| dc.description.abstract | To fully understand the physiological and biochemical roles of the forkhead transcription factors FOXO1, FOXO3, FOXO4, and FOXO6 in fat cells, we stably cloned NIH3T3, 3T3-L1 and C3H10T1/2 fibroblasts with overexpression of the respective FOXO gene. Different FOXO cDNAs inserted into the pMSCV-neo vector (provided by Professor Shen-Liang Chen) were respectively transfected into murine GP+E-86 retrovirus package cells and then selected with G418 (600 μg/ml) for 2 weeks. The harvested retrovirus containing the inserted FOXO gene from the culture medium was directly transferred to NIH3T3, 3T3-L1, or C3H10T1/2 cells and G418 (600 ~ 800 μg/ml) was added to the medium 2 d after the initial infection. After an additional 2-week selection with G418, total RNA isolated from the stable clones was verified with the expression levels of FOXO gene using the methods of RT-PCR and Western blotting. We successfully cloned the NIH3T3, 3T3-L1, and C3H10T1/2 fibroblasts overexpressing with the following wild types and mutants of FOXO transcription factors: the wild types of human FOXO1 (hFOXO1-WT), mouse FoxO3 (mFoxO3-WT), mouse FoxO4 (mFoxO4-WT) , and mouse FoxO6 (mFoxO6-WT); the constitutively active mutants of human FOXO1-AAA (hFOXO1-AAA), human FOXO3-AAA (hFOXO3-AAA), human FOXO4-AAA (hFOXO4-AAA), and mouse FoxO6-S184A (mFoxO6-S184A); and a DNA binding-deficient type of human FOXO1 (hFOXO1-H215R). In NIH3T3 cells, expression with hFOXO1-WT, hFOXO1-AAA, mFoxO3-WT, hFOXO3-AAA, mFoxO6-WT, or mFoxO6-S184A reduced the cell number during a 5-day period of incubation. In 3T3-L1 preadipocytes, expression with either hFOXO1-WT、mFoxO3-WT or hFOXO3-AAA inhibited cell growth, while expression with mFOXO4-WT, or mFoxO6-WT stimulated cell growth. In C3H10T1/2 preadipocytes, expression with hFOXO1-WT, hFOXO1-AAA, hFOXO1-H215R, mFoxO4-WT, or mFoxO6-WT inhibited cell growth, while expression with either mFoxO3-WT, mFoxO4-AAA-FLAG or mFoxO6-S184A stimulated cell growth. The morphology of each clone and parental cell was observed and photographed with no significant change. Using RT-PCR, we further observed that levels of adipogenic resistin, adiponectin, and aP2 mRNAs were altered in the FOXO1-overexpressed NIH3T3, 3T3-L1 and C3H10T1/2 cells when compared to those in the empty pMSCV-neo vector-transfected cells. As NIH3T3, 3T3-L1 and C3H10T1/2 fibroblasts can be differentiated into adipocytes, results of this study suggest the different physiological and biochemical roles of the distinct FOXO transcription factors on preadipocyte growth and adipocytokine gene expression.
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