Yonsei Med J.  2015 Nov;56(6):1590-1596. 10.3349/ymj.2015.56.6.1590.

The Role of Foxo3 in Leydig Cells

• Choi YS
• Song JE
• Kong BS
• Hong JW
• Novelli S
• Lee EJ

• KMID: 2345887
• DOI: http://doi.org/10.3349/ymj.2015.56.6.1590

Abstract

PURPOSE
Foxo3 in female reproduction has been reported to regulate proliferation of granulose cells that form follicles. There are no reports so far that discuss on the role of Foxo3 in males. This study was designed to outline the role of Foxo3 in the testes.
MATERIALS AND METHODS
Testes from mice at birth to postpartum week (PPW) 5 were isolated and examined for the expression of Foxo3 using immunostaining. To elucidate role of Foxo3 in Leydig cells, R2C cells were treated with luteinizing hormone (LH) and the phosphorylation of Foxo3. Testosterone and steroidogenic acute regulatory (StAR) protein levels were measured after constitutive active [triple mutant (TM)] human FOXO3 adenovirus was transduced and StAR promoter assay was performed.
RESULTS
Foxo3 expression in the testicles started from birth and lasted until PPW 3. After PPW 3, most Foxo3 expression occurred in the nuclei of Leydig cells; however, at PPW 5, Foxo3 was expressed in both the nucleus and cytoplasm. When R2C cells were treated with luteinizing hormone, Foxo3 phosphorylation levels by AKT increased. After blocking the PI3K pathway, LH-induced phosphorylated Foxo3 levels decreased, indicating that LH signaling regulates Foxo3 localization. When active FOXO3-TM adenovirus was introduced into a Leydig tumor cell line, the concentrations of testosterone and StAR protein decreased. When FOXO3 and a StAR promoter vector were co-transfected into HEK293 cells for a reporter assay, FOXO3 inhibited the StAR promoter.
CONCLUSION
FOXO3 affects testosterone synthesis by inhibiting the formation of StAR protein. LH hormone, meanwhile, influences Foxo3 localization, mediating its function.

Keyword

Foxo3; Leydig cell; testosterone; StAR

MeSH Terms

Animals
Cell Aging/*physiology
Cell Nucleus/metabolism
Cytoplasm/metabolism
Forkhead Transcription Factors/*metabolism
HEK293 Cells
Humans
Leydig Cells/*drug effects/*enzymology/metabolism
Luteinizing Hormone/blood
Male
Mice
Phosphatidylinositol 3-Kinases
Phosphoproteins/metabolism
Phosphorylation
Signal Transduction/drug effects
Testosterone/blood/*metabolism
Forkhead Transcription Factors
Luteinizing Hormone
Phosphatidylinositol 3-Kinases
Phosphoproteins
Testosterone

Figure

• 

  Fig. 1 Foxo3 is detected in Leydig cells. In C57/BL6 male mice, testes were isolated at postpartum days (PPD) 1 and 5 and postpartum weeks (PPW) 3, 4, 5, and 12. Double-immunostaining was performed using each rabbit polyclonal Foxo3 with goat anti-GATA4 and observed under a laser scanning microscope, ×400 magnification. Scale bar indicates 200 um. FITC, fluorescein isothiocyanate; DAPI, 4',6-diamidino-2-phenylindole.

• 

  Fig. 2 LH/hCG regulates Foxo3 localization. (A) R2C cells were cultured in the serum-starved condition for 24 hr and stimulated with 10 IU of hCG for 10, 30, and 60 minutes. (B) To block the PI3K pathway, 20 uM of LY294002 was pretreated for 2 hr and then 10 IU of hCG was added for 30 min. Cell lysates were extracted and western blots were performed. The grape data was presented as the mean (±SEM) of triplicated experiments from three independent experiments and are presented as fold changes related to 0 min treatment in (A) and no-treatment in (B). Significant differences between control are denoted as *p<0.05, **p<0.01, †p<0.05, and ††p<0.01 compared btween 30 min and 60 min. Similar results were obtained in three independent experiments. LH, luteinizing hormone; hCG, human chorionic gonadotropin; PI3K, phosphoinositide 3-kinase; SEM, standard error of the mean; NS, not significant.

• 

  Fig. 3 FOXO3 over-expression decreases testosterone levels in Leydig cell lines. (A) We confirmed the Foxo3 levels in MA10 and R2C cells. To elucidate the role of Foxo3 in the nucleus, we constructed a constitutively active form of human FOXO3 by replacing the three AKT phosphorylation sites (T32, S253, and S315) with alanine. (B) Testosterone levels were measured in R2C cells infected with Ad-LacZ (empty), WT-FOXO3, or TM-FOXO3 adenovirus (5 pfu/cell for 48 hr). Testosterone levels decreased by WT-FOXO3 infection and more significantly by TM-FOXO3. *p<0.05, **p<0.01 vs. empty or WT-FOXO3 infection sample. Similar results were obtained in three independent experiments. WT, wild type; TM, triple mutant.

• 

  Fig. 4 Constitutively active FOXO3 downregulates StAR protein levels by directly inhibiting StAR gene expression. (A) StAR protein levels were measured in R2C cells infected with Ad-LacZ (empty) or TM-FOXO3 (1 or 5 pfu/cell) for 48 hr. Constitutively active FOXO3 decreases StAR protein level in a dose-dependent manner in R2C cells. Similar results were obtained from three independent experiments. (B) FOXO3 down-regulates mStAR promoter activity. mStAR promoter activity was measured by co-transfecting mStAR-Luc plasmid with empty, WT-FOXO3, or TM-FOXO3 vectors in HEK293 cells (*p<0.05 and **p<0.01 vs. empty, †p<0.05 compared between 30 min and 60 min). Similar results were obtained in three independent experiments. StAR, steroidogenic acute regulatory; WT, wild type; TM, triple mutant; mStAR, mouse steroidogenic acute regulatory protein.

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