Figure 1.
Analysis of SF-1 RNAi +/− cAMP-treated H295R cells.
(A) Experimental design. (B) qPCR of Control and siSF ± cAMP-treated cells show approximately 70% efficiency of SF-1 RNAi-mediated knockdown on mRNA level. (C) Western blot showing almost complete knockdown of SF-1 protein after RNAi-treatment. StAR, a protein under transcriptional control of SF-1, is also depleted, showing efficiency of knockdown on target gene level. (D) Venn diagram showing overlap between the different groups of differentially expressed genes.
Table 1.
Genes changed by both siSF-1 and cAMP treatment.
Figure 2.
Confirmation of steroidogenic target genes differentially expressed in microarrays and promoter assays of StAR, DAX-1 and SULT2A1 proximal promoters after cAMP treatment and flow-cytometry assay of siSF-1 treated cells.
(A) qPCR showing mRNA levels of six steroidogenic target genes. (B) Luciferase reporter assays of StAR, DAX-1 and SULT2A1 promoters after 16 h of ± cAMP treatment show that the down regulation of DAX-1 and SULT2A1 mRNA levels after cAMP-treatment cannot be recreated with the proximal 1 kb of the promoters alone. (C) Expression targets of DAX-1 differentially expressed in the Control vs. siRNF31 microarray as identified by Pathway Studio. Error bars show standard deviation. Statistical analysis done with Student's t-test (*p<0.05; **p<0.01; ***p<0.001).
Figure 3.
Analysis of differentially expressed genes in siRNF31 ± cAMP-treated H295R cells.
(A) qPCR showing approximately 60% efficiency of RNF31 RNAi-treatment on mRNA level. (B) Western blot showing efficient knockdown of RNF31 protein and upregulation of RNF31 target StAR in RNF31 RNAi-treated H295R cells. (C) Venn diagram showing overlap of differentially expressed genes in siSF-1 and siRNF31 microarrays. (D) Venn diagram overlap among the differentially expressed genes in all cAMP-treated samples.
Table 2.
Genes changed by both siSF-1 and siRNF31 treatment.
Table 3.
Top 20 GO biological processes in functional analysis of siRNF31 microarray results.
Table 4.
Selected GO biological processes from siSF-1 pathway-enrichment analysis.
Figure 4.
Putative SF-1 targets in the Wnt/β-catenin-signaling pathway.
(A) qPCR data of three genes in the Wnt/β-catenin pathway that are changed after siSF-1 knockdown. (B) Change in β-catenin responsive reporter plasmid TopFlash activity with increasing amounts of SF-1. Values shown are fold change compared to reporter plasmid luciferase activity without transfected β-catenin. (C) Schematic Wnt/β-catenin pathway with genes changed by siSF-1 marked in red (upregulation). Error bars show standard deviation. Statistical analysis done with Student's t-test (* p<0.05; **p<0.01;***p<0.001).
Figure 5.
Putative SF-1 target genes in TGFβ signalling pathways identified from pathway enrichment analysis in the DAVID online resource, siSF-1 effect on H295R cell proliferation.
(A) qPCR showing the effect on mRNA level of SF-1 knockdown ± cAMP treatment proteins in the TGFβ-pathway. (B) Flow-cytometry analysis of cells treated with TGFβ2 show significantly more cells in S-phase and less in G1/G0 after TGFβ2 treatment. (C) Schematic representation of the TGFβ-pathway with genes changed by SF-1 knockdown marked red for upregulation and green for downregulation. (D) Flow cytometry analysis of siSF-1 or control treated cells show more cells in G1/G0-phase and less in S-phase after SF-1 knockdown. (E) The SF-1 gene expression profile can classify malignant from benign adrenocortical tumors in clinical samples. The heatmap shows relative expression of genes differentially expressed in our siSF-1 microarray experiments in a study cohort of 58 ACCs and 34 ACAs [53]. Up- and downregulation is indicated by red and green, respectively, scaled across rows. The top bar illustrates the significant (p<0.01) separation of carcinomas (red) from adenomas (blue) by hierarchical clustering. Error bars show standard deviation. Statistical analysis done with Student's t-test (* p<0.05; **p<0.01; ***p<0.001).
Figure 6.
Hypothesis of SF-1 mechanism acting both in cis and in trans.
(A) Classical SF-1 action on steroidogenic enzyme gene promoters. SF-1 binds promoters in cis and recruit coactivators and the general transcription machinery to activate transcription. Raised intracellular cAMP levels due to ACTH (in the adrenal) activation of MC2R activates the CREB/CREM transcription factors that work synergistically to further increase transcription rates. Input from Wnt/β-catenin signalling through direct binding of β-catenin to SF-1 can also increase transcription. (B) Possible mechanisms of SF-1 dependent repression of Wnt/β-catenin signaling. 1. SF-1 binds in cis to promoters but due to post-translational modifications and/or specific corepressor recruitment represses instead of activates target gene transcription. 2. SF-1 binds in trans to transcription factor complex and directs corepressors to the site to repress transcription. This could also be mediated by post-translational modifications like SUMOylation. A third option is that transcription factors or corepressors whose expression is activated by SF-1 acts as repressors of the TGFβ and Wnt/β-catenin signalling making SF-1 an indirect regulator.