Akt-FOXO3a Signaling Affects Human Endothelial Progenitor Cell Differentiation

153 Hypertens Res Vol.31 (2008) No.1 p.153-159 Original Article Akt-FOXO3a Signaling Affects Human Endothelial Progenitor Cell Differentiation Masaki MOGI1),2), Kenneth WALSH2), Masaru IWAI1), and Masatsugu HORIUCHI1) Here we address the effect of Akt signaling on endothelial progenitor cells (EPCs). Human peripheral blood mononuclear cells (PBMCs) were cultured on fibronectin-coated dishes in EPC differentiation medium. PBMCs differentiated in a series of three steps: proliferation for foci formation, tight attachment to the dishes in the early stages of differentiation, and maturation in the late stages. In Western blot analysis, Akt expression was attenuated in the early stages of differentiation and was gradually upregulated during EPC maturation. Forkhead box–containing protein, class O 3a (FOXO3a), an Akt downstream target, was downregulated through phosphorylation in the late stages of EPC differentiation. Adenovirus-mediated overexpression of activated FOXO3a in PBMCs markedly increased the number of cell foci but reduced the number of DiI-acetyl LDL EPCs that appear at later time points. These data suggest that Akt/FOXO3a signaling is an important regulator of EPC maturation. (Hypertens Res 2008; 31: 153–159) Key Words: Akt, FOXO3a, endothelial progenitor cells, mononuclear cells, differentiation Introduction The serine-threonine protein kinase Akt (also known as protein kinase B, or PKB) functions downstream from phosphatidylinositol 3-kinase (PI3K) as an important regulator of cell survival, growth, and glucose metabolism in many cell types (1). Previously, we reported the role of Akt in hematopoietic stem cells using an Akt-gene knockout mouse model (2). Bone marrow from Akt1-deficient mice exhibits a reduced side-population (SP) fraction due to attenuated translocation of Bcrp1 by Akt. PI3K/Akt signaling has also been implicated in the differentiation of a variety of cell types, including hematopoietic cells (3–5). However, the regulation and expression of Akt during differentiation are unclear. Forkhead box–containing protein, O subfamily (FOXO) factors are downstream effectors of Akt that play a pivotal role in the regulation of cell-cycle progression and cell survival (6). FOXOs also respond to extracellular cues via changes in Akt signaling to control the differentiation and transformation of many cell types. Three FOXO factors (1, 3a, and 4) are substrates of the Akt protein kinase. They are inactivated through phosphorylation, which results in sustained nuclear exclusion (7, 8). FOXO factors are reported to be developmentally regulated during embryogenesis and myoblast differentiation (6). Although FOXO3a (FKHRL1) is reported to be expressed in hematopoietic progenitors (9, 10) and to affect hematopoiesis by controlling apoptosis signaling (11), FOXO3a’s effects on stem-progenitor cell differentiation remain unclear. Recently, FOXO1 (FKHR)-deficient mice were reported to display abnormal angiogenesis, indicating that FOXO1 plays a critical role in normal vascular development in rodents (12) (13). However, little is known about the function of FOXO3a in endothelial progenitor cell (EPC) development. Therefore, to elucidate the intracellular signaling mechanisms that control EPCs during differentiation, we examined the detailed differentiation steps of EPCs, the expression of Akt and From the 1)Department of Molecular Cardiovascular Biology and Pharmacology, Ehime University Graduate School of Medicine, Toon, Japan; and 2) Molecular Cardiology/Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, USA. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to M.H. and M.M.); from the Suzuken Memorial Foundation (to M.M.); and by the NIH (no. HL081587; to K.W.). Address for Reprints: Masatsugu Horiuchi, M.D., Ph.D., Department of Molecular Cardiovascular Biology and Pharmacology, Ehime University Graduate School of Medicine, Toon 791–0295, Japan. E-mail: Received April 22, 2007; Accepted in revised form August 5, 2007. 154 Hypertens Res Vol. 31, No. 1 (2008) Day 14 A g h Day 0 a f c B d e a b Attached ring formation Central cluster C Day 2 Circulating form a DAPI DAPI Mac-1 d l h Merge o Flk-1 Merge DAPI eNOS Mac-1 vWF w Flk-1 t Merge DAPI v s p Merge u r eNOS Mac-1 vWF q n k Day 8 Sprouting form DAPI DAPI eNOS g Flk-1 m j f c Day 5 Focus-formation i e b b vWF x Merge Merge Mogi et al: Akt-FOXO3a Signaling in EPC Differentiation FOXO3a, and the effects of enforced FKHRL1 activation in EPCs. Here, we demonstrated that the Akt-FOXO3a signaling axis can control the differentiation of progenitor cells. Methods Endothelial Progenitor Assay EPCs were cultured as described previously (14). Human peripheral blood monocytes (PBMCs) from healthy volunteers were isolated with Histopaque 1077 (Sigma, St. Louis, USA). PBMCs were cultured on fibronectin (Sigma)-coated 10-cm dishes or 6-well plates (BD Falcon, Franklin Lakes, USA) in EGM-2 medium (Clonetics, Walkersville, USA) containing 10% FBS, endothelial cell growth supplement, and antibiotics (Invitrogen, Carlsbad, USA) without corticosteroid. EPCs were defined 7 days after culture by staining with both fluorescein isothiocyanate (FITC)–labeled lectin (Sigma) and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanineperchlorate–labeled acetylated low-density lipoprotein (LDL) [DiI-acLDL] (Biomedical Technologies, Stoughton, USA) in three different fields. 155 (15). The following primary antibodies were used: anti-Akt (Santa Cruz Biotechnology), anti-phospho-Akt (Cell Signaling Technology, Beverly, USA), anti-FOXO3a (rabbit polyclonal IgG, Upstate Biotechnology), anti-phospho-FOXO3a (Ser253) (rabbit polyclonal IgG, Upstate Biotechnology), anti-phospho-FOXO3a (Thr32) (rabbit polyclonal IgG, Upstate Biotechnology). Adenovirus Gene Transfer A green fluorescent protein (GFP)–containing adenoviral vector of FOXO3a-AAA triple mutant (TM-FOXO3a), which was not phosphorylatable because three phosphorylation sites (Thr32, Ser253, and Ser315) were replaced by alanine residues, was used as described previously (16). Transfection efficiency estimated by GFP was usually expressed in 50 to 70% of the cells. Statistical Analysis Data are shown as mean±SEM All other data were evaluated with the two-tailed, unpaired Student’s t-test or compared by one-way analysis of variance. Immunofluorescent Staining Immunofluorescent staining was performed as described previously (2). Attached cells on fibronectin-coated dishes were washed and then fixed with ethanol and methanol solution (1:1 mixed) and permeabilized with 0.1% saponin prior to incubation with primary antibody. The following primary antibodies were used: anti–macrophage antigen-1 (Mac-1) and anti–von Willebrand factor (vWF) (BD Pharmingen, Franklin Lakes, USA), anti-flk-1 and anti–endothelial nitricoxide sy (...truncated)