Contributions of steroidogenic factor 1 to the transcription landscape of Y1 mouse adrenocortical tumor cells

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Abstract

The contribution of steroidogenic factor 1 (SF-1) to the gene expression profile of Y1 mouse adrenocortical cells was evaluated using short hairpin RNAs to knockdown SF-1. The reduced level of SF-1 RNA was associated with global changes that affected the accumulation of more than 2000 transcripts. Among the down-regulated transcripts were several with functions in steroidogenesis that were affected to different degrees—i.e., Mc2r > Scarb1 > Star  Hsd3b1 > Cyp11b1. For Star and Cyp11b1, the different levels of expression correlated with the amount of residual SF-1 bound to the proximal promoter regions. The knockdown of SF-1 did not affect the accumulation of Cyp11a1 transcripts even though the amount of SF-1 bound to the proximal promoter of the gene was reduced to background levels. Our results indicate that transcripts with functions in steroidogenesis vary in their dependence on SF-1 for constitutive expression. On a more global scale, SF-1 knockdown affects the accumulation of a large number of transcripts, most of which are not recognizably involved in steroid hormone biosynthesis.

Introduction

Steroidogenic factor 1 (SF-1) is considered to be a master transcription factor essential for the coordinate and tissue-specific expression of genes with functions in adrenal steroidogenesis (Schimmer and White, 2010). This role of SF-1 is based largely on reporter gene assays using limited promoter/regulatory DNA, which, as noted previously, may overemphasis the importance of SF-1 at the expense of more distal regulatory elements (Milstone et al., 1992). Indeed, manipulating SF-1 gene dosage in vivo raises some doubts about a master regulatory role for SF-1 in the expression of genes involved in adrenal steroidogenesis.

In SF-1+/− heterozygous mice, the levels of SF-1 are reduced and the adrenal glands are hypoplastic; however, the adrenal cells from these animals paradoxically exhibit increased levels of Cyp11a1, Star and Mc2r—i.e., transcripts putatively under SF-1 control (Bland et al., 2000b, Bland et al., 2004). In humans, heterozygous SF-1 mutations that disrupt its DNA binding activity sometimes cause adrenal insufficiency (Achermann et al., 1999, Achermann et al., 2002, Biason-Lauber and Schoenle, 2000); in other cases, however, they have no effect on adrenal function while at the same time impairing gonadal function (Lin et al., 2007, Kohler et al., 2008). Thus, the requirement for SF-1 in the expression of genes required for adrenal steroidogenesis appears not to be dosage sensitive either in mice or in humans.

Similarly, manipulating SF-1 dosage in H295R human adrenocortical tumor cells by over-expressing wild-type SF-1 (Doghman et al., 2007), by transformation with a dominant negative SF-1 mutant (Li et al., 2004) or by knocking down SF-1 with siRNA (Doghman et al., 2007, Ye et al., 2009) has little or no effect on key enzymes in steroidogenesis such as STAR and CYP11A1. Transcripts encoding HSD3B2 are only modestly reduced in the presence of dominant negative SF-1, but are not affected (Ye et al., 2009) or paradoxically decreased (Doghman et al., 2007) in cells over-expressing SF-1. Taken together, these results suggest that SF-1 may not be an obligatory regulator of adrenal steroidogenesis in vivo.

Given the concerns about the relative importance of SF-1 in situ discussed above, the present study was undertaken to determine the consequences of SF-1 knockdown on the steroidogenic potential of Y1 adrenocortical tumor cells. As reviewed elsewhere (Rainey et al., 2004), the Y1 mouse adrenocortical cell line has been used widely to study adrenal steroidogenesis and was used quite extensively in the reporter gene assays that gave rise to the hypothesis that SF-1 regulates the expression of genes with functions in steroidogenesis. We report that transcripts with functions in steroidogenesis vary in their dependence on SF-1 for constitutive expression. Transcripts encoding Cyp11a1 and Cyp11b1 are relatively resistant to SF-1 knockdown, whereas transcripts encoding StAR, Mc2r, Hsd3b1 and Scarb1 are considerably more sensitive. To determine if SF-1 has a more global role in the adrenal cortex extending beyond its role in steroidogenesis, we also have examined the genome-wide consequences of SF-1 knockdown in these cells. We observe that SF-1 knockdown alters the transcription landscape of Y1 adrenal cells by affecting the expression of slightly more than 2000 transcripts, most of which are not recognizably involved in steroid hormone biosynthesis. These latter results strongly suggest that SF-1 has additional functions in the adrenal cortex.

Section snippets

Oligonucleotides and plasmids

Gene-specific oligonucleotides were synthesized by Invitrogen Canada Inc. (Burlington, ON; Appendix A). pRNAT-CMV3.1/Neo vectors (Genscript Corp., Piscataway, NJ) expressing shRNAs targeted to three regions of the mouse SF-1 transcript (nucleotides 151–171, 565–585 and 1375–1395 respectively) were prepared as described previously (Rui et al., 2008). A control shRNA vector, with nucleotides 151–171 from SF-1 in scrambled order, was obtained from Genscript Corp. The SF-1 expression plasmid

The levels of SF-1 and other transcripts with functions in steroidogenesis in shRNA-transformed clones

Ten G418-resistant clones, isolated from Y1 cells following transfection with plasmids encoding SF-1 shRNA, were screened at first passage for SF-1 transcripts by quantitative RT-PCR. Eight of these clones had transcript levels that were reduced by at least 75% (i.e., from 9% to 23% of the levels in untransfected cells; Fig. 1). These eight included isolates transformed with each of the three shRNAs tested, indicating that each vector was capable of knocking down SF-1. A control plasmid with a

Discussion

This study demonstrates that SF-1 shRNAs targeting the SF-1 transcript greatly diminished the accumulation of transcripts encoding Mc2r (the ACTH receptor), Scarb1 (the HDL receptor that participates in cholesterol uptake), Star (a transport protein that carries cholesterol across mitochondrial membranes) and Hsd3b1 (a steroid reductase that converts pregnenolone to progesterone) in Y1 mouse adrenal cells, while reducing the accumulation of SF-1 transcripts by at least 75% (Fig. 2). These

Acknowledgments

This work was supported by a research grant from the Canadian Institutes of Health to B.P.S. and by NIDDK NIH Research Grant R01-DK062027 to Dr. Gary D. Hammer. We also acknowledge Dr. Hammer for helpful discussions. The plasmid pcDNA-Zeo+ was a gift from Dr. James Woodgett, Department of Medical Biophysics, University of Toronto, Toronto, Canada.

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