Akt Is a Major Downstream Target of PI3-Kinase Involved in Angiotensin II–Induced Proliferation

Abstract

Abstract-Different signal transduction cascades have been implicated in angiotensin II (Ang II)-mediated cell growth, such as the extracellular signal-regulated kinase 1/2 (ERK1/2) and the phosphatidylinositol 3-kinase (PI3K) pathways.To identify the downstream targets of PI3K involved in Ang II-induced proliferation, we used both rat aortic smooth muscle (RASM) cells and a CHO cell line stably expressing the rat AT 1A receptor. The ERK1/2 and PI3K pathways are independently activated and implicated in Ang II-mediated DNA synthesis and cell number increase in these 2 cell lines. In addition, a specific inhibitor of Akt inhibited Ang II-induced Akt phosphorylation, DNA synthesis and proliferation in CHO-AT 1A or RASM cells. A dominant-negative mutant of Akt was also found to selectively block Ang II-induced proliferation of CHO-AT 1A cells. To further elucidate the signaling events leading to Akt activation, we used an AT 1 receptor mutant (AT 1A D74E), deficient for Gq protein coupling, and the intracellular calcium chelator BAPTA-AM. Although altered Akt and ERK1/2 activation was observed in the CHO-AT 1A D74E cell line, blockade of intracellular calcium elevation did not affect phosphorylation of these kinases. These results provide the first evidence of a specific and necessary role of Akt in Ang II-induced proliferation through a Gq protein-dependent calcium-independent pathway. (Hypertension. 2003;41:882-890.)

Akt Down-Regulates ERK1/2 Nuclear Localization and Angiotensin II-induced Cell Proliferation through PEA-15

“…Twenty-four clones expressing various levels of HA-Akt1 were isolated and analyzed. As shown in Figure 1A (right), one representative cell line of this clone In both cell lines, phosphorylated Akt was inhibited by the PI3K inhibitor LY294002 ( Figure 1A, left) but unaffected by the MEK inhibitor U0126 (our unpublished data), as already shown for endogenous Akt (Dugourd et al, 2003). These results indicate that Akt overexpression led to an increased Akt activation upon AngII stimulation that is PI3K dependent and MEK/ERK1/2 independent.…”

“…sion did not alter AngII-induced ERK1/2 activation that remained MEK-dependent but PI3K-independent, as observed previously in wild-type CHO-AT 1A cells (Dugourd et al, 2003). We then analyzed the functional consequences of PEA-15 overexpression on the activation of the nuclear targets of ERK1/2, such as Elk-1.…”

“…For DNA synthesis quantification, [ 3 H]thymidine incorporation experiments were performed as described previously (Dugourd et al, 2003). After G 0 arrest, cells were stimulated with 100 nM AngII for 16 h and labeled with 1 Ci/ml [ 3 H]thymidine for 2 h. After washing with trichloroacetic acid, radioactivity was measured by liquid scintillation counting.…”

“…We have previously shown that endogenous PI3K/Akt and ERK1/2 pathways are both necessary for mediating proliferation in CHO cells stably expressing the rat type 1 AngII receptor (CHO-AT 1A ) (Dugourd et al, 2003). The modification of the precise balance between these two endogenous pathways remains poorly investigated even though it could lead to dramatic changes in the cellular response.…”

“…We have previously reported that angiotensin II (AngII) stimulates cell proliferation through the angiotensin type 1A receptor (AT 1A ) by activation of the endogenous PI3K and ERK1/2 pathways in transfected Chinese hamster ovary (CHO) cells (CHO-AT 1A ) and in rat aortic smooth muscle cells (Dugourd et al, 2003). In this process, endogenous Akt and ERK1/2 were independently stimulated but were both necessary for AngII-induced cell proliferation.…”