Angiotensin II up-regulates angiotensin I-converting enzyme (ACE), but down-regulates ACE2 via the AT1-ERK/p38 MAP kinase pathway

Abstract

The recent discovery of the angiotensin II (Ang II)-breakdown enzyme, angiotensin I converting enzyme (ACE) 2, suggests the importance of Ang II degradation in hypertension. The present study explored the signaling mechanism by which ACE2 is regulated under hypertensive conditions. Real-time PCR and immunohistochemistry showed that ACE2 mRNA and protein expression levels were high, whereas ACE expression levels were moderate in both normal kidney and heart. In contrast, patients with hypertension showed marked ACE up-regulation and ACE2 down-regulation in both hypertensive cardiopathy and, particularly, hypertensive nephropathy. The inhibition of ACE2 expression was shown to be associated with ACE up-regulation and activation of extracellular regulated (ERK)1/2 and p38 mitogen-activated protein (MAP) kinases. In vitro, Ang II was able to up-regulate ACE and down-regulate ACE2 in human kidney tubular cells, which were blocked by an angiotensin II (AT)1 receptor antagonist (losartan), but not by an AT2 receptor blocker (PD123319). Furthermore, blockade of ERK1/2 or p38 MAP kinases by either specific inhibitors or a dominant-negative adenovirus was able to abolish Ang II-induced ACE2 down-regulation in human kidney tubular cells. In conclusion, Ang II is able to up-regulate ACE and down-regulate ACE2 expression levels under hypertensive conditions both in vivo and in vitro. The AT1 receptor-mediated ERK/p38 MAP kinase signaling pathway may be a key mechanism by which Ang II down-regulates ACE2 expression, implicating an ACE/ACE2 imbalance in hypertensive cardiovascular and renal damage.

Figures

Figure 1

ACE and ACE2 expression in normal and hypertensive heart tissues. Serial sections of normal and hypertensive human hearts are stained with antibodies to ACE and ACE2. A–C: Normal human heart tissue stained with antibodies to ACE2 (A), ACE (B), or a non-immune isotype antibody as a negative control (C). D–F: A hypertensive human heart tissue stained with antibodies to ACE2 (D), ACE (E), or a non-immune isotype antibody as a negative control (F). G: Semiquantitative analysis. Results represent for the mean ± SEM for a group of 8 normal tissues or 12 hypertensive heart tissues. H: Results of real-time PCR analysis. Each bar represents the mean ± SEM for a group of six tissues. *P < 0.05, ***P < 0.001 compared to normal. Original magnification ×200.

Figure 2

Immunohistochemistry demonstrates that hypertensive nephropathy is associated with an increase in ACE and a decrease in ACE2 expression as well as an increase in ERK1/2 and p38 MAP kinase activation. Serial sections of normal human kidneys and human hypertensive nephropathy are stained with antibodies to ACE, ACE2, phospho-ERK1/2, and phospho-p38 MAP kinases. A–E: A normal human kidney tissue stained with antibodies to ACE2 (A), ACE (B), phospho-ERK1/2 (C), phospho-p38 (D), or a non-immune isotype antibody as a negative control (E). F–J: A hypertensive human kidney tissue stained with antibodies to ACE2 (F), ACE (G), phospho-ERK1/2 (H), phospho-p38, (I) or a non-immune isotype antibody as a negative control (J). It should be noted that a decrease in ACE2 (F) and an increase in ACE (G) are associated with a marked activation of ERK1/2 and p38 MAP kinases as evidenced by a nuclear staining pattern of phospho-ERK1/2 (H) and phospho-p38 (I). A clear nuclear staining pattern of both phospho-ERK1/2 and phospho-p38 is further illustrated in the inserted picture (C, D, H, I). Original magnification ×200.

Figure 3

Quantitative analysis of immunohistochemistry and real-time PCR demonstrates that hypertensive nephropathy is associated with an increase in ACE and a decrease in ACE2 expression, as well as an increase in ERK1/2 and p38 MAP kinase activation. A: Semiquantitative analysis of ACE and ACE2 immunohistochemical staining. B: Detection of renal ACE and ACE2 mRNA expression by real-time PCR. C: Semiquantitative analysis of phosphor-p38 and ERK1/2 within the kidney. Each bar represents the mean ± SEM for a group of 8 normal tissues or 12 hypertensive tissues. *P < 0.05, **P < 0.01, ***P < 0.001 compared to normal.

Figure 4

Ang II induces ACE expression and down-regulates ACE2 expression by HK-2 cells in a time-dependent manner. Real-time PCR (A, B) and Western blot analyses (C; quantification D, E) demonstrate that Ang II (1 μmol/L) is able to up-regulate ACE, but down-regulate ACE2 mRNA and protein expression in a time-dependent manner. Data shown represent the mean ± SEM for three independent experiments. Note that in the absence of Ang II stimulation (control) there is no significant change in ACE or ACE2 expression in the entire time course. *P < 0.05, **P < 0.01 compared to the time 0, respectively.

Figure 5

Ang II induces ACE expression and down-regulates ACE2 expression by HK-2 cells in a dose-dependent manner. Real-time PCR (A, B) and Western blot analyses (C) demonstrate that addition of Ang II is able to up-regulate ACE, but down-regulate ACE2 mRNA (at 24 hours) and protein (at 48 hours) expression in a dose-dependent manner. Note that no further change in ACE and ACE2 mRNA expression is observed beyond 1 μmol/L of Ang II (A, B). Data shown represent the mean ± SEM for five independent experiments for mRNA expression and three independent experiments for protein expression. *P < 0.05, **P < 0.01, ***P < 0.001 as compared with time 0, respectively.

Figure 6

Signaling mechanisms of Ang II-induced up-regulation of ACE and down-regulation of ACE2 at the mRNA levels. Real-time PCR demonstrates that addition of Ang II (1 μmol/L) for 24 hours is able to up-regulate ACE (A) and down-regulate ACE2 (B) at the mRNA levels, which is blocked by the AT1 receptor antagonist losartan (1 μmol/L), but not by the AT2 receptor antagonist PD 123319 (1 μmol/L). Interestingly, blockade of p38 MAP kinase and ERK1/2 MAP kinase with SB203580 (10 μmol/L) and PD98059 (20 μmol/L) has no effect on Ang II induced up-regulation of ACE mRNA, but abolishes Ang II-induced down-regulation of ACE2 mRNA. Results are expressed as the mean ± SEM for three independent experiments.*P < 0.05, **P < 0.01 compared to non-Ang II control (the first bar); #P < 0.05, ##P < 0.01 compared to Ang II stimulation alone (the second bar).

Figure 7

Signaling mechanisms of Ang II-induced up-regulation of ACE and down-regulation of ACE2 at the protein levels. Western blot analysis demonstrates that addition of Ang II (1 μmol/L) for 48 hours is able to up-regulate ACE and down-regulate ACE2 at the protein levels, which is blocked by the AT1 receptor antagonist losartan (1 μmol/L), but not by the AT2 receptor antagonist PD 123319 (1 μmol/L). Blockade of p38 MAP kinase and ERK1/2 MAP kinase with SB203580 (10 μmol/L) and PD98059 (20 μmol/L) has no effect on Ang II-induced up-regulation of ACE, but abolishes Ang II-induced down-regulation of ACE2. Results are expressed as the mean ± SEM for three independent experiments.*P < 0.05, **P < 0.01 compared to non-Ang II control (the first bar, respectively); #P < 0.05 compared to Ang II stimulation alone (the second bar, respectively).

Figure 8

Blockade of ERK1/2 MAP kinase and p38-MAP kinase by dominant negative adenovirus abolishes Ang II-induced down-regulation of ACE2 mRNA. Real-time PCR demonstrates that blockade of ERK1/2 and P38 MAP kinases by Adv-DN-ERK and Adv-DN-P38 (MOI of 30) abolishes Ang II (1 μmol/L)-induced down-regulation of ACE2 (B), but has no effect on Ang II-induced up-regulation of ACE mRNA (A) at 24 hours. Results are expressed as mean ± SEM for three independent experiments. **P < 0.01, ***P < 0.001 compared to non-Ang II control (the first bar, respectively); ##P < 0.01 compared to Ang II stimulation alone (the second bar, respectively).

Figure 9

Blockade of ERK1/2 MAP kinase and p38-MAP kinase by dominant negative adenovirus abolishes Ang II-induced down-regulation of ACE2 protein. Western blot analysis demonstrates that blockade of ERK1/2 and P38 MAP kinases by Adv-DN-ERK and Adv-DN-P38 (MOI of 30) abolishes addition of Ang II (1 μmol/L)-induced down-regulation of ACE2 protein, but has no effect on Ang II-induced up-regulation of ACE at 24 hours. Results are expressed as mean ± SEM for three independent experiments. **P < 0.01, ***P < 0.001 compared to non-Ang II control (the first bar, respectively); #P < 0.05 compared to Ang II stimulation alone (the second bar, respectively).

Figure 10

Scheme for Ang II autoregulatory feedback loop leading to its biological effects. Ang II signals through the AT1 receptor to up-regulate the ACE-dependent Ang II generating pathway and down-regulate the ACE2-mediated Ang II degradation pathway, ultimately leading to the elevation of Ang II levels and hypertension.