Angiotensin-(1-7) inhibits epidermal growth factor receptor transactivation via a Mas receptor-dependent pathway

Abstract

Background and purpose: The transactivation of the epidermal growth factor (EGF) receptor appears to be an important central transduction mechanism in mediating diabetes-induced vascular dysfunction. Angiotensin-(1-7) [Ang-(1-7)] via its Mas receptor can prevent the development of hyperglycaemia-induced cardiovascular complications. Here, we investigated whether Ang-(1-7) can inhibit hyperglycaemia-induced EGF receptor transactivation and its classical signalling via ERK1/2 and p38 MAPK in vivo and in vitro.

Experimental approach: Streptozotocin-induced diabetic rats were chronically treated with Ang-(1-7) or AG1478, a selective EGF receptor inhibitor, for 4 weeks and mechanistic studies performed in the isolated mesenteric vasculature bed as well as in primary cultures of vascular smooth muscle cells (VSMCs).

Key results: Diabetes significantly enhanced phosphorylation of EGF receptor at tyrosine residues Y992, Y1068, Y1086, Y1148, as well as ERK1/2 and p38 MAPK in the mesenteric vasculature bed whereas these changes were significantly attenuated upon Ang-(1-7) or AG1478 treatment. In VSMCs grown in conditions of high glucose (25 mM), an Src-dependent elevation in EGF receptor phosphorylation was observed. Ang-(1-7) inhibited both Ang II- and glucose-induced transactivation of EGF receptor. The inhibition of high glucose-mediated Src-dependant transactivation of EGF receptor by Ang-(1-7) could be prevented by a selective Mas receptor antagonist, D-Pro7-Ang-(1-7).

Conclusions and implications: These results show for the first time that Ang-(1-7) inhibits EGF receptor transactivation via a Mas receptor/Src-dependent pathway and might represent a novel general mechanism by which Ang-(1-7) exerts its beneficial effects in many disease states including diabetes-induced vascular dysfunction.

Figure 1

Diabetes-induced phosphorylation of EGF receptor occurs at multiple tyrosine residues that can be attenuated by chronic treatment with Ang-(1-7) or AG1478, a selective inhibitor of EGF receptor, in the mesenteric bed vasculature of STZ-induced diabetic rats. (A) A representative Western blot showing the levels of phosphorylated EGF receptor (P-EGFR) at the indicated tyrosines Y992, Y1068, Y1086 and Y1148, total EGF receptor (EGFR) and β-actin in the isolated mesenteric bed from normal controls (C), diabetic (D) and diabetic animals treated for 4 weeks with Ang-(1-7) (A1-7) or AG1478 (AG). (B–F) Densitometry histograms showing levels of total EGF receptor normalized to actin (B) and levels of phosphorylated EGF receptor at the stated tyrosine residue normalized to total EGF receptor (C–F). n = 6; mean ± SD. *Indicates significantly different (P < 0.05) mean values from normal non-diabetic rats (C), whereas # indicates significantly different mean values (P < 0.05) from diabetic rats (D).

Figure 2

The effect of chronic treatment with Ang-(1-7) or AG1478 on ERK1/2 and p38 MAPK signalling in the isolated mesenteric bed of STZ-induced diabetic rats. (A) Representative Western blot showing the levels of phosphorylated ERK1/2 and p38 MAPK (p P38) in the isolated mesenteric bed from normal controls (C), diabetic (D) and diabetic animals treated for 4 weeks with Ang-(1-7) [A1-7]or AG1478 [AG]. (B–C) Densitometry histograms showing levels of phosphorylated (p-) ERK1/2 (B) and p38 MAPK (C) normalized to actin. n = 6; Mean ± SD. *Indicates significantly different (P < 0.05) mean values from normal non-diabetic rats (C), whereas # indicates significantly different mean values (P < 0.05) from diabetic rats (D).

Figure 3

Ang-(1-7) is a dose-dependent inhibitor of high glucose-induced transactivation of epidermal growth factor (EGF) receptor at Y1068 and phosphorylation of p38 MAPK and ERK1/2 in a manner similar to AG1478 in VSMC. (A) Representative Western blot showing the levels of phosphorylated EGF receptor (Y1068), p38MAPK (p-P38) and ERK1/2 (p-ERK1/2) in VSMC grown in normal (5.5 mM) d-glucose (NG), high glucose (25.5 mM d-glucose; HG) or HG co-treated with increasing doses of AG1478 (AG) or Ang-(1-7) (labelled as A1-7). (B–D) Densitometry histograms showing levels of phosphorylated EGF receptor normalized to total EGF receptor (B) and levels of phosphorylated p38 MAPK (C) or ERK1/2 (D) normalized to actin. (E) and (F) Representative Western blot and histogram, respectively, showing that in contrast to 25 mM d-glucose (HG), the osmotic control, L-glucose (19.5 mM L-glucose + 5.5 mM d-glucose) had no significant effect on EGF receptor phosphorylation compared with normal glucose (5.5 mM glucose) in VSMC. n = 6; mean ± SD. *Indicates significantly different (P < 0.05) mean values from VSMC grown in normal 5.5 mM d-glucose (NG), whereas # indicates significantly different mean values (P < 0.05) from those grown in high glucose, 25.5 mM d-glucose (HG).

Figure 4

Ang-(1-7) inhibits Ang II-mediated transactivation of EGF receptor and signalling via p38 MAPK and ERK1/2 in VSMC in a manner similar to that of an AT₁ receptor antagonist, losartan, and AG1478, a selective inhibitor of EGF receptor phosphorylation. (A) Representative Western blot showing the levels of phosphorylated EGF receptor (Y1068), p38MAPK (p-P38) and ERK1/2 (p-ERK1/2), total EGF receptor (EGFR) and actin in VSMC grown in normal (5.5 mM) d-glucose (NG), or treated with 1 µM Ang II, 1 µM losartan (LOS), 100 µM AG1478 (AG) or 1 µM Ang-(1-7) (labelled as A1-7); (B–D) Densitometry histograms showing levels of phosphorylated EGF receptor normalized to total EGF receptor (B) and levels of phosphorylated p38 MAPK (C) or ERK1/2 (D) normalized to actin. n = 6; mean ± SD. *Indicates significantly different (P < 0.05) mean values from VSMC grown in normal 5.5 mM d-glucose (NG), whereas # indicates significantly different mean values (P < 0.05) from those grown in high glucose 25.5 mM d-glucose (HG).

Figure 5

High glucose-mediated EGF receptor transactivation occurs via Src-depedent pathway. (A) Representative Western blot showing the levels of phosphorylated Src at Y436 (p-Src), total Src (Src), phosphorylated EGF receptor at Y1068 (p-EGFR), total EGF receptor (EGFR) and phosphorylated ERK1/2 in VSMC grown in normal (5.5 mM) d-glucose (NG), high glucose (25.5 mM) d-glucose (HG) or HG treated with increasing doses (2 and 25 µM) of Src selective inhibitor, SU6656 (lanes labelled as HG+ SU 2 and HG+ SU 25, respectively). (B–D) Densitometry histograms showing levels of phosphorylated Src (Y416) normalized to total Src (B), phosphorylated EGF receptor normalized to total EGF receptor (B) and levels of phosphorylated ERK1/2 (D) normalized to actin. (E) Representative Western Blot and (F) densitometry histogram showing that in contrast to 25 mM d-glucose (HG), the osmotic control, L-glucose (19.5 mM L-glucose + 5.5 mM d-glucose) had no significant effect on Src phosphorylation compared with normal glucose (5.5 mM glucose) in VSMC. n = 6; mean ± SD. *Indicates significantly different (P < 0.05) mean values from VSMC grown in normal 5.5 mM d-glucose (NG), whereas # indicates significantly different mean values (P < 0.05) from those grown in high glucose 25.5 mM d-glucose (HG).

Figure 6

The effect of Ang-(1-7) and the selective Mas receptor inhibitor [D-Pro7-Ang-(1-7)] on glucose-induced Src phosphorylation and subsequent signalling via EGF receptor/ERK1/2 pathway in VSMC. (A) Representative Western blot showing the levels of phosphorylated Src at Y416 (p-Src), total Src (Src), phosphorylated EGF receptor at Y1068 (p-EGFR), total EGF receptor (EGFR) and phosphorylated ERK1/2 in VSMC grown in normal (5.5 mM) d-glucose (NG), high glucose (25.5 mM) d-glucose (HG) or HG treated with 1 µM Ang-(1-7) alone or together with 10 µM D-Pro7-Ang-(1-7) (lanes labelled as HG+A1-7 and HG+A1-7 + D-PRO, respectively). (B–D) Densitometry histograms showing levels of phosphorylated Src normalized to total Src (B), phosphorylated EGF receptor normalized to total EGF receptor (C), and levels of phosphorylated ERK1/2 (D) normalized to actin. n = 6; mean ± SD. *Indicates significantly different (P < 0.05) mean values from VSMC grown in normal 5.5 mM d-glucose (NG), whereas # indicates significantly different mean values (P < 0.05) from those grown in high glucose 25.5 mM d-glucose (HG).

Figure 7

Model of how Ang-(1-7) via its Mas receptor might inhibit diabetes or high glucose-induced EGF receptor signalling and prevent vascular complications associated with diabetes and/or hyperglycaemia. The pharmacological inhibitors used in the present study are also indicated. (See text for further explanation.)