POD-1 binding to the E-box sequence inhibits SF-1 and StAR expression in human adrenocortical tumor cells

Abstract

Pod-1/Tcf21 is expressed at epithelial-mesenchymal interaction sites during development of many organs. Different approaches have demonstrated that Pod-1 transcriptionally inhibits Sf-1/NR5A1 during gonadal development. Disruption of Sf-1 can lead to disorders of adrenal development, while increased dosage of SF-1 has been related to increased adrenal cell proliferation and tumorigenesis. In this study, we analyzed whether POD-1 overexpression inhibits the endogenous Sf-1 expression in human and mouse adrenocortical tumor cells. Cells were transiently transfected with luciferase reporter gene under the control of Sf-1 promoter and with an expression vector encoding Pod-1. Pod-1 construct inhibited the transcription of the Sf1/Luc reporter gene in a dose-dependent manner in mouse Y-1 adrenocortical carcinoma (ACC) cells, and inhibited endogenous SF-1 expression in the human H295R and ACC-T36 adrenocortical carcinoma cells. These results were validated by chromatin immunoprecipitation assay with POD-1-transfected H295R cells using primers specific to E-box sequence in SF-1 promoter region, indicating that POD-1 binds to the SF-1 E-box promoter. Moreover, POD-1 over-expression resulted in a decrease in expression of the SF-1 target gene, StAR (Steroidogenic Acute Regulatory Protein). Lastly, while the induced expression of POD-1 did not affect the cell viability of H295R/POD-1 or ACC-T36/POD-1 cells, the most significantly enriched KEGG pathways for genes negatively correlated to POD-1/TCF21 in 33 human ACCs were those associated with cell cycle genes.

Fig. 1

Luciferase reporter gene assay in Y1 adrenocortical tumor cell line and TM3 Leydig cells transfected with 0.5 μg or 1 μg of pDNA3/Pod-1 and 1 μg pDNA3Sf-1/Luc. Cells were assayed for both Firefly and Renilla by using Dual-Luciferase reporter assay system. Statistical significance was tested by paired T test on 3 pairs.

Fig. 2

Expression data for POD-1 and SF-1 from arrays run on human adrenocortical tissues. POD-1 was lower in ACA than normal (NL, p = 1 × 10⁻⁷, average fold-change = 0.51) and even lower in ACC compared to ACA (p = 3 × 10⁻⁸, average fold-change = 0.60, both tests from a one-way ANOVA model fit to the three groups).

Fig. 3

SF-1 was negatively correlated to POD-1 in public array data for 33 ACCs (r = −0.54; p = 0.0012). In ACAs the correlation was negative but not significant (r = −0.25; p = 0.26).

Fig. 4

RT-qPCR analysis of relative gene expression of POD-1 and SF-1 in H295R and ACC-T36 cells transiently transfected with (A) empty vector pcDNA3 versus pcDNA3Pod1 and (B) empty vector pCMVMyc versus pCMVMycPod1. Statistical significance was tested by paired T test on 3 pairs.

Fig. 5

Chromatin enrichment was confirmed by PCR amplification of the E-box region of SF-1 promoter from anti-Myc immunoprecipated H295pCMVMycPod1 DNA. The positions of the amplicons in relation to the Transcriptional Start Sites (TSSs – represented by arrows) are shown. Black and open bars represent, respectively, the exon and a different E-box sequence. Androgen Receptor (AR), Input (Inp) 0.1% DNA, Anti-MYC-IP NCIpCMVMycPod1 (IP), anti-IgG (IgG).

Fig. 6

Viability assay of NCI-H295R cell line (A and B) and ACC-T36 adrenocortical cell culture (C and D) transiently transfected with pcDNA3Pod1 (A and C) or pCMVMycPod1 (B and D) versus respective empty vectors. The MTS assay was initiated 48 h after cell transfection. Results are expressed as mean ± SD. Statistical significance two-way ANOVA with Tukey (post-test); n = 3.

Fig. 7

RT-qPCR analysis of relative gene expression of StAR in H295R and ACC-T36 cells transiently transfected with pCMVMycPod1 or with the empty vector pCMVMyc. Statistical significance was tested by paired T test on 3 pairs.