ang II in combination with inhibitors for 24 h. (A) MicroRNA (miRNA) expression was revealed with q-PCR. Control (DMSO), Gαq-i (Gαq inhibitor/YM254890, 10 nM), Mek-i (Mek1 inhibitor/UO126, 10 µM), p38-i (p38 inhibitor/SB203580, 10 µM) and Jnk-i (Jnk inhibitor/ SP600125, 10 µM). The expression is normalized against the stably expressed miR-17 and miR-191. q-PCR values are illustrated with values for cells treated with Ang II or vehicle. (B) q-PCR values of Ang II treatments were divided by their respective vehicle expression value to achieve fold change values. Note the difference in fold change at the y-axis. Treatments were tested for significance by one-way anova, *P < 0.05 (n = 3).
Figure 4
Figure 4
Angiotensin II (Ang II) and [Sar1, Ile4, Ile8] Ang II (SII Ang II)-regulated expression of miRNAs in primary neonatal cardiomyocytes and adult cardiac fibroblasts. (A) Rat cardiac fibroblasts and myocytes were treated with Ang II (100 nM) or SII Ang II (18.7 µM) for 48 h and miRNA expression was evaluated by q-PCR. The expression is normalized against the stably expressed miRNAs miR-17 and miR-191. Effect of treatment was tested for significance by paired two-tailed t-tests, *P < 0.05 (n = 3). (B) Representative Western blots of Erk1/2 phosphorylation (p-Erk1/2) in adult rat cardiac fibroblasts treated with 100 nM Ang II, 18.5 µM SII Ang II or 0.15 nM phorbol myristate acetate (PMA; positive control). Total Erk1/2 (t-Erk1/2) is shown as a loading control.
Figure 5
Figure 5
Angiotensin II (Ang II)-mediated microRNA (miRNA) expression in primary cardiac fibroblasts is dependent on Erk1/2. Cells were treated with Ang II (100 nM) in combination with inhibitors for 48 h. Control (DMSO), Gαq-i (Gαq inhibitor/YM254890, 10 nM), Mek-i (Mek1 inhibitor/UO126, 10 µM), p38-i (p38 inhibitor/SB203580, 10 µM) and Jnk-i (Jnk inhibitor/ SP600125, 10 µM). (Left-hand coumns) The expression is normalized against the stably expressed let-7f. q-PCR values are illustrated with values for cells treated with Ang II or vehicle. (Right-hand columns) q-PCR values of Ang II treatments were divided with the respective vehicle expression value to achieve fold change values. *P < 0.05 tested with Student's two-tailed t-test (n = 5).
Figure 6
Figure 6
Expression profile of AT1aR, AT1bR and AT2R in cardiac myocytes and fibroblasts. SYBR green based real-time q-PCR was used to detect and quantify mRNA expression of the three ATR subtypes. As a positive control we used pooled mRNA extracted from three embryonic rat hearts. The expression was normalized against the stably expressed mRNAs for Rpl13a and β-actin (n = 3).
Figure 7
Figure 7
Losartan inhibits the up-regulation of angiotensin II (Ang II)-induced miRNAs in cardiac fibroblasts. Rat cardiac fibroblasts were treated with 1 µM losartan 30 min prior to Ang II stimulation for 48 h and miRNA expression was evaluated by q-PCR. The expression is normalized against the stably expressed let-7f. Effect of treatment was tested for significance by a paired one-tailed t-test, *P < 0.05 (n = 5).
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