Histone deacetylase inhibitors reduce steroidogenesis through SCF-mediated ubiquitination and degradation of steroidogenic factor 1 (NR5A1)

Abstract

Histone deacetylase (HDAC) inhibitors such as trichostatin A and valproic acid modulate transcription of many genes by inhibiting the activities of HDACs, resulting in the remodeling of chromatin. Yet this effect is not universal for all genes. Here we show that HDAC inhibitors suppressed the expression of steroidogenic gene CYP11A1 and decreased steroid secretion by increasing the ubiquitination and degradation of SF-1, a factor important for the transcription of all steroidogenic genes. This was accompanied by increased expression of Ube2D1 and SKP1A, an E2 ubiquitin conjugase and a subunit of the E3 ubiquitin ligase in the Skp1/Cul1/F-box protein (SCF) family, respectively. Reducing SKP1A expression with small interfering RNA resulted in recovery of SF-1 levels, demonstrating that the activity of SCF E3 ubiquitin ligase is required for the SF-1 degradation induced by HDAC inhibitors. Overexpression of exogenous SF-1 restored steroidogenic activities even in the presence of HDAC inhibitors. Thus, increased SF-1 degradation is the cause of the reduction in steroidogenesis caused by HDAC inhibitors. The increased SKP1A expression and SCF-mediated protein degradation could be the mechanism underlying the mode of action of HDAC inhibitors.

FIG. 1.

HDAC inhibitors reduce SF-1 and CYP11A1 levels in both mouse Y1 and human H295 adrenocortical tumor cell lines. (A and B) TSA reduces SF-1 and CYP11A1 levels in a dose- (A) and time (B)-dependent manner. Y1 cells were treated with 12.5 (1×), 25 (2×), 50 (4×), 100 (8×), and 200 (16×) ng/ml of TSA for 24 h (A) or with 100 ng/ml TSA for the indicated times (B). In the recovery (R) experiment, after a 24-h exposure to TSA, cells were replaced in fresh medium for another 24 h. The levels of SF-1, CYP11A1, acetyl-tubulin (Ac-tub), and HSP70 (loading control), determined by immunoblotting, are shown. (C) Y1 and H295 adrenocortical cell lines were treated for 24 h without (control [CTRL]) or with 100 ng/ml TSA, 2 mM VPA, or 2 mM NaB. The levels of SF-1, CYP11A1, acetyl-tubulin, acetyl-histone H3 (Ac-H3), and HSP70, determined by immunoblotting, are shown.

FIG. 2.

HDAC inhibitors diminish steroidogenesis through modulating SF-1 level. (A) Normal Y1 cells and stable clones (18 and 55) were treated for 24 h without (CTRL) or with 100 ng/ml TSA or 2 mM VPA. Levels of SF-1, SF-1-HA, CYP11A1, CYP21, and HSP70, as determined by immunoblotting analysis, are shown. The asterisk denotes nonspecific cross-reacting material (CRM). (B and C) The progesterone (B) and corticosterone (C) levels in the medium of panel A were determined by enzyme immunoassay or radioimmunoassay and normalized to the corresponding cell numbers. All values represent the results of at least three separate experiments, with error bars representing standard deviations. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared to control cells in each Y1 clone). N.D., not detected.

FIG. 3.

HDAC inhibitors downregulate the expression of Cyp11a1 but not Sf-1. (A) Quantitative real-time RT-PCR analysis of mRNA levels for Cyp11a1 and Sf-1 from Y1 cells treated for 24 h without (CTRL) or with 100 ng/ml TSA, 2 mM VPA, or 2 mM NaB. Results were normalized to GAPDH expression and are expressed as values relative to ethanol-treated Y1 cells. **, P < 0.01; ***, P < 0.001 (compared to the control cells). (B) Expression of luciferase reporters driven by promoters of Cyp11a1 (2.3 kb), Sf-1 (0.7 kb), SV40, TK, and CMV in Y1 cells. After treatment without (CTRL) or with 100 ng/ml TSA for 24 h, luciferase activities were determined and normalized to total protein levels of each transfectant and are expressed as activity relative to the value for the control. All values show the results from at least three separate experiments, with error bars representing standard deviations.

FIG. 4.

TSA increases SF-1 turnover. (A) Levels of FLAG-SF-1 (F-SF-1) and FLAG-PKAc (F-PKAc) at different time points in a pulse-chase experiment. The expression plasmid for F-SF-1 or F-PKAc was transfected into Y1 cells. After 24 h, cells were pulse-labeled with [³⁵S]methionine for 1 h and chased in the absence (CTRL) or presence of 100 ng/ml TSA for the indicated times. ³⁵S-F-SF-1 and ³⁵S-F-PKAc were immunoprecipitated by anti-FLAG, separated by gel electrophoresis, and detected by autoradiography. (B) The relative amounts of F-SF-1 and F-PKAc in cells treated without (CTRL) or with 100 ng/ml TSA are shown. The graphs show the quantitative results of three independent experiments. t_1/2, half-life of SF-1. **, P < 0.01 compared to the control cells.

FIG. 5.

TSA and VPA induce polyubiquitination and proteasome-dependent degradation of SF-1. (A) Y1 cells were treated for 12 h without (CTRL) or with 100 ng/ml TSA or 2 mM VPA in the presence of 10 mM MG132 or vehicle control (dimethyl sulfoxide [DMSO]). Levels of SF-1 and HSP70, determined by immunoblotting analysis, are shown. (B) The FLAG-SF-1 and FLAG-PKAc subunits were expressed in Y1 cells. The cells were treated for 12 h with 100 ng/ml TSA (T) or 2 mM VPA (V) in the presence of 10 mM MG132. The anti-FLAG immunoprecipitates were analyzed by immunoblotting (IB) with antibodies against ubiquitin (α-Ub), SF-1 (α-SF-1), or FLAG (α-FLAG).

FIG. 6.

Silencing of Skp1a restores the HDAC inhibitor-reduced SF-1 level. (A) The mRNA levels of Skp1a, Ube2D1, and Ube2L6 from Y1 cells treated for 24 h without (CTRL) or with 100 ng/ml TSA, 2 mM VPA, or 2 mM NaB were determined by quantitative real-time PCR. Results were normalized to GAPDH expression and are expressed relative to the values for control cells. **, P < 0.01; ***, P < 0.001 (compared to control cells). (B) SKP1A levels were determined by immunoblotting analysis of whole-cell lysates of Y1 and Y1-SF-1 55 cells that had been treated for 24 h as in panel A. (C) Y1 and Y1-SF-1 55 cells were transfected with scrambled (Scram) or Skp1a-targeted siRNA oligonucleotides three times at 24-h intervals. In the third transfection, the cells were also treated without (CTRL) or with 100 ng/ml TSA or 2 mM VPA. The protein levels of SKP1A, SF-1, and HSP70 were determined by immunoblotting analysis.