Protein Kinase A-α Directly Phosphorylates FoxO1 in Vascular Endothelial Cells to Regulate Expression of Vascular Cellular Adhesion Molecule-1 mRNA

Abstract

Forkhead box O1 (FoxO1) is a member of the FoxO transcription factor family that plays important roles in regulation of glucose homeostasis, cellular proliferation, differentiation, and vascular homeostasis in response to insulin and other growth factors (1-3). Mice lacking FoxO1 die in utero from improper vascular development (4). Overexpression of FoxO1 in primary endothelial cells impairs cell migration and tube formation, whereas knockdown of FoxO1 using siRNA enhances angiogenic function (5). Moreover, siRNA knockdown of FoxO1 in human coronary artery endothelial cells reduces VEGF-induced vascular cellular adhesion molecule-1 (VCAM-1) 2 expression and monocyte adhesion to endothelial cells (6). FoxO1 function is regulated, in part, by post-translational modifications including phosphorylation, acetylation, and ubiquitination (7-9). Phosphorylation of FoxO1 at a number of specific regulatory sites results in translocation of FoxO1 from the nucleus to the cytosol that impairs its transcriptional activity (2). Akt, a serine/threonine kinase downstream from PI3K in insulin signaling pathways, phosphorylate FoxO1 at Thr 24 , Ser 256 , and Ser 319 to promote nuclear exclusion of FoxO1. Thus, insulin negatively regulates FoxO1 functions via phosphorylation by Akt (10, 11). In addition to Akt, other kinases including SGK phosphorylate FoxO1 to regulate its function in a similar manner. For example, SGK phosphorylates FoxO1 at Thr 24 and Ser 319 (11,12). Similar to Akt, SGK is activated by a variety of growth and survival factors including insulin (13,14). cAMP-dependent protein kinase (PKA) is a key regulator of many processes involved with cell growth and development. PKA is activated when cAMP binds to the regulatory subunit of PKA, resulting in release of the catalytic subunit that then phosphorylates a variety of protein substrates including ion channels, key metabolic enzymes, and transcription factors (15).In a previous study, we reported that dihydroepiandrosterone treatment of primary endothelial cells acutely increases phosphorylation of FoxO1 in a PKA-dependent manner to reduce expression of ET-1 by interfering with the binding of FoxO1 to the human ET-1 promoter (3). Therefore, in the present study, we tested the hypothesis that FoxO1 is a novel direct substrate for PKA-␣ that helps to regulate endothelial function in response to activation of PKA-␣.

Apolipoprotein A-I Increases Insulin Secretion and Production From Pancreatic β-Cells via a G-Protein-cAMP-PKA-FoxO1–Dependent Mechanism

“…Because PKA can also directly phosphorylate FoxO1 in endothelial cells, 36 we asked whether this might explain the previously reported apoA-I-mediated increase in insulin synthesis in MIN6 cells 10 and in Ins-1E cells in the present study. The result in Figure 4 showing that apoA-I excludes FoxO1 from the nucleus of Ins-1E cells is consistent with this notion.…”

Increased Atherosclerosis and Endothelial Dysfunction in Mice Bearing Constitutively Deacetylated Alleles of Foxo1 Gene

“…We (38) and others (32) have shown that Icam and Vcam are FoxO1 targets. In primary endothelial cells derived from Foxo1 KR/KR mice, we observed increased expression of Icam1 and decreased expression of Vcam1, consistent with our previous observations that the effects of the FoxO1 deacetylated mutant are target genespecific (15) (Fig.…”

Forkhead box O transcription factors as possible mediators in the development of major depression

“…There is interplay between the PI3K/Akt/FoxO and cAMP/PKA signaling pathways (Chen et al, 2007). FoxO1 is a direct substrate for PKA and PKA phosphorylates FoxO1 at Thr24, Ser256, and Ser319, three known Akt phosphorylation sites (Lee et al, 2011). Epac is an exchange protein activated by cAMP.…”