Regulation of StAR by the N-terminal Domain and Coinduction of SIK1 and TIS11b/Znf36l1 in Single Cells

Abstract

The cholesterol transfer function of steroidogenic acute regulatory protein (StAR) is uniquely integrated into adrenal cells, with mRNA translation and protein kinase A (PKA) phosphorylation occurring at the mitochondrial outer membrane (OMM). The StAR C-terminal cholesterol-binding domain (CBD) initiates mitochondrial intermembrane contacts to rapidly direct cholesterol to Cyp11a1 in the inner membrane (IMM). The conserved StAR N-terminal regulatory domain (NTD) includes a leader sequence targeting the CBD to OMM complexes that initiate cholesterol transfer. Here, we show how the NTD functions to enhance CBD activity delivers more efficiently from StAR mRNA in adrenal cells, and then how two factors hormonally restrain this process. NTD processing at two conserved sequence sites is selectively affected by StAR PKA phosphorylation. The CBD functions as a receptor to stimulate the OMM/IMM contacts that mediate transfer. The NTD controls the transit time that integrates extramitochondrial StAR effects on cholesterol homeostasis with other mitochondrial functions, including ATP generation, inter-organelle fusion, and the major permeability transition pore in partnership with other OMM proteins. PKA also rapidly induces two additional StAR modulators: salt-inducible kinase 1 (SIK1) and Znf36l1/Tis11b. Induced SIK1 attenuates the activity of CRTC2, a key mediator of StAR transcription and splicing, but only as cAMP levels decline. TIS11b inhibits translation and directs the endonuclease-mediated removal of the 3.5-kb StAR mRNA. Removal of either of these functions individually enhances cAMP-mediated induction of StAR. High-resolution fluorescence in situ hybridization (HR-FISH) of StAR RNA reveals asymmetric transcription at the gene locus and slow RNA splicing that delays mRNA formation, potentially to synchronize with cholesterol import. Adrenal cells may retain slow transcription to integrate with intermembrane NTD activation. HR-FISH resolves individual 3.5-kb StAR mRNA molecules via dual hybridization at the 3'- and 5'-ends and reveals an unexpectedly high frequency of 1:1 pairing with mitochondria marked by the matrix StAR protein. This pairing may be central to translation-coupled cholesterol transfer. Altogether, our results show that adrenal cells exhibit high-efficiency StAR activity that needs to integrate rapid cholesterol transfer with homeostasis and pulsatile hormonal stimulation. StAR NBD, the extended 3.5-kb mRNA, SIK1, and Tis11b play important roles.

Keywords: PCR; Sik1; StAR; Tis11b; fluorescence in situ hybridization.

Figure 1

Characterization of N-terminal sequence changes in the 3′UTR of mouse StAR mRNA. (A) Design for the transfection of MA-10 cells with dUTR–StAR and stimulation by Br-cAMP (a). Diagram showing empty vector and StAR expression vector (StAR dUTR) (b). Comparison of the N-terminal cleavage and phosphorylation of natively expressed and transfected StAR dUTR. Each is stimulated for 0, 30, and 60 min (c). (B) Diagram showing empty vector and StAR expression vectors with different 3′UTRs (a). Effects of StAR 3′UTR on basal expression in StAR cDNA constructs. Western blot of StAR protein in MA-10, Y-1, and COS-1 cells transfected with these vectors (b) (46).

Figure 2

NTD cleavage in COS-1 cells parallels changes in primary bovine adrenal cells. (A) Effects of aminoglutethimide (0.5 mM) and cycloheximide (0.2 mM) on cholesterol transfer in ACTH-stimulated cultured bovine adrenal cells (47) are shown in (a) mitochondrial cholesterol and (b) determination of cholesterol–CYP11A1 complex. Data are represented as mean ± SEM, ***P < 0.001. (B) Multispecies sequence comparison of StAR NTD that shares conserved MMP cleavage sites A and B. The typically used mouse and bovine identification of products is compared to the average M.W determined by mass spectrometry for bovine products. (C) Mutation of conserved cleavage sites (M28, M30) in the NTD of bovine StAR. The blue lines indicate the predicted cleavage sites for the production of the 30- and 28-kDa form of StAR. Expression of CMV bovine StAR vectors in COS-1 cells. Effects of mutations as observed by immunoblotting for StAR (48). (D) 2D gel determinations of NTD cleavage products in BAC cells (20). Decreases (p28, #1–2) and increases (p28, #3–4; p30, #5–6) of NTD cleavage products are shown in response to dibutryl cAMP. Data are represented as mean ± SEM, ***P < 0.001.

Figure 3

PCR determination of acute activation of StAR transcription in Y-1 adrenal cells. (A) Design of PCR primers for StAR p-RNA and sp-RNA/mRNA. (B) PCR time course for Y-1 cells [p-RNA (E1/I1) versus sp-RNA (E5/E6)] (0–30 min every 3 min). (C) PCR time course for E7-S and E7-L (0–15 min). (D) HR-FISH 20-mer fluorescent probes for StAR p-RNA and sp-RNA/mRNA. (E) Co-expression of p-RNA and sp-RNA at StAR loci in Y-1 cells after 60 min of stimulation. (F) Locus changes and cytoplasmic mRNA in Y-1 cells under basal conditions and after acute activation for 15 min (p-RNA versus sp-RNA) (sensitivity is high for mRNA detection).

Figure 4

Spatiotemporal regulation of StAR mRNA in Y-1 cells. (A) Time course of 0–120 min for the stimulation of StAR RNA as determined by q-PCR to detect the 3′UTR (E7-S, E7-L) and translated sequence (E5/E6). (B) p-RNA and sp-RNA at StAR loci in multiple cells; sensitivity is sufficient to detect RNA concentrated at loci but not cytoplasmic mRNA. Lower expression of mRNA requires a higher sensitivity setting for the microscope. (C) Loci in representative nuclei (0–180 min). p-RNA, sp-RNA, and merged. (D) Nuclear positioning for Z-sections a, b, and c. The positioning of sp-RNA versus 3′EU HR-FISH probes. StAR loci are found near the nuclear midline; cytoplasmic StAR mRNA is primarily observed between slice b and the adherent plasma membrane (37). (E) Z-projections of HR-FISH for StAR RNA at high sensitivity (N-SIM microscope) after stimulation for 60 and 120 min (dual addition of 3′EU and sp-RNA). StAR loci (yellow, due to probe overlap), sp-RNA (red), and 3′EU (green) visualize cytoplasmic mRNA. DAPI (blue) visualizes nuclear DNA. (F) Measurements of the nucleus, locus, and individual message. Scale bar, 1 μm. (G) N-SIM mRNA images after 120 min in the lower portion of the cell (slice c) with dual labeling by sp-RNA and 3′EU. Enlargement resolution of dual labeling with StAR mRNA and sp-RNA or 3′EU. (H) HR-FISH of StAR mRNA (sp-RNA) and immunochemistry of StAR proteins after 60 and 180 min of stimulation employing N-SIM microscopy. The StAR protein is present in the matrix of all Y-1 mitochondria (38). (I) Enlarged region after 60 min of stimulation showing the pairing of StAR mRNA and mitochondrial matrix-localized StAR proteins. Diagram of the spatial relationship between matrix proteins and OMM-associated StAR mRNA.

Figure 5

Induction and mobilization of SIK1 in Y-1 cells. (A) Diagram of SIK1 protein kinase sites; T182/LKB, S577 PKA. Short and long mRNAs differing in terms of their 3′UTR. (B) Stimulation of SIK1 p-RNA and long mRNA (4.4 kb) by Br-cAMP. Data are represented as mean ± SEM, *P < 0.05, **P < 0.01, and ***P < 0.001. (C) Br-cAMP (1 mM) affects the relocation of SIK1–GFP (initially nuclear) and CRTC2–GFP (initially cytoplasmic) (38). (D) Induction by the SIK1 inhibitor staurosporine; concentration (a), time-dependence (b), stimulation of RNA and sp-RNA at the gene loci after 60 min (c), and synergy lowering the E50 for each (d). (E) Br-cAMP and staurosporine induce SIK1 to a similar extent, suggesting that SIK1 exhibits feedback control.

Figure 6

Inhibition of basal and induced expression of StAR by PKA-resistant SIK1–S577A–GFP. (A) Nuclear location of SIK1–S577A–GFP with and without Br-cAMP. (B) Suppression of Br-cAMP induction (180 min) of StAR RNA at the loci or in the cytoplasm in cells that transfected with SIK1–S577A–GFP versus GFP. (C) Quantitative expression in transfected Y-1 cells versus neighboring untransfected cells (a, image #1 and image #2). Associated mRNA intensity in 20 transfected cells versus 6 adjacent untransfected cells (b).

Figure 7

Selective targeting of extended StAR 3′UTR by transfected TIS11b/Znf36l1. (A) The structure of TIS11b/Znf36li with RNA binding Zn-finger domains and kinase sites. (B) Other genes exhibiting RNA target sites (TATTTATT) (51). (C) Tis11b targeted sequences are conserved in mouse and human StAR 3′UTRs, specifically the terminal cleavage/polyadenylation site. (D) The impact of TIS11b on StAR mRNA and protein expression according to terms of the 3′UTR length: CMV–StAR vectors; StAR protein expression (immunoblot) and mRNA (q-PCR relative to actin) with and without Tis11b transfection (34).

Figure 8

Suppressive effects of Tis11b in both Y-1 and MA-10 cells. (A) Induction of TIS11b in Y-1 and MA-10 cells by 1 mM Br-cAMP, measured by q-PCR (34). (B) Schematic for the suppression of TIS11b using siRNA and shRNA in the indicated cell lines. (C) Time course for the selective stimulation of 3.5-kb StAR mRNA following the suppression of Tis11b (34). (D,E) Effects of Tis11b (shRNA) suppression on the dose–response stimulation of StAR mRNA levels. (F,G) Effects of Tis11b (cell line) suppression on the dose–response stimulation of Tis11b mRNA and StAR protein levels.

Figure 9

Nuclear/cytoplasmic distribution of Tis11b and activity. (A) TIS11b protein expression in the nuclei and cytoplasm of Y-1 and MA-10 cells under basal conditions and after stimulation (6 h/400 μM Br-cAMP). (B) Effects of stimulation time on TIS11b immunohistochemistry in MA-10 cells. (C) Stimulation of StAR sp-RNA/mRNA in MA-10 cells transfected with either mock CMV–GFP or CMV–Tis11b–GFP. (D) Quantitation of StAR sp-RNA/mRNA in MA-10 cells transfected with either mock CMV–GFP or CMV–Tis11b–GFP.

Figure 10

Modulation of StAR control of cholesterol availability via the N-terminal StAR regulatory domain, SIK1, and TIS11b. (A) Modulation of the cholesterol transfer activity of the C-terminal StAR domain by the N-terminal regulatory domain. Three functional compartments are distinguishable: green: StAR transfer from OMM cholesterol-binding activity and transfer of pp37 from the OMM to the IMM, where proteolytic processing occurs with possible supplemental activity in adrenal cells. Brown: cholesterol transfer at sites of membrane contact, possibly directed by mPTP modulation. Blue: factors modulating the partnership between StAR and cholesterol, including VDACs, TSPO, GTPases (OPA1, MFN2), and the activation effects of membrane fusion (ER, inter-into). (B) (a) schematic detailing the activities of SIK1 and CRTC2 in mediating the effects of cAMP and PKA on StAR mRNA expression in comparison to Tis11b and (b) modulation of StAR mRNA availability to the mitochondria by SIK1/CRTC2 (transcription and splicing) and Znf36l1/Tis11b (regulation of 3.5-kb StAR mRNA location/3′UTR processing).