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. 2011 Jun;178(6):2665-81.
doi: 10.1016/j.ajpath.2011.02.006.

Angiotensin II-induced MMP-2 activity and MMP-14 and basigin protein expression are mediated via the angiotensin II receptor type 1-mitogen-activated protein kinase 1 pathway in retinal pigment epithelium: implications for age-related macular degeneration

Marianne Pons  1 , Scott W Cousins, Oscar Alcazar, Gary E Striker, Maria E Marin-Castaño
Affiliations

Angiotensin II-induced MMP-2 activity and MMP-14 and basigin protein expression are mediated via the angiotensin II receptor type 1-mitogen-activated protein kinase 1 pathway in retinal pigment epithelium: implications for age-related macular degeneration

Marianne Pons et al. Am J Pathol. 2011 Jun.

Abstract

Accumulation of various lipid-rich extracellular matrix (ECM) deposits under the retinal pigment epithelium (RPE) has been observed in eyes with age-related macular degeneration (AMD). RPE-derived matrix metalloproteinase (MMP)-2, MMP-14, and basigin (BSG) are major enzymes involved in the maintenance of ECM turnover. Hypertension (HTN) is a systemic risk factor for AMD. It has previously been reported that angiotensin II (Ang II), one of the most important hormones associated with HTN, increases MMP-2 activity and its key regulator, MMP-14, in RPE, inducing breakdown of the RPE basement membrane, which may lead to progression of sub-RPE deposits. Ang II exerts most of its actions by activating the mitogen-activated protein kinase (MAPK) signaling pathway. Herein is explored the MAPK signaling pathway as a potential key intracellular modulator of Ang II-induced increase in MMP-2 activity and MMP-14 and BSG protein expression. It was observed that Ang II stimulates phosphorylation of extracellular signal-regulated kinase (ERK) and p38 MAPK in RPE cells and ERK/p38 and Jun N-terminal kinase (JNK) in mice. These effects were mediated by Ang II type 1 receptors. Blockade of ERK or p38 MAPK abrogated the increase in MMP-2 activity and MMP-14 and BSG proteins in ARPE-19 cells. A better understanding of the molecular events by which Ang II induces ECM dysregulation is of critical importance to further define its contribution to the progression of sub-RPE deposits in AMD patients with HTN.

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Figures

Figure 1
Figure 1
Ang II activates MAPKs in ARPE-19 cells incubated with increasing concentrations of Ang II (10−11 to 10−7 mol/L) for 5 minutes. Phosphorylation dose-response of ERK (A), p38 MAPK (B), and JNK (C). Top panels: Representative Western blots for evaluation of phosphorylated protein in cell lysates. Numbers on the left represent protein molecular weight in kilodaltons. GAPDH protein is shown as the loading control. Bottom panels: Bar graphs corresponding to mean results of three independent experiments normalized to their corresponding loading controls. Results are expressed as mean ± SEM. *P < 0.05 and **P < 0.01 compared with untreated control cells.
Figure 2
Figure 2
Phosphorylation time course of MAPKs ERK, p38, and JNK in ARPE-19 cells stimulated with Ang II. A–C: Time-dependent ERK phosphorylation (A), p38 phosphorylation (B), and JNK phosphorylation (C) assessed using Western blot analysis on cell lysates from ARPE-19 cells treated with Ang II (10−7 mmol/L) for various periods. Top panels: Western blots from a representative experiment. Numbers on the left represent protein molecular weight in kilodaltons. GAPDH protein is shown as the loading control. Bottom panels: Bar graphs corresponding to the mean results of three independent experiments normalized to their corresponding loading controls. Results are expressed as percentage of control, and are the mean ± SEM of three independent experiments run in duplicate. *P < 0.05 compared with untreated control cells.
Figure 3
Figure 3
AT1 blocks Ang II-induced ERK and p38 phosphorylation in ARPE-19 cells. ARPE-19 cells were stimulated with Ang II in the presence of Ang II. p-ERK protein (A) and p-p38 protein (B) assessed using Western blot analysis. Top panels: Representative Western blots for p-ERK and p-p38. Numbers on the left represent protein molecular mass in kilodaltons. p-ERK and p-p38 protein expression was normalized to GAPDH. Bottom panels: Ratios of p-ERK/GAPDH or p-p38/GAPDH. Data are expressed as percentage of control, and are the mean ± SEM of three independent experiments run in duplicate. *P < 0.05 compared with untreated control cells; **P < 0.05 versus Ang II alone.
Figure 4
Figure 4
Representative immunofluorescent double staining of phosphorylated ERK and p38 MAPK (green) and nuclei (blue) in Ang II–stimulated ARPE-19 cells in the presence of Ang II in the presence or absence of Ang II receptor blockers AT1 and AT2. Figure is a composite image demonstrating the immunostaining of nontreated (basal) (A–E and K–O) and treated (Ang II) (F–J and P–T) cells in the absence (control) (B, G, L, and Q) or presence of candesartan (AT1 blocker) (C, H, M, and R) or PD123319 (AT2 blocker) (D, I, N, and S) or both (AT1/2 blockers) (E, J, O, and T). Negative controls were generated by omission of the primary antibody. Sections were analyzed using confocal microscopy (×40 magnification).
Figure 5
Figure 5
Ang II up-regulated BSG protein expression through AT1 activation in RPE-19 cells. A: RPE-derived BSG protein expression in the presence of various concentrations of Ang II for 24 hours. B: BSG protein expression in the presence of candesartan (CD) alone, PD123319 (PD) alone, or a combination of both Ang II receptor blockers before treatment with Ang II. Top panels: Representative Western blots for BSG. Numbers on the left represent protein molecular weight in kilodaltons. Bottom panels: Ratios of BSG/GAPDH. Data are expressed as percentage of control, and are the mean ± SEM of three independent experiments run in duplicate. *P < 0.05 and **P < 0.01 versus control. ***P < 0.05 versus Ang II alone.
Figure 6
Figure 6
Inhibition of ERK and p38 MAPK blocks Ang II–induced MMP-2 activity and MMP-14 and BSG protein expression in ARPE-19 cells. A: Inhibition of Ang II–induced MMP-2 activity by the selective inhibitor of ERK PD98059 (PD), and p38α and p38β MAPK isoforms, SB203580 (SB). Top: Representative gelatin zymogram. Numbers on the left represent protein molecular weight in kilodaltons. Bottom: Bar graph corresponding to the mean results of three independent experiments. B: Inhibition of Ang II–induced MMP-14 protein expression by PD and SB. Top: Representative Western blot for MMP-14. Numbers on the left represent protein molecular weight in kilodaltons. Bottom: Ratio of MMP-14/GAPDH. C: Ang II effect on expression of BSG in ARPE-19 cells in the presence or absence of MAPKs inhibitors. Top: Representative Western blot for BSG. Numbers on the left represent protein molecular weight in kilodaltons. Bottom: Ratio of BSG/GAPDH. Data are expressed as percentage of control, and are the mean ± SEM of three independent experiments run in duplicate. *P < 0.05 versus control; **P < 0.05 versus Ang II alone.
Figure 7
Figure 7
Effect of ERK and p38 MAPK gene silencing on ERK and p38 protein expression. ARPE-19 cells were transiently transfected with either a scrambled sequence (Sc) or a specific siRNA against ERK or p38 MAPK (p38). A: Reduction in endogenous ERK1/2 protein expression by siRNA against ERK MAPK. B: Reduction in endogenous p38 MAPK protein expression by siRNA against p38 MAPK. Efficiency of siRNA against ERK or p38 MAPK in reducing endogenous ERK or P38 protein was confirmed using Western blot analysis. Top panels: Representative Western blots for ERK (A) or p38 MAPK (B). Numbers on the left represent protein molecular mass in kilodaltons. ERK and p38 MAPK protein expression was normalized to GAPDH. Bottom panels: Mean results of four independent experiments run in duplicate on ARPE-19 cells. Data are expressed as percentage of control. *P < 0.05 compared with nontransfected control cells.
Figure 8
Figure 8
Effect of ERK and p38 MAPK gene silencing on Ang II–induced MMP-2 activity and MMP-14 and BSG protein expression in ARPE-19 cells. A: Effect of ERK and p38 MAPK siRNA on Ang II–induced MMP-2 activity. Top: Gelatin zymogram from a representative experiment. Numbers on the left represent protein molecular mass in kilodaltons. Bottom: Bar graph corresponding to mean results of three independent experiments. B: Effect of ERK and p38 MAPK siRNA on Ang II–induced MMP-14 protein expression. C: Effect of ERK and p38 MAPK siRNA on Ang II–induced BSG protein expression. Top: Representative Western blot for MMP-14 (B) and BSG (C). Numbers on the left represent protein molecular weight in kilodaltons. MMP-14 and BSG protein expression was normalized to GAPDH. Bottom: Ratio of MMP-14/GAPDH (B) or BSG/GAPDH (C). Results are expressed as percentage of control, and are the mean ± SEM of four independent experiments. *P < 0.05 compared with nontransfected control cells; **P < 0.05 versus nontransfected Ang II–treated cells.
Figure 9
Figure 9
A: Systolic blood pressure in response to administration of Ang II alone for 1, 7, 14, and 30 days and Ang II in combination with the AT1 antagonist candesartan (CD) or the AT2 antagonist PD123319 (PD) for 30 days in C57BL/6 mice. *P < 0.05 and **P < 0.01 versus their respective basal blood pressure; †P < 0.01 versus Ang II administration for 30 days. B: Systolic blood pressure and (C) plasma renin activity (PRA) in 1k1C mice and in the corresponding sham-operated control group (s1K1C). Data are expressed as mean ± SEM. **P < 0.01 compared with corresponding control and sham-operated control groups.
Figure 10
Figure 10
Regulation of phosphorylated ERK MAPK in dissected RPE sheets from mice treated with Ang II infused subcutaneously via osmotic minipump. Proteins were extracted from RPE sheets (n = 6 eyes per group). A: Phosphorylated p-ERK protein expression evaluated using Western blot analysis in RPE from mice treated with Ang II for 1, 7, and 14 days. Top: Western blot from a representative experiment. Numbers on the left represent protein molecular mass in kilodaltons. Bottom: Ratio of p-ERK/GAPDH. Data are the mean ± SEM results from six eyes expressed as percentage of control. **P < 0.01 versus control. B: RPE-derived p-ERK protein expression evaluated using Western blot analysis in RPE sheets from mice treated with saline solution, Ang II, or Ang II in combination with candesartan (CD) or PD123319 (PD) for 30 days. Protein was extracted from RPE sheets (n = 6 eyes per group). Top: Western blot from a representative experiment. Numbers on the left represent protein molecular mass in kilodaltons. Bottom: Ratio of p-ERK/GAPDH. Data are expressed as percentage of control. Shown are mean ± SEM values. ***P < 0.001 versus control mice. †P < 0.001 versus mice treated with Ang I. C: RPE-derived p-ERK protein expression evaluated using Western blot analysis in RPE sheets from 1K1C mice with renovascular HTN and control 1K1C mice (s1K1C) underwent sham clipping and right nephrectomy. Top: p-ERK protein expression evaluated using Western blot analysis from a representative experiment. Numbers on the left represent protein molecular mass in kilodaltons. Bottom: Ratio of p-ERK/GAPDH. Data are expressed as percentage of control. Shown are mean ± SEM values.
Figure 11
Figure 11
Regulation of phosphorylated p38 (p-p38) MAPK in dissected RPE sheets from mice treated with Ang II. A: Expression of p-p38 protein evaluated using Western blot analysis in RPE sheets from mice treated with Ang II for 1, 7, and 14 days. Top: Western blot from a representative experiment. Numbers on the left represent protein molecular mass in kilodaltons. Bottom: Ratio of p-p38/GAPDH protein. Data are the mean results from six eyes and are expressed as percentage of control. Shown are mean ± SEM values. *P < 0.05 and **P < 0.01 versus control. B: RPE-derived p-p38 protein expression evaluated using Western blot analysis in RPE sheets from mice treated with saline solution, Ang II alone, or Ang II in combination with candesartan (CD) or PD123319 (PD) for 30 days. Protein was extracted from RPE sheets (n = 6 eyes per group). Top: Expression of p-p38 protein evaluated using Western blot analysis from a representative experiment. Numbers on the left represent protein molecular mass in kilodaltons. Bottom: Ratio of p-p38/GAPDH protein expression. Data are expressed as percentage of control. Shown are mean ± SEM values. **P < 0.01 versus control. †P < 0.01 versus mice treated with Ang II. C: RPE-derived p-p38 protein expression evaluated using Western blot analysis in RPE sheets from 1K1C mice with renovascular HTN and control 1K1C mice (s1K1C) that underwent sham clipping and right nephrectomy. Top: Ratio of p-p38/GAPDH protein expression evaluated using Western blot analysis from a representative experiment. Numbers on the left represent protein molecular mass in kilodaltons. Bottom: Data are expressed as percentage of control. Shown are mean ± SEM values.
Figure 12
Figure 12
Regulation of phosphorylated JNK MAPK in dissected RPE sheets from mice treated with Ang II infused subcutaneously using an osmotic minipump. Proteins were extracted from RPE sheets (n = 6 eyes per group). A: Phosphorylated JNK (p-pJNK) protein expression evaluated using Western blot analysis in RPE from mice treated with Ang II for 1, 7, and 14 days. Top: Western blot from a representative experiment. Numbers on the left represent protein molecular mass in kilodaltons. Bottom: Ratio of p-JNK/GAPDH. Data are the mean results from six eyes expressed as percentage of control. Shown are mean ± SEM values. *P < 0.05 and **P < 0.01 versus control. B: RPE-derived p-JNK protein expression evaluated using Western blot analysis in RPE sheets from mice treated with saline solution, Ang II, or Ang II in combination with candesartan (CD) or PD123319 (PD). Protein was extracted from RPE sheets (n = 6 eyes per group). Top: Western blot from a representative experiment. Numbers on the left represent protein molecular mass in kilodaltons. Bottom: Ratio of p-JNK/GAPDH. Data are expressed as percentage of control. Shown are mean ± SEM values. **P < 0.01 versus control; †P < 0.01 versus mice treated with Ang II. C: RPE-derived p-JNK protein expression evaluated using Western blot analysis in RPE sheets from 1K1C mice with renovascular HTN and control 1K1C mice (s1K1C) that underwent sham clipping and right nephrectomy. Top: Ratio of p-JNK/GAPDH protein expression evaluated using Western blot analysis from a representative experiment. Numbers on the left represent protein molecular mass in kilodaltons Bottom: Data are expressed as percentage of control. Shown are mean ± SEM values.
Figure 13
Figure 13
MMP-14 protein expression in isolated RPE sheets from control and treated mice. A: Effect on MMP-14 protein expression after 1, 7, and 14 days of treatment with saline solution or Ang II. B: RPE-derived MMP-14 protein expression in mice treated with saline solution, Ang II, or Ang II in combination with candesartan (CD) or PD123319 (PD) for 30 days. C: MMP-14 protein expression in RPE sheets from 1K1C mice and from the corresponding sham-operated control group (s1K1C). Mice were sacrificed at 30 days after initiation of renovascular HTN. Top panels: Ratio of MMP-14/GAPDH protein expression evaluated using Western blot analysis from a representative experiment. Numbers on the left represent protein molecular mass in kilodaltons. Bottom panels: Mean densitometry results. Data are expressed as percentage of control. Shown are mean ± SEM values. *P < 0.05 versus control; †P < 0.05 versus Ang II.
Figure 14
Figure 14
BSG protein expression in isolated RPE sheets from control and treated mice. A: RPE BSG protein expression in mice treated with saline solution or Ang II for 1, 7, and 14 days. B: BSG protein expression in mice treated with saline solution, Ang II, or Ang II in combination with candesartan (CD) or PD123319 (PD) for 30 days. C: BSG protein expression in RPE sheets from 1K1C mice and from the corresponding sham-operated control group (s1K1C). Mice were sacrificed at 30 days after initiation of renovascular HTN. Top panels: Ratio of BSG/GAPDH protein expression evaluated using Western blot analysis from a representative experiment. Numbers on the left represent protein molecular mass in kilodaltons. Bottom panels: Mean densitometry results. Data are expressed as percentage of control. Shown are mean ± SEM values. *P < 0.05; **P < 0.01 versus control; †P < 0.01 versus Ang II.
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