FoxO1 Protein Cooperates with ATF4 Protein in Osteoblasts to

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  • FoxO1 Protein Cooperates with ATF4 Protein in Osteoblaststo Control Glucose Homeostasis*SReceived for publication, July 14, 2011, and in revised form, January 6, 2012 Published, JBC Papers in Press, February 1, 2012, DOI 10.1074/jbc.M111.282897

    Aruna Kode, Ioanna Mosialou, Barbara C. Silva, Sneha Joshi, Mathieu Ferron, Marie Therese Rached1,and Stavroula Kousteni2

    From the Department of Medicine, Division of Endocrinology and the Department of Genetics and Development, College ofPhysicians and Surgeons, Columbia University, New York, New York 10032 and the Department of Physiology and CellularBiophysics, College of Physicians and Surgeons, New York, New York 10032

    Background: The skeleton regulates glucose metabolism and energy expenditure.Results: Two transcription factors interact to regulate the activity of an osteoblast-secreted hormone favoring energymetabolism.Conclusion: The skeleton utilizes an intricate transcriptional machinery to maintain energy homeostasis.Significance: Transcription factor-mediated regulation of energy metabolism by the skeleton has potential applications indiseases of abnormal glucose metabolism.

    The Forkhead transcription factor FoxO1 inhibits through itsexpression in osteoblasts -cell proliferation, insulin secretion,and sensitivity. At least part of the FoxO1 metabolic functionsresult from its ability to suppress the activity of osteocalcin, anosteoblast-derived hormone favoring glucose metabolism andenergy expenditure. In searching formechanismsmediating themetabolic actions of FoxO1, we focused on ATF4, because thistranscription factor also affects glucose metabolism throughits expression in osteoblasts. We show here that FoxO1 co-lo-calizes with ATF4 in the osteoblast nucleus, and physicallyinteracts with and promotes the transcriptional activity ofATF4. Genetic experiments demonstrate that FoxO1 and ATF4cooperate to increase glucose levels and decrease glucose toler-ance. These effects result from a synergistic effect of the twotranscription factors to suppress the activity of osteocalcinthrough up-regulating expression of the phosphatase catalyzingosteocalcin inactivation. As a result, insulin production by-cells and insulin signaling in the muscle, liver and white adi-pose tissue are compromised and fat weight increases by theFoxO1/ATF4 interaction. Taken together these observationsdemonstrate that FoxO1 and ATF4 cooperate in osteoblasts toregulate glucose homeostasis.

    FoxO1, one of the four FoxO isoforms of the Forkhead familyof transcription factors, is highly expressed in insulin-respon-sive tissues, including pancreas, liver, skeletal muscle, and adi-pose tissue. In all these tissues FoxO1 orchestrates the tran-scriptional cascades regulating glucose metabolism, in part bybeing a major target of insulin signaling. In most cells insulinsignaling favors FoxO1 phosphorylation; this results in FoxO1

    nuclear exclusion, thereby preventing its transcriptional activ-ity. In itsmost recently discoveredmode of action in the controlof energy metabolism, FoxO1 was shown to act as a transcrip-tional link between the skeleton and the pancreas as well asinsulin target tissues by regulating the novel endocrine functionof the skeleton in energy homeostasis (15). Indeed, through itsexpression in osteoblasts FoxO1 decreases -cell proliferationand function, resulting in a decrease in insulin secretion (1). Italso suppresses insulin sensitivity in insulin-target tissues suchas adipose tissue, the liver, and the muscle. These effects com-promise glucose metabolism and increase blood glucose levels.This function of FoxO1 is due to its ability to promote carbox-ylation and inactivation of osteocalcin, an osteoblast-secretedhormone that favors, insulin secretion, and sensitivity andenergy expenditure (1). Adding another level of complexity tothis function in a feedback mode of regulation, FoxO1 is also atarget of insulin signaling in osteoblasts (3). Insulin suppressesthe activity of osteoblastic FoxO1, thus, promoting osteocalcinbioactivity.In the context of whole body physiology it is remarkable that

    the exact same transcriptional mediator of insulin actions in allperipheral insulin-sensitive target organs also regulates themetabolic activity of osteocalcin and its insulin-up-regulatingas well as insulin-sensitizing functions. This property estab-lishes FoxO1 as a common unifying link of insulin signalingamong all glucose-regulating organs. At the same time it raisesthe question of how such a ubiquitously expressed transcrip-tion factor could fulfill in osteoblasts a function that it does notfulfill in other cell types; that is, to affect the glucose-regulatingfunction of other organs. To address this question we searchedfor osteoblast-specific or osteoblast-enriched transcription fac-tors that could be effectors or co-regulators of FoxO1 signalingin its metabolic functions in osteoblasts.ATF4 is a transcription factor that accumulates predomi-

    nantly in osteoblasts and acts through them to affect glucosemetabolism and insulin sensitivity (6). Analysis ofAtf4/miceshowed that these animals had a metabolic phenotype similarto that of mice lacking FoxO1 in osteoblasts and characterized

    * This work was supported, in whole or in part, by National Institutes of HealthGrants R01-AR055931, 3-R01-AR055931-02S1, and P01-AG032959.

    S This article contains supplemental Table S1.1 Present address: MRC Clinical Sciences Center, Imperial College London

    Hammersmith Campus, Du Cane Road, London W12 0NN, U.K.2 To whom correspondence should be addressed: The Russ Berrie Medical

    Sciences Pavilion, 1150 Saint Nicholas Ave., Rm. 411, New York, NY 10032.Tel.: 212-851-5223; Fax: 212-851-5225; E-mail: [email protected]

    THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 12, pp. 87578768, March 16, 2012 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.


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  • by enhanced insulin secretion and insulin sensitivity in the liver,fat, and muscle. Thus, we examined whether FoxO1-mediatedregulation of glucose homeostasis occurs through its interac-tion with ATF4. Here we show that FoxO1 engages in a func-tional complex with ATF4 in osteoblasts. In this complex thetwo transcription factors synergize to regulate glucose meta-bolism, insulin production, and insulin sensitivity.


    MiceAll the protocols and experiments were conductedaccording to the guidelines of the Institute of ComparativeMedicine, Columbia University. Generation of FoxO1fl/fl,1(I)Collagen-Cre (1(I)Col-Cre), and Atf4/ mice has previ-ously been reported (710). Mice with osteoblast specific dele-tion of FoxO1 (FoxO1osb/) were generated by crossingFoxO1fl/fl mice with transgenic mice expressing Cre under thecontrol of the osteoblast-specific collagen type 1A1 promoter(1(I) Collagen-Cre) as previously described (1). Genotypingwas performed at weaning stage by PCR analysis of genomicDNA. In each experiment the mice used were of the samegenetic background as they were all littermates. In all experi-ments data presented were obtained from male animals.Histological Analysis of Pancreatic Islets, White Adipose Tis-

    sue, and Liver SectionsHistological analysis was performed aspreviously described (1). Briefly, fat and pancreata were col-lected, fixed overnight in 10% neutral formalin solution,embedded in paraffin, sectioned at 4 m, and stained withhematoxylin and eosin. Pancreatic sections were immuno-stained for cells using guinea pig anti-swine insulin polyclonalantibody (Dako). -Cell proliferation was assessed using anantibody recognizing Ki67 antigen, the prototypic cell cycle-related nuclear protein expressed by proliferating cells in allphases of the active cell cycle.-Cell area represents the surfacepositive for insulin immunostaining divided by the total pan-creatic surface. -Cell mass was calculated as -cell area mul-tiplied by pancreatic weight. Livers were cryoembedded, sec-tioned at 5 m, and stained with Oil red O (Crystalgen).Cell CulturesPrimary osteoblasts were prepared from cal-

    varia of 5-day-old pups as previously described (2) and werecultured in freshMEMand 10%FBS. TheOB-6 bonemarrow-derived osteoblastic cell line has been described (11) and wascultured under the same conditions as calvaria.Transient Transfections and Luciferase AssaysCos-7 cells

    were seeded in 48-well plates at a density of 104 cells/well. Oneday after plating, cells were transfectedwith Lipofectamine Plus(Invitrogen) according to themanufacturers protocol.We car-ried out co-transfections of the FoxO1 or ATF4 expressionplasmids with either a FoxO-reporter construct or anOsteocal-cin-reporter construct or an ESP-reporter construct (50 ng).The Esp-luc, which bears mutations in the FoxO1 and ATF4site, was generated by site-directed mutagenesis using theQuikChange site-directedmutagenesis kit (Stratagene) accord-ing to the manufacturers instructions. Nucleotide substitu-tions at positions 3, 4, and 5 of these elements are indicated initalic: Foxo1, 5-TGGGGTT-3 (WT: TGTTTTT) and ATF4,5-ACGGAA-3 (WT: ACATCA). The total amount of DNAwas adjusted to 270 ng/well with pCMV5 or pcDNA controlvectors. Transfection was stopped by adding 20% FBS. Lucifer-

    ase assays were carried out using the Dual Luciferase ReporterAssay System (Promega), and luciferase activity was quantifiedusing Fluostar Omega. pRL-CMV Renilla luciferase controlvector (20 ng) (Promega) was cotransfected as an internalstandard to normalize for transfection efficiency. Normalizedluciferase activity is presented as -fold induction over the emptyvector control (EV,3 considered 1