FKHR is a member of the forkhead domain family, which encodes transcription factors containing a
conserved DNA-binding motif related to the Drosophila region-specific homeotic gene 'forkhead.
NCBI Summary:
This gene belongs to the forkhead family of transcription factors which are characterized by a distinct forkhead domain. The specific function of this gene has not yet been determined; however, it may play a role in myogenic growth and differentiation. Translocation of this gene with PAX3 has been associated with alveolar rhabdomyosarcoma. [provided by RefSeq, Jul 2008]
General function
Nucleic acid binding, DNA binding, Transcription factor
FoxO1a can alter cell cycle progression by regulating the nuclear localization of p27kip in granulosa cells.
Cunningham MA, et al .
Forkhead transcription factors of the FOXO family are important downstream targets of the PI 3-kinase pathway, which has been shown to play a critical role in cell proliferation and cell survival. Activation of FOXOs can block cellular proliferation and drive cells into a quiescent state. In certain cell types, this cell cycle arrest is dependent on the transcriptional induction of the cell-cycle inhibitor p27(kip) (1). In granulosa cells, which go through an exponential growth phase during development of the ovarian follicle, we find that FoxO1a is a key regulator of the G1/S transition in these cells. Overexpression of a dominant negative version of FoxO1a (Foxo1a-Delta256; a C-terminal truncation mutant that possesses a functional DNA-binding domain, but lacks a transactivation domain) causes a dramatic increase in S-phase cells (>8-fold increase by both DNA content and BrdU incorporation assays). Surprisingly, this is not dependent on transactivation of the p27(kip) gene. We provide evidence that when FoxO1a activity is impeded, p27(kip) protein is largely localized to the cytosol, suggesting that FoxO1a blocks cell cycle entry by altering the compartmentalization of p27(kip) within the cell, increasing its concentration in the nucleus. These studies demonstrate for the first time that FoxO1a can regulate p27(kip) nuclear localization.
Genes in sterol/steroid and lipid biosynthetic pathways are targets of FSH and FOXO1 regulation in granulosa cells: evidence from cells expressing mutant forms of FOXO1. Liu Z et al. The forkhead box transcription factor FOXO1 is highly expressed in granulosa cells of growing follicles but is down-regulated by FSH in culture or by LH-induced luteinization in vivo. To analyze the function of FOXO1, we infected rat and mouse granulosa cells with adenoviral vectors expressing two FOXO1 mutants: a gain-of-function mutant FOXOA3 that has three serine residues mutated to alanines rendering this protein constitutively active and nuclear and FOXOA3-mDBD in which the DNA binding domain is mutated. The infected cells were then treated with vehicle or FSH for specific time intervals. Infection of the granulosa cells was highly efficient, caused only minimal apoptosis and maintained FOXO1 protein at levels of the endogenous protein observed in cells prior to exposure to FSH. RNA was prepared from control and adenoviral infected cells exposed to vehicle or FSH for 12 and 24h. Affymetrix microarray and data-base analyses identified, and real time RT-PCR verified, that genes within the lipid, sterol and steroidogenic biosynthetic pathways (Hmgcs1, Hmgcr, Mvk, Sqle, Lss, Cyp51, Tm7sf2, Dhcr24 and Star, Cyp11a1 and Cyp19), including two key transcriptional regulators Srebf1 and Srebf2 of cholesterol biosynthesis and steroidogenesis (Nr5a1, Nr5a2) were major targets induced by FSH and suppressed by FOXOA3 and FOXOA3-mDBD in the cultured granulosa cells. By contrast, FOXOA3 and FOXOA3-mDBD induced expression of Cyp27a1 mRNA that encodes an enzyme involved in cholesterol catabolism to oxysterols. The genes up-regulated by FSH in cultured granulosa cells were also induced in granulosa cells of preovulatory follicles and corpora lutea collected from immature mice primed with FSH (eCG) and LH (hCG), respectively. Conversely, Foxo1 and Cyp27a1 mRNAs were reduced by these same treatments. Collectively, these data provide novel evidence that FOXO1 may play a key role in granulosa cells to modulate lipid and sterol biosynthesis, thereby preventing elevated steroidogenesis during early stages of follicle development.
Role of the PI3-Kinase and ERK Pathways in the Induction of HIF-1 Activity and the HIF-1 Target VEGF in Ovarian Granulosa Cells in response to Follicle Stimulating Hormone. Alam H et al. FSH stimulation of granulosa cells (GCs) results in increased hypoxia-inducible factor (HIF)-1alpha protein levels and HIF-1 activity that is necessary for up-regulation of certain FSH target genes including vascular endothelial growth factor (VEGF). We report that the role of the phosphatidylinositol (PI)-3-kinase/AKT pathway in increasing HIF-1alpha protein in FSH-stimulated GCs extends beyond an increase in mammalian target of rapamycin (mTOR)-stimulated translation. FSH increases phosphorylation of the AKT target mouse double minute 2 (MDM2); a phosphomimetic mutation of MDM2 is sufficient to induce HIF-1 activity. The PI3-kinase/AKT target forkhead box-containing protein O subfamily 1 (FOXO1) also effects the accumulation of HIF-1alpha as evidenced by the ability of a constitutively active FOXO1 mutant to inhibit the induction by FSH of HIF-1alpha protein and HIF-1 activity. Activation of the PI3-kinase/AKT pathway in GCs by IGF-1 is sufficient to induce HIF-1alpha protein but surprisingly not HIF-1 activity. HIF-1 activity also appears to require a PD98059-sensitive protein (kinase) activity stimulated by FSH that is both distinct from mitogen-activated extracellular signal-regulated kinase (ERK) kinase1/2 or 5 and independent of the PI3-kinase/AKT pathway. These results indicate that FSH-stimulated HIF-1 activation leading to up-regulation of targets such as VEGF requires not only PI3-kinase/AKT-mediated activation of mTOR as well as phosphorylation of FOXO1 and possibly MDM2 but also a protein (kinase) activity that is inhibited by the classic ERK kinase inhibitor PD98059 but is not ERK1/2 or 5. Thus, regulation of HIF-1 activity in GCs by FSH under normoxic conditions is complex and requires input from multiple signaling pathways.
Identification of Sheep Ovary Genes Potentially Associated with Off-season Reproduction. Chen L et al. Off-season reproduction is a favorable economic trait for sheep industry. Hu sheep, an indigenous Chinese sheep breed, demonstrates a higher productivity of lambs and displays year-around oestrous behavior under proper nutrition and environment. The genetic basis behind these traits, however, is not well understood. In order to identify genes associated with the off-season reproduction, we constructed a suppression subtractive hybridization (SSH) cDNA library using pooled ovary mRNAs of 6 oestrous Hu females as a tester and the pooled ovary mRNAs of 6 non-oestrous Chinese Merino females as a driver. A total of 382 resulting positive clones were obtained after the SSH. We identified 114 differentially up-regulated genes in oestrous Hu sheep by using subsequent screening and DNA sequencing, of which 8 were previously known, 93 were reported for the first time in sheep, and 13 were novel with no significant homology to any sequence in the DNA databases. Functions of the genes identified are related to cell division, signal transduction, structure, metabolism, or cell defense. To validate the results of SSH, 6 genes (Ntrk2, Ppap2b, Htra1, Nid1, Serpine2 and Foxola) were selected for conformational analysis using quantitative real-time PCR (qRT-PCR), and two of them (Htral and Foxo1a) were verified by Northern blot. All of the 6 genes were differentially up-regulated in the ovary of oestrous Hu. It is obvious that off-season reproduction is a complex trait involving multiple genes in multiple organs. This study helps to provide a foundation for the final identification of functional genes involved in the sheep ovary.
Expression regulated by
FSH, LH, Steroids
Comment
FOXO1 mediates hypoxia-induced G0/G1 arrest in ovarian somatic granulosa cells by activating the TP53INP1-p53-CDKN1A pathway. Li C et al. (2021) The development of ovarian follicles constitutes the foundation of female reproduction. The proliferation of granulosa cells (GCs) is a basic process required to ensure normal follicular development. However, the mechanisms involved in controlling GC cell cycle are not fully understood. Here, by performing gene expression profiling in the domestic pig (Sus scrofa), we showed that cell cycle arrest at G0/G1 phase is highly correlated with pathways associated with hypoxic stress and FOXO signalling. Specifically, the elevated proportion of GCs at the arrested G0/G1 phase was accompanied by increased nuclear translocation of FOXO1 under conditions of hypoxia both in vivo and in vitro. Furthermore, phosphorylation of 14-3-3 by the JNK kinase is required for hypoxia-mediated FOXO1 activation and the resultant G0/G1 arrest. Notably, a FOXO1 mutant without DNA-binding activity failed to induce G0/G1 arrest of GCs during hypoxia. Importantly, we identified a new target gene of FOXO1, namely TP53INP1, which contributes to suppression of the G1-S cell cycle transition in response to hypoxia. Furthermore, we demonstrated that the inhibitory effect of the FOXO1-TP53INP1 axis on the GC cell cycle is mediated through a p53-CDKN1A-dependent mechanism. These findings could provide avenues for the clinical treatment of human infertility caused by impaired follicular development.//////////////////Cunningham MA, et al 2003 reported that Follicle stimulating hormone promotes nuclear exclusion of the forkhead transcription factor FoxO1a via PI 3-kinase in porcine granulosa cells.
The forkhead family of transcription factors are conserved in evolution and known to play critical roles in the regulation of cellular differentiation and proliferation in many systems. The current studies demonstrate for the first time that FKHR (FoxO1a) is expressed in porcine granulosa cells, and follicle-stimulating hormone (FSH) stimulates FKHR phosphorylation and regulates its subcellular localization in this system. RT-PCR and Western blot studies demonstrated that FKHR is expressed in this cell system and showed no change in FKHR message or protein levels in response to FSH (0-6 h). However, 32p-orthophosphate labeling of cultured granulosa cells revealed robust phosphorylation after FSH treatment for 30min. In addition, FSH caused nuclear exclusion of FKHR in these cells, apparently through the PI 3-kinase signal transduction pathway. The cytosolic accumulation of FKHR protein that was observed in FSH-treated cells both by Western blot and immunohistochemistry was blocked when the cells were pre-incubated with the PI 3-kinase inhibitor LY294002. Our data also demonstrate that Akt/PKB, an established kinase for FKHR, is phosphorylated in response to FSH treatment. Interestingly, while FKHR was phosphorylated by 30min after FSH treatment, the time course for Akt phosphorylation was relatively delayed and sustained. While these studies do not preclude Akt involvement in FSH-stimulated FKHR phosphorylation, they do suggest that other kinases may contribute to rapid signaling to FKHR. Since FKHR has been shown to activate genes involved in apoptosis and growth inhibition, FSH may promote growth and survival by initiating the phosphorylation of FKHR, causing its nuclear exclusion, and reducing its effect as a cell cycle arrest or death-promoting transcription factor.
Regulation of FoxO1 Transcription Factor by Nitric Oxide and Cyclic GMP in Cultured Rat Granulosa Cells Li X, et al .
FoxO1 is a transcription factor implicated in a multitude of physiological processes including cell cycle progression, apoptosis and insulin signaling. Recent findings indicate that FoxO1 is a key regulator during the proliferation and maturation of granulosa cells. Over the past several years, it has become evident that nitric oxide (NO) and cGMP modulate ovarian function. There has been no information, however, about whether NO-cGMP affects FoxO1 expression or about the relationship between NO-cGMP and FoxO1. In the present study, we used immunoblot analysis to determine whether NO and cGMP affect FoxO1 expression in cultured granulosa cells. Our results clearly showed that FSH suppressed FoxO1 expression in a time-dependent manner, and that NO-cGMP stimulated FoxO1 expression in cultured granulosa cells. In addition, this stimulatory effects of NO and cGMP can be blocked by FSH in cultured granulosa cells. These findings demonstrate that NO and cGMP influence FoxO1 expression possibly through antagonizing the action of FSH in cultured granulosa cells. Results of both immunoblot analysis and immunohistochemistry also show that estradiol implantation do not affect the expression of FoxO1 in rat granulosa cells as gonadotrophins do, indicating that mechanism of estradiol on granulosa cells is different from gonadotrophins. Together, our experiments suggest that expression of FoxO1 in rat granulosa cells can be regulated by gonadotrophins and the NO/cGMP signaling pathway.
Ovarian localization
Granulosa
Comment
Richards JS, et al 2002 reported the expression of FKHR, FKHRL1, and AFX Genes in the Rodent
Ovary and Evidence for Regulation by IGF-I, Estrogen, and the
Gonadotropins.
This study
was undertaken to determine if during ovarian follicular development FSH regulates putative targets of PKB and Sgk,
namely specific Forkhead transcription factor family members. Using in vivo and in vitro mouse and rat models, the authors show 1)
that FKHR (Forkhead homolog of rhabdomysarcoma = Forkhead box binding protein (Foxo1), FKHRL1 (Forkhead-like
protein-1 = Foxo3), and AFX (a Forkhead transcription factor = Foxo4); all defined according to the Human and Mouse
Gene Nomenclature Committee) are expressed in the rodent ovary and 2) that FSH regulates transcription of the FKHR gene
as well as phosphorylation of FKHR protein. Specifically, FSH/PMSG (primarily via E2) enhance expression of the FKHR
gene in granulosa cells of developing follicles.
Furthermore, E2 enhances expression of other IGF-I pathway components
(IGF-1R?and Glut-1), and IGF-I enhances expression of ER? indicating that these two hormones comprise an autocrine
regulatory network within growing follicles. In contrast, FSH and LH/human CG (via cAMP, PKA, and PI3K pathways) terminate FKHR expression as granulosa cells differentiate to luteal cells. In na? granulosa cells, both FSH and IGF-I
stimulate rapid phosphorylation of FKHR at multiple sites causing its redistribution from the nucleus to the cytoplasm in a PI3K-dependent manner. However, the effects of FSH and IGF-I differ markedly in differentiated granulosa cells in which
FSH (but not IGF-I) induces Sgk and enhances phosphorylation of FKHR, PKB, and Sgk. The elevated expression of FKHR
in granulosa cells of growing follicles indicates that FKHR may be linked to the proliferation of granulosa cells and that its
phosphorylation by FSH, IGF-I, and other factors may impact its functional activity in this process. Thus, as a target of FSH
(cAMP), E2 and IGF-I signaling in granulosa cells, FKHR likely coordinates numerous cell survival mechanisms.
Differential expression of genes for transcription factors in theca and granulosa cells following selection of a dominant follicle in cattle. Zielak AE et al. Transcription factors inhibit or assist RNA polymerases in the initiation and maintenance of transcription; however, the cell specific expression and roles of transcription factors within bovine ovarian follicles during development are unknown. The aim of present study was to determine if the expression of transcription factors in theca and granulosa cells differ between the dominant and the largest subordinate follicles at different stages of the follicle wave. We used a bovine cDNA microarray to screen granulosa and theca cells from dominant and subordinate follicles for differential expression of genes coding for transcription factors. Expression was confirmed using reverse transcription polymerase chain reaction and differences in mRNA abundance further examined at Emergence, Selection and Dominance stages of the follicle wave. We have identified five genes encoding for transcription factors that have not been previously described in developing follicles with greater mRNA abundance in subordinate compared to dominant follicles. The genes (and their putative roles) are CEBP-beta (responsible for luteinization), SRF (cell survival), FKHRL1 (stimulates apoptosis), NCOR1 (modulation of the actions of the oestradiol receptor) and Midnolin (control of development via regulation of mRNA transport in cells). Mol. Reprod. Dev. (c) 2007 Wiley-Liss, Inc.
Follicle stages
Secondary, Antral, Preovulatory
Comment
Relationship between FoxO1 protein levels and follicular development, atresia, and luteinization in the rat ovary.
Shi F,et al 2003 .
FoxO1 is a transcription factor implicated in a growing number of physiological processes, including apoptosis, cell cycle progression, and insulin signaling. Recent findings indicate that FSH and growth factors influence ovarian functions in part through regulation of FoxO1. The present study utilized immunohistochemical analysis to determine the ovarian localization and regulation of FoxO1 protein levels in neonatal rats, immature rats during gonadotropin-induced follicular development, ovulation, and luteinization, and in spontaneously developing ovarian cysts of aging rats. In postnatal rats, FoxO1 immunoreactivity was very faint in ovaries of 5- and 10-day-old females. In contrast, strong immunoreactivity was observed in granulosa cells of larger developing follicles at 25 days of age. To stimulate follicle development, immature female rats received equine chorionic gonadotropin (eCG) followed 52 h later by an ovulatory dose of human chorionic gonadotropin (hCG). Prior to gonadotropin treatment, moderate FoxO1 immunoreactivity was observed in granulosa cells of small follicles. Subsequently, treatment with eCG markedly decreased FoxO1 protein levels in granulosa cells of healthy antral and preovulatory follicles. Interestingly, FoxO1 staining was observed in cumulus and antral, but not mural granulosa cells of preovulatory follicles. Induction of ovulation and luteinization with hCG further decreased ovarian FoxO1 levels, with no staining evident in corpora lutea. At all time points, the most intensive FoxO1 staining was observed in granulosa cells of atretic follicles, with predominantly nuclear localization. Similarly, while FoxO1 levels were low in granulosa cells of preovulatory follicles in proestrous rats, FoxO1 staining was intense in granulosa cells of spontaneously developing cystic follicles in aged, acyclic females. Together, these findings indicate that FoxO1 is expressed in a regulated, cell-specific manner during ovarian follicular development, atresia and luteinization, suggesting roles in these physiological processes.
Phenotypes
POF (premature ovarian failure)
Mutations
1 mutations
Species: human
Mutation name: None
type: naturally occurring fertility: subfertile Comment: Mutational screening of FOXO3A and FOXO1A in women with premature ovarian failure. Watkins WJ et al. FOXO3A and FOXO1A are excellent candidate genes for the development of premature ovarian failure and have not been analyzed previously in POF patients. Potentially causal mutations in FOXO3A (2/90; 2.2%) and FOXO1A (1/90; 1.1%) were identified in POF patients; however, the pathological role of these mutations will be determined only by screening increased numbers of patients and controls, or by functional studies.