A single cell level measurement of StAR expression and activity in adrenal cells

Abstract

The Steroidogenic acute regulatory protein (StAR) directs mitochondrial cholesterol uptake through a C-terminal cholesterol binding domain (CBD) and a 62 amino acid N-terminal regulatory domain (NTD) that contains an import sequence and conserved sites for inner membrane metalloproteases. Deletion of the NTD prevents mitochondrial import while maintaining steroidogenesis but with compromised cholesterol homeostasis. The rapid StAR-mediated cholesterol transfer in adrenal cells depends on concerted mRNA translation, p37 StAR phosphorylation and controlled NTD cleavage. The NTD controls this process with two cAMP-inducible modulators of, respectively, transcription and translation SIK1 and TIS11b/Znf36l1. High-resolution fluorescence in situ hybridization (HR-FISH) of StAR RNA resolves slow RNA splicing at the gene loci in cAMP-induced Y-1 cells and transfer of individual 3.5 kb mRNA molecules to mitochondria. StAR transcription depends on the CREB coactivator CRTC2 and PKA inhibition of the highly inducible suppressor kinase SIK1 and a basal counterpart SIK2. PKA-inducible TIS11b/Znf36l1 binds specifically to highly conserved elements in exon 7 thereby suppressing formation of mRNA and subsequent translation. Co-expression of SIK1, Znf36l1 with 3.5 kb StAR mRNA may limit responses to pulsatile signaling by ACTH while regulating the transition to more prolonged stress

Monitoring of Dual CRISPR/Cas9-Mediated Steroidogenic Acute Regulatory Protein Gene Deletion and Cholesterol Accumulation Using High-Resolution Fluorescence In Situ Hybridization in a Single Cell

“…The 3D spatial distribution of StAR loci, mRNA, and protein which is central to the sm-FISH assessment of gene expression was best seen in MA-10 cells. Thus, StAR protein and mRNA after 3 h of stimulation showed very characteristic 3D organizations ( 38 ) (Figure 4 B). The StAR protein in these mitochondria is located in the matrix and is inactive in promoting cholesterol transfer to Cyp11a1.…”

The potent roles of salt-inducible kinases (SIKs) in metabolic homeostasis and tumorigenesis

“…34 On the other hand, SIKs also transcriptionally regulate the Na + ,K + -ATPase, either by directly inhibiting the entry of CRTC into the nucleus to transcript ATP1B1 gene, which encodes a Na + , K + -ATPase subunit, 2 or by indirectly repressing the hormonesinduced Na + ,K + -ATPase expression, 74 via an increased CYP11A and StAR mRNA levels to promote the adrenocorticotropic hormone production. 55,[75][76][77] Other downstream signaling pathways TGFβ-Smad pathway. In normal epithelial cells, TGFβ-Smad signaling pathway is considered to play an anti-cancer role by inducing cell cycle arrest and apoptosis.…”

“…115 Subsequent studies demonstrated that SIK1 repressed the efficient operation of the CREB transcription activation complex, thereby inhibiting the CREdriven transcription of the CYP11A gene and the StAR gene in Y1 cells. 55,[75][76][77] Du et al 116 found that, similar to SIK1, SIK2 can also repress the expression of lipogenic genes (FAS, ACC2 and SCD1), and this effect can be reversed by depleting SREBP1. In addition, SIK2 promotes fatty acid synthesis by upregulating SREBP1c expression, thus promoting the transcription of Fasn in ovarian cancer cells.…”

“… 34 On the other hand, SIKs also transcriptionally regulate the Na + ,K + -ATPase, either by directly inhibiting the entry of CRTC into the nucleus to transcript ATP1B1 gene, which encodes a Na + ,K + -ATPase subunit, 2 or by indirectly repressing the hormones-induced Na + ,K + -ATPase expression, 74 via an increased CYP11A and StAR mRNA levels to promote the adrenocorticotropic hormone production. 55 , 75 – 77 …”

“… 115 Subsequent studies demonstrated that SIK1 repressed the efficient operation of the CREB transcription activation complex, thereby inhibiting the CRE-driven transcription of the CYP11A gene and the StAR gene in Y1 cells. 55 , 75 – 77 …”