Apelin-13 induces proliferation, migration, and collagen I mRNA expression in human RPE cells via PI3K/Akt and MEK/Erk signaling pathways

Abstract

Purpose: Our previous study showed that apelin was increased in the vitreous and fibrotic membranes of patients with proliferative diabetic retinopathy (PDR) in vivo, which suggested that apelin may be involved in the development of PDR. In this study, we investigated whether the expression of apelin was upregulated in human retinal pigment epithelial (RPE) cells in vitro under high glucose conditions. Furthermore, to explore the role of apelin in RPE cells, we investigated the effect of exogenous recombinant apelin on proliferation, migration, and collagen I (a major component of extracellular matrix molecules, associated with PDR) expression and investigated the signaling pathways involved in these processes.

Methods: Real-time PCR and western blot were performed to determine the apelin expression in ARPE-19 cells under high glucose conditions. Exogenous recombinant apelin was used to study the effect of apelin on ARPE-19 cells in vitro. Cell proliferation, migration, and collagen I expression were assessed using an MTT assay, a transwell assay, and real-time PCR analysis. LY294002 (an inhibitor of phosphatidylinositol 3-kinase) and PD98059 (an inhibitor of mitogen-activated protein kinase) were used to help to determine the apelin signaling mechanism.

Results: High glucose upregulated apelin expression in RPE cells. Exogenous recombinant apelin activated protein kinase B (Akt) and extracellular signal-regulated kinase (Erk) phosphorylation and promoted proliferation, migration, and collagen I expression in RPE cells. Pretreatment with LY294002 and PD98059 abolished apelin-induced activation of Akt and Erk, proliferation, and collagen I expression. Apelin-induced migration was partially blocked by pretreatment with LY294002 and PD98059.

Conclusions: The expression of apelin was upregulated under high glucose conditions in RPE cells in vitro. Exogenous recombinant apelin increased the biologic activity of RPE cells, as well as the expression of collagen I. Apelin promoted proliferation, migration, and collagen I expression through the PI3K/Akt and MEK/Erk signaling pathways in RPE cells. From these results, we revealed the role of apelin in regulating proliferation, migration, and collagen I expression in RPE cells and the signaling mechanism under these processes, which suggested that apelin may play a profibrotic role in the development of PDR.

Figure 1

Effect of high glucose concentration on apelin expression in RPE cells. A: Real-time PCR analysis of apelin mRNA expression. RPE cells were exposed to normal glucose (NG) and high glucose (HG) for 48 h. Compared with the NG, the expression of apelin was upregulated in response to HG. B: Western blot analysis of apelin protein expression. RPE cells were exposed to NG and HG for 48 h. C: The expression of apelin on protein increased under HG conditions compared with NG. NG and HG represent the normal glucose group and the high glucose group. The data shown represent the mean±standard deviation (SD) of three independent experiments, *p<0.05 versus NG.

Figure 2

Apelin-induced phosphorylation of Erk and Akt. A, B, C: RPE cells were incubated with 10⁻⁸ M, 10⁻⁷ M, and 10⁻⁶ M apelin for 30 min for assay of Erk and Akt phosphorylation, levels of phosphorylated and total Akt and Erk were determined with western blot analysis, respectively. RPE cells were pretreated with 10 μM LY294002 (D, E) or 20 μM PD98059 (F, G) for 30 min and then incubated with 10⁻⁷ M apelin for 30 min for assay of Akt and Erk phosphorylation. Levels of phosphorylated Akt and Erk were determined with western blot analysis. The data represent the mean±standard deviation (SD) of three independent experiments, *p<0.001 versus untreated control.

Figure 3

Apelin-induced proliferation, migration, and collagen I expression in RPE cells. A: RPE cell proliferation was determined with MTT after 48 h incubation with concentrations of 10⁻⁸ M, 10⁻⁷ M, and 10⁻⁶ M apelin. The 10⁻⁸ M and 10⁻⁷ M apelin treatment increased RPE cell proliferation. *p<0.05 versus untreated control. B, C: RPE cell migration in response to apelin treatment was measured using a transwell assay (a: control; b: 10⁻⁸ M apelin; c: 10⁻⁷ M apelin; d: 10⁻⁶ M apelin. 200X magnification). The values were assessed by the mean number of migrated cells. The number of migrated cells per high power field (HPF) is shown. *p<0.001 versus untreated control. D: Collagen I expression in RPE cells was determined with real-time PCR. Time-dependent induction of collagen I expression after stimulation with 10⁻⁷ M apelin. *p<0.05 versus untreated control. The data are expressed as means±standard deviation (SD), and the experiments were performed independently three times.

Figure 4

Distinct intracellular signaling pathways mediate apelin-induced proliferation, migration, and collagen I production. A: RPE cells were treated with various kinase inhibitors in the presence of 10⁻⁷ M apelin. Blocking apelin-induced RPE cell proliferation by pretreatment with 10 μM LY294002 and 20 μM PD98059 for 30 min, as measured with MTT assay after 48 h incubation. *p<0.05 versus untreated control. #p<0.05 versus apelin-treated alone. B, C: Inhibition of apelin-induced migration by pretreatment with 10 μM LY294002 and 20 μM PD98059 for 30 min, as measured with a transwell assay after 6 h incubation (a: control; b: 10⁻⁶ M apelin; c: LY294002+10⁻⁶ M apelin; d: PD98059+10⁻⁶ M apelin. 200× magnification). The number of migrated cells per HPF is shown. *p<0.001 versus untreated control. #p<0.01 versus apelin-treated alone. D: Complete inhibition of apelin-induced collagen I expression by 10 μM LY294002 and 20 μM PD98059. RPE cells were stimulated with 10⁻⁷ M apelin with or without pretreatment with kinase inhibitors for 30 min. After 3 h incubation, RNA was extracted, and collagen I expression was measured with real-time PCR. *p<0.05 versus untreated control. #p<0.05 versus apelin-treated alone. The data shown represent the mean±standard deviation (SD) of three independent experiments.