SCAP/SREBP pathway is required for the full steroidogenic response to cyclic AMP

Contributed by Joseph A. Beavo, July 19, 2016 (sent for review May 14, 2016; reviewed by Marco Conti, Donald Maurice, and Timothy Osborne)
September 6, 2016
113 (38) E5685-E5693

Significance

Luteinizing hormone stimulates production of testosterone and other steroids largely through a surge in the second messenger cAMP and subsequent activation of protein kinase A (PKA) in target cells. Rates of steroidogenesis are also dependent on the availability of cholesterol, a steroid building block. We propose, based on our results, that cAMP/PKA coordinates the functions of multiple pathways to regulate cellular cholesterol handling and synthesis and downstream steroid output. Activation of the cholesterol-sensing SCAP–SREBP2 pathway plays an important role in cAMP/PKA coordination of steroidogenesis. These cAMP/PKA-induced pathways are likely to be major regulators of sterol biosynthesis and cholesterol recharging in steroid hormone synthetic and other tissues. Cyclic nucleotide phosphodiesterases can be targeted to promote steroidogenesis and cholesterol metabolism.

Abstract

Luteinizing hormone (LH) stimulates steroidogenesis largely through a surge in cyclic AMP (cAMP). Steroidogenic rates are also critically dependent on the availability of cholesterol at mitochondrial sites of synthesis. This cholesterol is provided by cellular uptake of lipoproteins, mobilization of intracellular lipid, and de novo synthesis. Whether and how these pathways are coordinated by cAMP are poorly understood. Recent phosphoproteomic analyses of cAMP-dependent phosphorylation sites in MA10 Leydig cells suggested that cAMP regulates multiple steps in these processes, including activation of the SCAP/SREBP pathway. SCAP [sterol-regulatory element-binding protein (SREBP) cleavage-activating protein] acts as a cholesterol sensor responsible for regulating intracellular cholesterol balance. Its role in cAMP-mediated control of steroidogenesis has not been explored. We used two CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR associated protein 9) knockout approaches to test the role of SCAP in steroidogenesis. Our results demonstrate that SCAP is required for progesterone production induced by concurrent inhibition of the cAMP phosphodiesterases PDE4 and PDE8. These inhibitors increased SCAP phosphorylation, SREBP2 activation, and subsequent expression of cholesterol biosynthetic genes, whereas SCAP deficiency largely prevented these effects. Reexpression of SCAP in SCAP-deficient cells restored SREBP2 protein expression and partially restored steroidogenic responses, confirming the requirement of SCAP–SREBP2 in steroidogenesis. Inhibitors of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase and isoprenylation attenuated, whereas exogenously provided cholesterol augmented, PDE inhibitor-induced steroidogenesis, suggesting that the cholesterol substrate needed for steroidogenesis is provided by both de novo synthesis and isoprenylation-dependent mechanisms. Overall, these results demonstrate a novel role for LH/cAMP in SCAP/SREBP activation and subsequent regulation of steroidogenesis.

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Acknowledgments

This work was supported in part by NIH Grants R01GM083926, R01GM083926-02S1, R01HL062887, P01HL092969, and R01HL126028 and the Viral Vector and Transgenic Mouse Core and the Quantitative and Functional Proteomics Core of the Diabetes Research Center at the University of Washington (P30DK017047). We acknowledge support from the NIH to the UW Keck Microscopy Center (S10 OD016240).

Supporting Information

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References

1
AT Bender, JA Beavo, Cyclic nucleotide phosphodiesterases: Molecular regulation to clinical use. Pharmacol Rev 58, 488–520 (2006).
2
DH Maurice, et al., Advances in targeting cyclic nucleotide phosphodiesterases. Nat Rev Drug Discov 13, 290–314 (2014).
3
LC Tsai, JA Beavo, The roles of cyclic nucleotide phosphodiesterases (PDEs) in steroidogenesis. Curr Opin Pharmacol 11, 670–675 (2011).
4
LC Tsai, M Shimizu-Albergine, JA Beavo, The high-affinity cAMP-specific phosphodiesterase 8B controls steroidogenesis in the mouse adrenal gland. Mol Pharmacol 79, 639–648 (2011).
5
M Shimizu-Albergine, LC Tsai, E Patrucco, JA Beavo, cAMP-specific phosphodiesterases 8A and 8B, essential regulators of Leydig cell steroidogenesis. Mol Pharmacol 81, 556–566 (2012).
6
V Vasta, M Shimizu-Albergine, JA Beavo, Modulation of Leydig cell function by cyclic nucleotide phosphodiesterase 8A. Proc Natl Acad Sci USA 103, 19925–19930 (2006).
7
N Oki, SI Takahashi, H Hidaka, M Conti, Short term feedback regulation of cAMP in FRTL-5 thyroid cells. Role of PDE4D3 phosphodiesterase activation. J Biol Chem 275, 10831–10837 (2000).
8
M Golkowski, M Shimizu-Albergine, HW Suh, JA Beavo, S-E Ong, Studying mechanisms of cAMP and cyclic nucleotide phosphodiesterase signaling in Leydig cell function with phosphoproteomics. Cell Signal 28, 764–778 (2016).
9
MS Brown, JL Goldstein, The SREBP pathway: Regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 89, 331–340 (1997).
10
TL Steck, Y Lange, SCAP, an ER sensor that regulates cell cholesterol. Dev Cell 3, 306–308 (2002).
11
LP Sun, J Seemann, JL Goldstein, MS Brown, Sterol-regulated transport of SREBPs from endoplasmic reticulum to Golgi: Insig renders sorting signal in Scap inaccessible to COPII proteins. Proc Natl Acad Sci USA 104, 6519–6526 (2007).
12
A Radhakrishnan, JL Goldstein, JG McDonald, MS Brown, Switch-like control of SREBP-2 transport triggered by small changes in ER cholesterol: A delicate balance. Cell Metab 8, 512–521 (2008).
13
AJ Brown, L Sun, JD Feramisco, MS Brown, JL Goldstein, Cholesterol addition to ER membranes alters conformation of SCAP, the SREBP escort protein that regulates cholesterol metabolism. Mol Cell 10, 237–245 (2002).
14
RA DeBose-Boyd, et al., Transport-dependent proteolysis of SREBP: Relocation of site-1 protease from Golgi to ER obviates the need for SREBP transport to Golgi. Cell 99, 703–712 (1999).
15
PJ Espenshade, D Cheng, JL Goldstein, MS Brown, Autocatalytic processing of site-1 protease removes propeptide and permits cleavage of sterol regulatory element-binding proteins. J Biol Chem 274, 22795–22804 (1999).
16
PJ Espenshade, WP Li, D Yabe, Sterols block binding of COPII proteins to SCAP, thereby controlling SCAP sorting in ER. Proc Natl Acad Sci USA 99, 11694–11699 (2002).
17
JD Horton, et al., Combined analysis of oligonucleotide microarray data from transgenic and knockout mice identifies direct SREBP target genes. Proc Natl Acad Sci USA 100, 12027–12032 (2003).
18
M Matsuda, et al., SREBP cleavage-activating protein (SCAP) is required for increased lipid synthesis in liver induced by cholesterol deprivation and insulin elevation. Genes Dev 15, 1206–1216 (2001).
19
TJ Grevengoed, EL Klett, RA Coleman, Acyl-CoA metabolism and partitioning. Annu Rev Nutr 34, 1–30 (2014).
20
JE Kanter, C Tang, JF Oram, KE Bornfeldt, Acyl-CoA synthetase 1 is required for oleate and linoleate mediated inhibition of cholesterol efflux through ATP-binding cassette transporter A1 in macrophages. Biochim Biophys Acta 1821, 358–364 (2012).
21
P Maloberti, et al., Silencing the expression of mitochondrial acyl-CoA thioesterase I and acyl-CoA synthetase 4 inhibits hormone-induced steroidogenesis. FEBS J 272, 1804–1814 (2005).
22
F Cornejo Maciel, et al., An arachidonic acid-preferring acyl-CoA synthetase is a hormone-dependent and obligatory protein in the signal transduction pathway of steroidogenic hormones. J Mol Endocrinol 34, 655–666 (2005).
23
SQ Tsai, et al., Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat Biotechnol 32, 569–576 (2014).
24
MS Brown, JL Goldstein, A proteolytic pathway that controls the cholesterol content of membranes, cells, and blood. Proc Natl Acad Sci USA 96, 11041–11048 (1999).
25
J Sakai, et al., Identification of complexes between the COOH-terminal domains of sterol regulatory element-binding proteins (SREBPs) and SREBP cleavage-activating protein. J Biol Chem 272, 20213–20221 (1997).
26
W Shao, PJ Espenshade, Sterol regulatory element-binding protein (SREBP) cleavage regulates Golgi-to-endoplasmic reticulum recycling of SREBP cleavage-activating protein (SCAP). J Biol Chem 289, 7547–7557 (2014).
27
JL Goldstein, RA DeBose-Boyd, MS Brown, Protein sensors for membrane sterols. Cell 124, 35–46 (2006).
28
M Lehto, VM Olkkonen, The OSBP-related proteins: A novel protein family involved in vesicle transport, cellular lipid metabolism, and cell signalling. Biochim Biophys Acta 1631, 1–11 (2003).
29
M Aridor, WE Balch, Kinase signaling initiates coat complex II (COPII) recruitment and export from the mammalian endoplasmic reticulum. J Biol Chem 275, 35673–35676 (2000).
30
DA Freeman, M Ascoli, Studies on the source of cholesterol used for steroid biosynthesis in cultured Leydig tumor cells. J Biol Chem 257, 14231–14238 (1982).
31
L Nagy, DA Freeman, Cholesterol movement between the plasma membrane and the cholesteryl ester droplets of cultured Leydig tumour cells. Biochem J 271, 809–814 (1990).
32
J Hu, Z Zhang, WJ Shen, S Azhar, Cellular cholesterol delivery, intracellular processing and utilization for biosynthesis of steroid hormones. Nutr Metab (Lond) 7, 47 (2010).
33
JL Bos, H Rehmann, A Wittinghofer, GEFs and GAPs: Critical elements in the control of small G proteins. Cell 129, 865–877 (2007).
34
D Meiri, et al., Modulation of Rho guanine exchange factor Lfc activity by protein kinase A-mediated phosphorylation. Mol Cell Biol 29, 5963–5973 (2009).
35
J Cherfils, M Zeghouf, Regulation of small GTPases by GEFs, GAPs, and GDIs. Physiol Rev 93, 269–309 (2013).
36
D Li, MB Sewer, RhoA and DIAPH1 mediate adrenocorticotropin-stimulated cortisol biosynthesis by regulating mitochondrial trafficking. Endocrinology 151, 4313–4323 (2010).
37
E Ikonen, Cellular cholesterol trafficking and compartmentalization. Nat Rev Mol Cell Biol 9, 125–138 (2008).
38
SK Halder, M Fink, MR Waterman, D Rozman, A cAMP-responsive element binding site is essential for sterol regulation of the human lanosterol 14alpha-demethylase gene (CYP51). Mol Endocrinol 16, 1853–1863 (2002).
39
PA Edwards, D Tabor, HR Kast, A Venkateswaran, Regulation of gene expression by SREBP and SCAP. Biochim Biophys Acta 1529, 103–113 (2000).
40
S Rome, et al., Microarray analyses of SREBP-1a and SREBP-1c target genes identify new regulatory pathways in muscle. Physiol Genomics 34, 327–337 (2008).
41
F Bergeron, G Nadeau, RS Viger, GATA4 knockdown in MA-10 Leydig cells identifies multiple target genes in the steroidogenic pathway. Reproduction 149, 245–257 (2015).
42
SM Eacker, et al., Hormonal regulation of testicular steroid and cholesterol homeostasis. Mol Endocrinol 22, 623–635 (2008).
43
C Holm, Molecular mechanisms regulating hormone-sensitive lipase and lipolysis. Biochem Soc Trans 31, 1120–1124 (2003).
44
BJ Clark, V Ranganathan, R Combs, Post-translational regulation of steroidogenic acute regulatory protein by cAMP-dependent protein kinase A. Endocr Res 26, 681–689 (2000).
45
MT Dyson, MP Kowalewski, PR Manna, DM Stocco, The differential regulation of steroidogenic acute regulatory protein-mediated steroidogenesis by type I and type II PKA in MA-10 cells. Mol Cell Endocrinol 300, 94–103 (2009).
46
DM Stocco, StAR protein and the regulation of steroid hormone biosynthesis. Annu Rev Physiol 63, 193–213 (2001).
47
DM Stocco, BJ Clark, Role of the steroidogenic acute regulatory protein (StAR) in steroidogenesis. Biochem Pharmacol 51, 197–205 (1996).
48
J Liu, H Li, V Papadopoulos, PAP7, a PBR/PKA-RIalpha-associated protein: A new element in the relay of the hormonal induction of steroidogenesis. J Steroid Biochem Mol Biol 85, 275–283 (2003).
49
MT Dyson, et al., Mitochondrial A-kinase anchoring protein 121 binds type II protein kinase A and enhances steroidogenic acute regulatory protein-mediated steroidogenesis in MA-10 mouse Leydig tumor cells. Biol Reprod 78, 267–277 (2008).
50
PV Hornbeck, et al., PhosphoSitePlus, 2014: Mutations, PTMs and recalibrations. Nucleic Acids Res 43, D512–D520 (2015).
51
C Poderoso, et al., A mitochondrial kinase complex is essential to mediate an ERK1/2-dependent phosphorylation of a key regulatory protein in steroid biosynthesis. PLoS One 3, e1443 (2008).

Information & Authors

Information

Published in

Go to Proceedings of the National Academy of Sciences
Go to Proceedings of the National Academy of Sciences
Proceedings of the National Academy of Sciences
Vol. 113 | No. 38
September 20, 2016
PubMed: 27601673

Classifications

Submission history

Published online: September 6, 2016
Published in issue: September 20, 2016

Keywords

  1. steroidogenesis
  2. cholesterol
  3. cAMP
  4. phosphodiesterase
  5. SCAP/SREBP

Acknowledgments

This work was supported in part by NIH Grants R01GM083926, R01GM083926-02S1, R01HL062887, P01HL092969, and R01HL126028 and the Viral Vector and Transgenic Mouse Core and the Quantitative and Functional Proteomics Core of the Diabetes Research Center at the University of Washington (P30DK017047). We acknowledge support from the NIH to the UW Keck Microscopy Center (S10 OD016240).

Authors

Affiliations

Masami Shimizu-Albergine
School of Medicine, Department of Pharmacology, University of Washington, Seattle, WA 98195;
School of Medicine, Department of Medicine, Division of Metabolism, Endocrinology and Nutrition, University of Washington, Seattle, WA 98109;
University of Washington Diabetes Institute, School of Medicine, University of Washington, Seattle, WA 98109;
Brian Van Yserloo
School of Medicine, Department of Medicine, Division of Metabolism, Endocrinology and Nutrition, University of Washington, Seattle, WA 98109;
University of Washington Diabetes Institute, School of Medicine, University of Washington, Seattle, WA 98109;
Martin G. Golkowski
School of Medicine, Department of Pharmacology, University of Washington, Seattle, WA 98195;
Shao-En Ong
School of Medicine, Department of Pharmacology, University of Washington, Seattle, WA 98195;
Joseph A. Beavo2,1 [email protected]
School of Medicine, Department of Pharmacology, University of Washington, Seattle, WA 98195;
Karin E. Bornfeldt2,1 [email protected]
School of Medicine, Department of Medicine, Division of Metabolism, Endocrinology and Nutrition, University of Washington, Seattle, WA 98109;
University of Washington Diabetes Institute, School of Medicine, University of Washington, Seattle, WA 98109;
School of Medicine, Department of Pathology, University of Washington Diabetes Institute, University of Washington, Seattle, WA 98109

Notes

2
To whom correspondence may be addressed. Email: [email protected] or [email protected].
Author contributions: M.S.-A., B.V.Y., M.G.G., S.-E.O., J.A.B., and K.E.B. designed research; M.S.-A., B.V.Y., M.G.G., and S.-E.O. performed research; M.S.-A., B.V.Y., M.G.G., S.-E.O., J.A.B., and K.E.B. analyzed data; and M.S.-A., J.A.B., and K.E.B. wrote the paper.
Reviewers: M.C., University of California, San Francisco; D.M., Queen’s University at Kingston; and T.O., Sanford Burnham Prebys Medical Discovery Institute.
1
J.A.B. and K.E.B. contributed equally to this work.

Competing Interests

The authors declare no conflict of interest.

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    SCAP/SREBP pathway is required for the full steroidogenic response to cyclic AMP
    Proceedings of the National Academy of Sciences
    • Vol. 113
    • No. 38
    • pp. 10449-E5697

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