Inhibition of NR5A1 Phosphorylation Alleviates a Transcriptional Suppression Defect Caused by a Novel NR0B1 Mutation

Abstract

Context: Mutations in the NR0B1 gene, also well-known as the DAX1 gene, are known to cause congenital adrenal hypoplasia associated with hypogonadotropic hypogonadism. The abnormal NR0B1 protein fails to suppress the transcription of promoters of steroidogenic enzymes, which are also targets of NR5A1 protein, also well-known as Ad4BP/SF-1 protein. Since NR5A1 and NR0B1 have antagonistic effects on steroidogenesis, the loss of function due to NR0B1 mutations may be compensated by inducing loss of function of NR5A1 protein.

Patient: A middle-aged man was diagnosed with congenital adrenal hypoplasia associated with hypogonadotropic hypogonadism and genetic analysis revealed him to have a novel NR0B1 mutation, c.1222C>T(p.Gln408Ter).

Methods: NR0B1 activity was evaluated in CLK1/4 inhibitor-treated 293T cells via immunoblotting and luciferase assays of the STAR promoter.

Results: TG003 treatment suppressed NR5A1 protein function to compensate for the mutant NR0B1 showing inhibited suppression of transcription. Immunoblotting analyses showed that the phosphorylation status of NR5A1 at Ser203 was attenuated by the CLK1/4 inhibitor.

Conclusion: The specific reduction of NR5A1 phosphorylation by a CLK1/4 inhibitor may alleviate developmental defects in patients with NR0B1 mutations.

Keywords: CLK1/4 inhibitor; NR0B1 mutation; NR5A1 phosphorylation; congenital adrenal hypoplasia.

Figure 1.

Functional features of the novel NR0B1 mutation. (A) Chromatogram of the c.1222C>T, p.Gln408Ter (Q408X) mutation in the case presented. (B) Scheme of the NR0B1 gene structure (upper) and resulting truncated protein of the Q408X mutation. (C) Immunoblot of wild-type and Q408X-mutant NR0B1 using anti-NR0B1 antibody. (D) Immunofluorescence of COS7 cells transfected with wild-type and Q408X-mutant NR0B1 using anti-NR0B1 antibody and merged via DAPI staining. (E) STAR promoter analyses of 293T cells transfected with NR5A1 along with wild-type and Q408X-mutant NR0B1 as indicated. One-way ANOVA, **P < 0.01, ****P < 0.0001, ns; not significant. Abbreviations/gene designations: NR0B1, dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1; NR5A1, Ad4-binding protein/steroidogenic factor-1; DAPI, 4′,6-diamidino-2-phenylindole; STAR, steroidogenic acute regulatory protein.

Figure 2.

Compensation of the transcriptional suppression defect of the NR0B1 mutant via treatment with TG003. STAR promoter analyses of 293T cells transfected with NR5A1 and NR0B1 wild-type or NR0B1 Q408X mutant treated with TG003 (0, 1, 10, or 50 μM) for 24 hours. One-way ANOVA, **P < 0.01, ***P < 0.001, ****P < 0.0001. Gene designations: NR0B1, dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1; NR5A1, Ad4-binding protein/steroidogenic factor-1; STAR, steroidogenic acute regulatory protein.

Figure 3.

(A, B) TG003-mediated suppression of STAR promoter activity in an NR0B1-independent manner. TG003 (0, 10, or 50 μM) did not alter the expression of NR0B1 wild-type or NR0B1 Q408X transiently expressed in 293T cells (A). TG003 (0, 10, or 50 μM) suppressed STAR promoter activity in the absence of NR0B1 and also where the NR0B1 Q408X mutant had a defect in transcriptional suppression of STAR promoter activity. One-way ANOVA, ****P < 0.0001 (B). NR0B1, dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1; STAR, steroidogenic acute regulatory protein. (C) Decreased phosphorylation of NR5A1 via TG003 treatment. Left panel shows representative western blot analysis of 3 independent assays in duplicate of NR5A1-transiently-transfected 293T cells treated with TG003 (0, 10, or 50 μM). Right graph shows the relative phosphorylation rate of NR5A1 (p-NR5A1) in TG003-treated cells compared to that in nontreated cells. One-way ANOVA, *P < 0.05. Gene designations: NR0B1, dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1; NR5A1, Ad4-binding protein/steroidogenic factor-1; STAR, steroidogenic acute regulatory protein.

Figure 4.

The molecular pathways and potential targets of TG003. (A) Transcriptional regulation of steroidogenic enzymes by NR0B1 and NR5A1. (B) Adrenal cortex zones on fetal adrenal, adrenal of NR0B1 normal, and adrenal of NR0B1 mutant. (C) The diagram of TG003 effects. Gene designations: NR0B1, dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1; NR5A1, Ad4-binding protein/steroidogenic factor-1.