Expression of the AT2 receptor developmentally programs extracellular signal-regulated kinase activity and influences fetal vascular growth

Abstract

Angiotensin II type 2 (AT2) receptor is abundantly expressed in vascular smooth muscle cells (VSMC) of the fetal vasculature during late gestation (embryonic day 15-20), during which the blood vessels undergo remodeling. To examine directly the influence of AT2 receptor expression in the developmental biology of VSMC, we studied cultures of VSMC from fetal and postnatal wild-type (Agtr2(+)) and AT2 receptor null (Agtr2(-)) mice. Consistent with in vivo data, AT2 receptor binding in cultured Agtr2(+) VSMC increased by age, peaking at embryonic day 20, and decreased dramatically after birth. Angiotensin II-induced growth in Agtr2(+) VSMC (embryonic day 20) was increased by the AT2 receptor blocker PD123319, indicating that the AT2 receptors are functional and exert an antigrowth effect in Agtr2(+) VSMC. Growth of VSMC in response to serum decreased age dependently and was higher in Agtr2(-) than in Agtr2(+), inversely correlating with AT2 receptor expression. However, serum-induced growth in Agtr2(+) and Agtr2(-) VSMC and the exaggerated Agtr2(-) VSMC growth was maintained even in the presence of PD123319 or losartan, an AT1 receptor blocker. Moreover, Agtr2(-) VSMC showed greater growth responses to platelet-derived growth factor and basic fibroblast growth factor, indicating that Agtr2(-) cells exhibit a generalized exaggerated growth phenotype. We studied the mechanism responsible for this phenotype and observed that extracellular signal-regulated kinase (ERK) activity was higher in Agtr2(-) VSMC at baseline and also in response to serum. ERK kinase inhibitor PD98059 inhibited both growth and ERK phosphorylation dose-dependently, while the regression lines between growth and ERK phosphorylation were identical in Agtr2(+) and Agtr2(-) VSMC, suggesting that increased ERK activity in Agtr2(-) VSMC is pivotal in the growth enhancement. Furthermore, the difference in ERK phosphorylation between Agtr2(+) and Agtr2(-) was abolished by vanadate but not by okadaic acid, implicating tyrosine phosphatase in the difference in ERK activity. These results suggest that the AT2 receptor expression during the fetal vasculogenesis influences the growth phenotype of VSMC via the modulation of ERK cascade.

Figure 1

AT1 and AT2 receptor binding densities in wild-type (Agtr2⁺) and AT2 receptor null (Agtr2^–) VSMC. Radioligand binding assay was performed using subconfluent Agtr2⁺ and Agtr2^– VSMC derived from embryonic day 15 (E15), day 18 (E18), and day 20 (E20) fetuses, and from mice of postnatal day 7 (7d) and day 28 (28d). Similar results were obtained in at least three different culture lines. The values are expressed as mean ± SEM (n = 4). ^§P < 0.05 vs. E15; *, **P < 0.05, 0.01 vs. 28d. AT1, angiotensin II type 1; AT2, angiotensin II type 2; VSMC, vascular smooth muscle cells.

Figure 2

Developmental change in wild-type (Agtr2⁺) and AT2 receptor null (Agtr2^–) VSMC growth. The numbers of VSMC derived from aortae of embryonic day 15 (E15), day 18 (E18), and day 20 (E20) fetuses and from aortae of postnatal day 7 (7d) and day 28 (28d) mice were counted at 1 day and 5 days after seeding, and then averaged for four wells. The growth of each cell culture line in response to 10% FBS was expressed as the fold increase in cell number from 1 day to 5 days. The values are expressed as mean ± SEM of five to nine independent experiments (the number is indicated in parenthesis), each of which consists of several Agtr2⁺ and Agtr2^– littermate culture lines. *P < 0.05 by paired t test; ^§P < 0.05 vs. E15 by Newman-Keuls’ test.

Figure 3

(a–c) Growth of wild-type (Agtr2⁺) and AT2 receptor null (Agtr2^–) VSMC derived from the aortae of embryonic day 20 fetuses, in response to angiotensin II (Ang II) (a), FBS (b), or other growth factors (c). (a) Subconfluent, quiescent second-passage cells were treated with vehicle, Ang II (0.3 μM), Ang II plus PD123319 (10 μM), or Ang II plus losartan (10 μM) for 3 days. The cell number is expressed as percent of the number before the treatment (baseline). Similar results were obtained in three different culture lines. The values are expressed as mean ± SEM (n = 6). *P < 0.05 vs. vehicle, Ang II, and Ang II plus losartan; † P < 0.05 vs. vehicle and Ang II plus losartan. (b) Subconfluent, quiescent second-passage cells were treated with vehicle, 10% FBS, 10% FBS plus PD123319 (10 μM), or 10% FBS plus losartan (10 μM) for 3 days. The cell number is expressed as percent of the number before the treatment (baseline). Similar results were obtained in three different culture lines. The values are expressed as mean ± SEM (n = 4). (c) DNA synthesis was assayed by measuring ³H-thymidine incorporation. Subconfluent, quiescent second-passage cells were treated with vehicle, 10% FBS, PDGF-BB (10 ng/ml), or bFGF (10 ng/ml). Similar results were obtained in three different culture lines. The values are expressed as mean ± SEM (n = 4). *, **P < 0.05, 0.01 vs. Agtr2⁺. bFGF, basic fibroblast growth factor; PDGF, platelet-derived growth factor.

Figure 4

ERK activity at baseline and in response to serum in wild-type (Agtr2⁺) and AT2 receptor null (Agtr2^–) VSMC derived from the aortae of embryonic day 20 fetuses. (a) Subconfluent, quiescent second-passage cells were treated with 10% FBS for the indicated time. The cell lysate was applied to the kinase assay for ERK and immunoblots for phospho-ERK and ERK. (b) Densitometric measurements of phospho-ERK (ERK-1 and ERK-2) at baseline and 15 min after serum stimulation. The values are expressed as mean ± SEM of four different pair of culture lines. ERK, extracellular signal-regulated kinase; MBP, myelin basic protein.

Figure 5

Effects of MEK inhibitor on DNA synthesis and ERK phosphorylation in wild-type (Agtr2⁺) and AT2 receptor null (Agtr2^–) VSMC derived from the aortae of embryonic day 20 fetuses. Subconfluent, quiescent cells were treated with 10% FBS plus 0–100 μM MEK inhibitor PD98059. (a) DNA synthesis was assayed as ³H-thymidine incorporation (TdR). The values are expressed as mean ± SEM (n = 6). *, **P < 0.05, 0.01 vs. PD98059 (–); ^§P < 0.05 vs. Agtr2⁺. (b and c) Immunoblotting for phospho-ERK and its densitometric analysis are shown. (d) Scatter diagram showing the correlation between DNA synthesis and ERK phosphorylation. The regression lines are identical in Agtr2⁺ and Agtr2^–. MEK, ERK kinase.

Figure 6

ERK dephosphorylation and MKP-1 protein level in wild-type (Agtr2⁺) and AT2 receptor null (Agtr2^–) VSMC. (a) Phosphorylated ERK was incubated with the lysates prepared from Agtr2⁺ and Agtr2^– VSMC and was detected by immunoblotting. The density of the band was compared with that treated without the sample lysate; then, dephosphorylation was calculated as percent decrease in ERK phosphorylation. (b) Protein levels of MKP-1 in the lysates were determined by immunoblotting using the anti–MKP-1 antibody. The values are expressed as mean ± SEM of four different pair of culture lines. MKP-1, MAP kinase phosphatase-1.

Figure 7

Effects of vanadate and okadaic acid on ERK phosphorylation in wild-type (Agtr2⁺) and AT2 receptor null (Agtr2^–) VSMC derived from the aortae of embryonic day 20 fetuses. Subconfluent VSMC were treated with vanadate (Va; 0–20 μM) or okadaic acid (Oka; 0 or 100 nM) in serum-free medium for 16 h. (a) Immunoblotting for phospho-ERK and ERK. (b) The densitometric analysis for phospho-ERK. The values are expressed as mean ± SEM of five (vanadate) or four (okadaic acid) different pair of culture lines.