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. 2011 Mar;22(3):212-8.
doi: 10.1016/j.jnutbio.2010.01.004.

Effects of Apigenin on Steroidogenesis and Steroidogenic Acute Regulatory Gene Expression in Mouse Leydig Cells

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Free PMC article

Effects of Apigenin on Steroidogenesis and Steroidogenic Acute Regulatory Gene Expression in Mouse Leydig Cells

Wei Li et al. J Nutr Biochem. .
Free PMC article

Abstract

Previous studies reported that the age-related decline in testosterone biosynthesis is associated with a decrease in the steroidogenic acute regulatory (StAR) protein which regulates the rate-limiting step of testosterone biosynthesis. To explore the possibility of delaying this decline using a dietary approach, we have examined the effect of a natural flavonoid, apigenin, on StAR gene expression in mouse Leydig cells. Incubation of these cells with the flavonoid enhanced cyclic adenosine monophosphate (cAMP)-induced steroidogenesis and StAR protein expression. The results from the analyses of StAR mRNA by reverse transcription-polymerase chain reaction and the luciferase assays of StAR promoter activity indicated that this flavonoid enhanced StAR gene expression at the level of transcription. Further studies showed that apigenin blocked the thromboxane A2 receptor and interrupted the signaling through the cyclooxygenase-2-thromboxane A synthase-thromboxane A2-receptor pathway, resulting in a reduction of DAX-1 (dosage sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene-1) protein, a transcriptional repressor of StAR gene expression. When DAX-1 protein was reduced, the sensitivity of the Leydig cells was dramatically enhanced, with sub-threshold level of cAMP being able to induce maximal levels of StAR protein expression and steroid hormone production. The present study suggests a potential application of apigenin to improve StAR protein expression and steroidogenic sensitivity of aging Leydig cells.

Figures

Fig. 1
Fig. 1
Effects of apigenin on steroidogenesis and StAR protein expression in mouse Leydig cells. A, MA-10 cells were cultured with or without 25 μM of 22(R)hydroxycholesterol (22R+ or 22R−), in the medium containing increasing concentrations of apigenin for 30 min. Then 0.1 mM dbcAMP was added to the culture for 6 h. The cells were collected and 20 μg of cell lysate protein was used for Western blot analysis of StAR protein. Progesterone concentration in the medium was assessed by RIA. **, p < 0.01; ***, p < 0.001; n=3; compared with 0.1 mM dbcAMP alone. B, MA-10 cells were cultured with 10μM apigenin for 30 min and then 10 ng/ml LH was added for 6 h. StAR protein and progesterone production were determined as described above. ***, p < 0.001; n=3; compared with 10 ng/ml LH alone. C, Leydig cells were isolated from mice and incubated with 5 μM apigenin for 30 min, followed by addition of 0.01 mM dbcAMP for 6 h. StAR protein and testosterone production were determined. **, p < 0.01; n=3; compared with 0.01 mM dbcAMP alone.
Fig. 2
Fig. 2
Effects of apigenin on StAR gene transcription in MA-10 mouse Leydig cells. MA-10 cells were cultured in serum-free Waymouth’s medium with increasing concentrations of apigenin for 30 min and then 0.1 mM dbcAMP was added to the culture for 6 h. A, the cells were collected for total RNA isolation and StAR mRNA was analyzed by RT-PCR with β-actin as an internal marker. B, MA-10 cells were transfected with a StAR promoter/luciferase plasmid (PGL2/StAR) and a pRLSV40 vector, a plasmid that constitutively expresses Renilla luciferase. The cells were then treated with dbcAMP and apigenin as described above and the cell lysate was used for luciferase assays using a Dual-Luciferase Reporter Assay System. The Relative Light Unit (RLU) was determined by dividing the reading from the PGL2/StAR promoter by the reading from Renilla luciferase. ***, p < 0.001, n=4; compared with 0.1 mM dbcAMP alone.
Fig. 3
Fig. 3
Apigenin increased steroidogenic sensitivity of MA-10 mouse Leydig cells to cAMP stimulation. MA-10 cells were cultured for 30 min in serum-free Waymouth’s medium with or without 10 μM of apigenin, and then increasing concentrations of dbcAMP were added to the culture for 6 h. A, the cells were collected and 20 μg of cell lysate protein was used for Western blot analysis of StAR protein. B, progesterone production in the culture medium was assessed by RIA. ***, p < 0.001, n=4, compared with the paired group stimulated with dbcAMP alone.
Fig. 4
Fig. 4
Apigenin blocked the thromboxane A2 receptor and reduced DAX-1 protein in MA-10 mouse Leydig cells. A, MA-10 cells were incubated with increasing levels of apigenin for 30 min. Then, 3H-SQ29548 was added to each well for 4 h. The cells were then rinsed and collected. The amount of 3H-SQ29548 bound to the cells was determined using a scintillation counter. Non-specific binding of 3H-SQ29548 was determined by co-incubation with 100 μM of unlabelled SQ29548. **, p < 0.01; ***, p < 0.001; n=4, compared with the total binding (100%). B and C, MA-10 cells were cultured in serum-free Waymouth’s medium with 10 μM of apigenin for 30 min and then 0.1 mM dbcAMP was added to the culture for 6 h. The cells were collected and 20 μg of cell lysate protein was used for Western blot analyses of DAX-1 protein and StAR protein.
Fig. 5
Fig. 5
Effects of protein kinase A and protein kinase C on DAX-1 protein and StAR mRNA levels in the apigenin-treated MA-10 mouse Leydig cells. A, MA-10 cells were cultured in serum-free Waymouth’s medium with 10 μM apigenin, 20 μM of PKC inhibitor GFX or 25 μM of PKA inhibitor H89 for 30 min, and then 0.1 mM dbcAMP was added to the culture for 6 h. The cells were collected and 20 μg of cell lysate protein was used for Western blot analysis of DAX-1 protein. B, the cells were treated as described above and collected for total RNA isolation. StAR mRNA was analyzed by RT-PCR with β-actin as an internal marker.
Fig. 6
Fig. 6
Effects of protein kinase A and protein kinase C on StAR protein expression and steroidogenesis in the apigenin-treated MA-10 mouse Leydig cells. A, MA-10 cells were cultured in serum-free Waymouth’s medium with 10μM apigenin, 20 μM of PKC inhibitor GFX or 25 μM of PKA inhibitor H89 for 30 min, and then 0.1 mM dbcAMP was added to the culture for 6 h. The cells were collected and 20 μg of cell lysate protein was used for Western blot analysis of StAR protein. B, progesterone production in the culture medium was assessed by RIA. ***, p < 0.001, n=4, compared with the group treated with apigenin and dbcAMP.

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