A cell-autonomous molecular cascade initiated by AMP-activated protein kinase represses steroidogenesis

Abstract

Steroid hormones regulate essential physiological processes, and inadequate levels are associated with various pathological conditions. In testosterone-producing Leydig cells, steroidogenesis is strongly stimulated by luteinizing hormone (LH) via its receptor leading to increased cyclic AMP (cAMP) production and expression of the steroidogenic acute regulatory (STAR) protein, which is essential for the initiation of steroidogenesis. Steroidogenesis then passively decreases with the degradation of cAMP into AMP by phosphodiesterases. In this study, we show that AMP-activated protein kinase (AMPK) is activated following cAMP-to-AMP breakdown in MA-10 and MLTC-1 Leydig cells. Activated AMPK then actively inhibits cAMP-induced steroidogenesis by repressing the expression of key regulators of steroidogenesis, including Star and Nr4a1. Similar results were obtained in Y-1 adrenal cells and in the constitutively steroidogenic R2C cells. We have also determined that maximum AMPK activation following stimulation of steroidogenesis in MA-10 Leydig cells occurs when steroid hormone production has reached a plateau. Our data identify AMPK as a molecular rheostat that actively represses steroid hormone biosynthesis to preserve cellular energy homeostasis and prevent excess steroid production.

FIG 1

Activation of AMPK by Fsk and time course of cAMP production in MA-10 Leydig cells. (A) P-AMPKα, AMPKα, P-ACC, and ACC levels were determined by Western blotting using whole-cell extracts from MA-10 Leydig cells treated for 2 h with either vehicle (DMSO), AICAR (1 mM), Fsk (10 μM), or Fsk plus AICAR. Representative blots from three independent experiments are shown. (B) Intracellular cAMP levels produced by MA-10 Leydig cells following Fsk treatment (10 μM) at different time points were quantified by ELISA (values are means plus standard errors of the means [SEM] [error bar] for 3 duplicate experiments). A P value of <0.05 was considered statistically significant using a one-way ANOVA test. Different letters indicate a statistically significant difference. (C) Same as panel B but with hCG stimulation (20 ng/ml). (D) MA-10 Leydig cells were treated as indicated for 24 h in serum-free medium before performing the cell viability 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RU, relative units; Ctrl, control.

FIG 2

Activation of AMPK blocks steroidogenesis in Leydig cell lines. (A) Progesterone secreted by MA-10 Leydig cells treated for 4 h with vehicle (−), AICAR (+), Fsk, Fsk plus AICAR, hCG, or hCG plus AICAR was quantified by ELISA. (B) Testosterone secreted by MLTC-1 Leydig cells treated for 4 h with vehicle, AICAR, Fsk, Fsk plus AICAR, hCG, or hCG plus AICAR was quantified by ELISA. (C) Progesterone secreted by MA-10 Leydig cells treated for 4 h with vehicle, AICAR, Fsk, Fsk plus AICAR, IBMX, IBMX plus AICAR, Fsk plus IBMX, or Fsk plus IBMX plus AICAR was quantified by ELISA. (D) Pde8a mRNA levels were determined by quantitative RT-PCR using total RNA isolated from MA-10 Leydig cells 32 h posttransfection of two distinct siRNAs against Pde8a. Values were normalized to Rpl19 levels, and results are shown as fold induction over siRNA control (values are means plus standard deviations [SD]). (E) MA-10 Leydig cells were transfected for 48 h with two different siRNAs against Pde8a and treated for 4 h with vehicle or Fsk, and progesterone production was quantified by ELISA. Values that are statistically significantly different (P < 0.05) are indicated by a bar and asterisk.

FIG 3

AMPK blocks steroidogenesis by targeting the cholesterol transport machinery. (A) Diagram representing the mode of action of 22(R)-hydroxycholesterol (OHC), which diffuses through the mitochondrial inner membrane bypassing the cholesterol transport machinery. MT, mitochondria; ER, endoplasmic reticulum. (B) Progesterone secreted by MA-10 Leydig cells treated for 4 h with vehicle, OHC, or OHC plus AICAR was quantified by ELISA. (C) Testosterone secreted by MLTC-1 Leydig cells treated for 4 h with vehicle, OHC, or OHC plus AICAR was quantified by ELISA.

FIG 4

AMPK blocks Fsk-induced steroidogenesis by targeting Star expression in MA-10 Leydig cells. (A) STAR and tubulin protein levels were determined by Western blotting using whole-cell extracts from MA-10 Leydig cells treated for 4 h with vehicle, AICAR, Fsk, or Fsk plus AICAR. (B) Star mRNA levels were determined by quantitative RT-PCR using total RNA isolated from MA-10 Leydig cells treated for 2.5 h with vehicle, Fsk, or Fsk plus AICAR. Values were normalized to Rpl19 levels, and results are shown as fold induction over the value for vehicle (mean plus SD). (C) Transient transfections of the murine Star promoter (bp −980 to +16) reporter were performed in MA-10 Leydig cells treated for 4 h with vehicle (DMSO), 8Br-cAMP (0.5 mM), or 8Br-cAMP plus AICAR. Previously characterized NR5A1 (bp −980, −135, −100, and −42) and NR4A1/5A1 (bp −100) elements are indicated. Results are shown as fold activation over the value for vehicle (mean plus SEM). (D) Progesterone secreted by MA-10 Leydig cells treated for 4 h with vehicle, metformin (10 mM), Fsk (10 μM), or Fsk plus metformin was quantified by ELISA. (E) MA-10 Leydig cells were treated for 4 h with vehicle, metformin, Fsk, or Fsk plus metformin. and STAR and tubulin protein levels were determined by Western blotting. (F) STAR, P-AMPKα, AMPKα, and tubulin protein levels were determined by Western blotting using whole-cell extracts from MA-10 Leydig cells treated for 2 to 24 h with vehicle or Fsk. (G) Progesterone secreted by MA-10 Leydig cells treated for 2 to 24 h with vehicle or Fsk was quantified by ELISA. (H) The Fsk-dependent induction of progesterone production is shown as a ratio of the progesterone levels in the presence of Fsk over vehicle, shown in panel G.

FIG 5

siRNA-mediated knockdown of AMPKα1 or LKB1 increases steroidogenesis and STAR expression in MA-10 Leydig cells. (A) AMPKα, LKB1, and tubulin protein levels were determined by Western blotting using whole-cell extracts from MA-10 Leydig cells 2 days posttransfection of the siRNAs against AMPKα1 or LKB1. (B) Progesterone secreted by MA-10 Leydig cells treated for 4 h with vehicle, Fsk, or Fsk plus AICAR was quantified by ELISA 2 days posttransfection of the siRNAs against AMPKα1 or LKB1. (C) Transient transfections of the murine Star promoter (bp −980 to +16) reporter were performed in MA-10 Leydig cells treated for 4 h with either vehicle (DMSO) or Fsk (10 μM) 24 h following transfection of siRNA against AMPKα1 or LKB1. Results are shown as fold activation over vehicle (mean plus SEM). (D) Star mRNA levels were determined by quantitative RT-PCR using RNA isolated from MA-10 Leydig cells treated for 2.5 h with vehicle or Fsk (10 μM) 2 days after transfection of the siRNAs against AMPKα1 or LKB1. Values were normalized to Rpl19 levels, and results are shown as fold activation over vehicle (mean plus SD). (E) STAR and tubulin protein levels were determined by Western blotting using whole-cell extracts from MA-10 Leydig cells treated with Fsk (10 μM) or not treated with Fsk 2 days posttransfection of the siRNAs against AMPKα1 or LKB1.

FIG 6

Activation of AMPK impairs the expression of several prosteroidogenic genes in MA-10 Leydig cells. (A) Heatmap showing the impact of AMPK activation on the transcriptome of MA-10 Leydig cells. The expression of 390 genes significantly affected by AICAR treatment in the presence of Fsk is shown (false discovery rate [FDR] of 0.05 and fold induction of ≥1.5 and less than or equal to −1.3). (B) Validation of genes affected by AICAR treatment was performed by quantitative RT-PCR using RNA isolated from MA-10 Leydig cells treated for 1.5 h with vehicle, AICAR, Fsk, or Fsk plus AICAR. Values were normalized to Rpl19 levels, and results are shown as fold activation over vehicle (mean plus SD) (n = 3). Values that are statistically significantly different are indicated by a bar and asterisks as follows: *, P < 0.05.

FIG 7

AMPK impairs Star expression by targeting the NR4A1/NR5A1 promoter element located at bp −100. (A) To locate the AICAR responsive element within the murine Star promoter, MA-10 Leydig cells were transiently transfected with a series of 5′ deletion constructs (bp −980, −195, −144, −120, −95 and −70 to +16) of the mouse Star promoter and treated with vehicle, 8Br-cAMP (0.5 mM), or 8Br-cAMP plus AICAR (1 mM) for 4 h. Results are shown as fold activation over vehicle (mean plus SEM). An asterisk indicates a statistically significant difference (P < 0.05). (B) MA-10 Leydig cells were transiently transfected with the bp −980 to +16 mouse Star promoter, either wild type or containing a mutation in the NR4A1/NR5A1 element at bp −100 (the mutation is indicated by a large X). Cells were then treated with vehicle, 8Br-cAMP (0.5 mM), or 8Br-cAMP plus AICAR (1 mM) for 4 h. Results are shown as activity relative to the activity of the wild-type reporter treated with vehicle, which was arbitrarily set at 1. The fold stimulation by cAMP is indicated.

FIG 8

AMPK impairs STAR expression by targeting NR4A1 in MA-10 Leydig cells. (A) NR4A1, NR5A1, and lamin B protein levels were determined by Western blotting using nuclear extracts from MA-10 Leydig cells treated for 2 h with vehicle, Fsk, or Fsk plus AICAR. (B) Nr4a1 mRNA levels were determined by quantitative RT-PCR using total RNA isolated from MA-10 Leydig cells treated for 1 h with vehicle, Fsk, or Fsk plus AICAR. Values were normalized to Rpl19 levels, and results are shown as fold induction over vehicle (mean plus SD). (C) Transient transfections of the murine Nr4a1 promoter (bp −747 to +50) were performed in MA-10 Leydig cells treated for 4 h with either vehicle, 8Br-cAMP (0.5 mM), or 8Br-cAMP plus AICAR. Results are shown as fold activation over vehicle (mean plus SEM). (D) Transient transfections of the murine Nr4a1 promoter (bp −747 to +50) reporter were performed in MA-10 Leydig cells treated for 4 h with either vehicle (DMSO) or Fsk (10 μM) 24 h following transfection of siRNA against AMPKα1 or LKB1. Results are shown as fold activation over vehicle (mean plus SEM). (E) Nr4a1 mRNA levels were determined by quantitative RT-PCR using RNA isolated from MA-10 Leydig cells treated for 1 h with vehicle or Fsk 2 days posttransfection of siRNAs against AMPKα1 or LKB1. Values were normalized to Rpl19 levels, and results are shown as fold induction over vehicle (mean plus SD) (n = 3 duplicates). (F) NR4A1/NR5A1 and lamin B protein levels were determined by Western blotting using nuclear extracts from MA-10 Leydig cells 2 days posttransfection of siRNAs against AMPKα1 or LKB1. (G) MA-10 Leydig cells were treated for 30 min with DMSO, Fsk, or Fsk plus AICAR (Fsk+A) in a serum-free medium, and a chromatin immunoprecipitation (IP) experiment was performed to assess recruitment of p300, P-CREB, and total CREB to the bp −284 to −3 region of the Nr4a1 promoter. An IgG was used as a negative control, and 1% of the sonicated extract was used as input. Ab, antibody. (H) Coimmunoprecipitation experiments were performed on nuclear extracts of MA-10 Leydig cells treated for 30 min with DMSO, Fsk, or Fsk plus AICAR (Fsk+A). Part (5%) of the nuclear extracts was used as input control.

FIG 9

AMPK impairs steroidogenesis by blunting STAR and NR4A1 expression in the Y-1 adrenal cell line. (A) P-AMPKα and AMPKα levels were determined by Western blotting using whole-cell extracts from Y-1 adrenal cells treated for 1 h with vehicle (DMSO), Fsk, or Fsk plus AICAR. (B) Progesterone secreted by Y-1 adrenal cells treated for 4 h with vehicle, Fsk, or Fsk plus AICAR was quantified by ELISA. (C) STAR and tubulin protein levels were determined by Western blotting using whole-cell extracts from Y-1 adrenal cells treated for 4 h with vehicle, Fsk, or Fsk plus AICAR. (D) Star mRNA levels were determined by quantitative RT-PCR using total RNA isolated from Y-1 adrenal cells treated for 2.5 h with vehicle, Fsk, or Fsk plus AICAR. Values were normalized to Rpl19 levels, and results are shown as fold induction over vehicle (mean plus SD). (E) Transient transfections of the murine Star promoter (bp −980 to +16) were performed in Y-1 adrenal cells treated for 4 h with vehicle, 8Br-cAMP (0.5 mM), or 8Br-cAMP plus AICAR. Results are shown as fold activation over vehicle (mean plus SEM). (F) NR4A1 and lamin B protein levels were determined by Western blotting using nuclear extracts from Y-1 adrenal cells treated for 2 h with vehicle, Fsk, or Fsk plus AICAR. (G) Nr4a1 mRNA levels were determined by quantitative RT-PCR using total RNA isolated from Y-1 adrenal cells treated for 1 h with vehicle, Fsk, or Fsk plus AICAR. Values were normalized to Rpl19 levels, and results are shown as fold induction over vehicle (mean plus SD). (H) Transient transfections of the murine Nr4a1 promoter (bp −747 to +50) were performed in Y-1 adrenal cells treated for 4 h with vehicle, 8Br-cAMP (0.5 mM), or 8Br-cAMP plus AICAR. Results are shown as fold activation over vehicle (mean plus SEM).

FIG 10

AMPK activation decreases steroid production in the constitutively steroidogenic R2C Leydig cells. (A) Progesterone secreted by the constitutively steroidogenic R2C Leydig cells treated for 0, 4, 8, or 24 h with vehicle (−) or AICAR (+) was quantified by ELISA. (B to D) Star (B), Nr4a1 (C), and Nr0b1 (D) mRNA levels were determined by quantitative RT-PCR using total RNA isolated from R2C Leydig cells treated for 8 h with vehicle or AICAR. Values were normalized to Rpl19 levels, and results are shown as fold induction over vehicle (mean plus SD).