Stimulation of StAR expression by cAMP is controlled by inhibition of highly inducible SIK1 via CRTC2, a co-activator of CREB

Abstract

In mouse steroidogenic cells the activation of cholesterol metabolism is mediated by steroidogenic acute regulatory protein (StAR). Here, we visualized a coordinated regulation of StAR transcription, splicing and post-transcriptional processing, which are synchronized by salt inducible kinase (SIK1) and CREB-regulated transcription coactivator (CRTC2). To detect primary RNA (pRNA), spliced primary RNA (Sp-RNA) and mRNA in single cells, we generated probe sets by using fluorescence in situ hybridization (FISH). These methods allowed us to address the nature of StAR gene expression and to visualize protein–nucleic acid interactions through direct detection. We show that SIK1 represses StAR expression in Y1 adrenal and MA10 testis cells through inhibition of processing mediated by CRTC2. Digital image analysis matches qPCR analyses of the total cell culture. Evidence is presented for spatially separate accumulation of StAR pRNA and Sp-RNA at the gene loci in the nucleus. These findings establish that cAMP, SIK and CRTC mediate StAR expression through activation of individual StAR gene loci.

Analysis of specific RNA in cultured cells through quantitative integration of q-PCR and N-SIM single cell FISH images: Application to hormonal stimulation of StAR transcription

“…6, we measured the number of spliced transcripts concentrated at the loci and identified them as individual species in the cytoplasm. Based on previous work (Lee et al, 2015), we expect the small cytoplasmic fluorescence sources of similar size to each correspond to a single mRNA. Their summation should correspond to the copy number per cell determined by RT-qPCR primed with 3’ PCR-specific primers on the macro-culture.…”

“…To quantify the StAR expression in single cells, we used synergistic binding of tandem 20-base oligomers to RNA (Lee et 2015). Fig.…”

“…Screening of multiple MA10 cells showed that separate sp-RNA mRNA were proportionately higher in sections proximal to the adherent surface. The mitochondria marked with mitotrackers have uniform and complete overlap with StAR protein immune-detection (Lee et al, 2015). The overlap of the sp-RNA with StAR protein, which is expressed only in mitochondria, is relatively localized.…”

“…cAMP regulates steroidogenic enzyme genes and steroid synthesis through collective modulation of chromatin and a shared cohort of transcription factors/cofactors that include SF1, GATA4, CREB/CBP, C/EBPβ, and NR4A1 (Clem et al, 2005; Di-Luoffo et al, 2015; Feng et al, 2000; Reinhart et al, 1999). Br-cAMP initiates a robust increase in StAR, beginning with primary RNA (p-RNA) transcripts (Lee et al, 2015). …”

“…In this study, we investigated the events that facilitate dynamic StAR transcription in response to cAMP stimulation in MA-10 Leydig cells, focusing on splicing anomalies at StAR gene loci. In particular, we visualized the coordinated regulation of StAR processing (Lee et al, 2015), which is synchronized by salt-inducible kinase (SIK1) and CREB-regulated transcription coactivator (CRTC2) (Screaton et al, 2004; Takemori et al, 2009; Uebi et al, 2010). The steps used for the processing of p-RNA to mature mRNA include capping, splicing, 3’UTR cleavage, and polyadenylation (Bentley, 2005; Hirose and Manley, 2000; Maniatis and Reed, 2002; Moore and Proudfoot, 2009), which involve multi-protein complexes organized around the C-terminus of RNA polymerase 2 (Pol 2) (Nojima et al, 2015).…”