SCAP/SREBP pathway is required for the full steroidogenic response to cyclic AMP.
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.
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;
Masami
M
Shimizu-Albergine
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
B
Van Yserloo
School of Medicine, Department of Pharmacology, University of Washington, Seattle, WA 98195;
Martin G
MG
Golkowski
School of Medicine, Department of Pharmacology, University of Washington, Seattle, WA 98195;
Shao-En
SE
Ong
School of Medicine, Department of Pharmacology, University of Washington, Seattle, WA 98195; beavo@uw.edu bornf@uw.edu.
Joseph A
JA
Beavo
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 beavo@uw.edu bornf@uw.edu.
Karin E
KE
Bornfeldt
D000818:Animals; D002352:Carrier Proteins; D002784:Cholesterol / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism*; D005786:Gene Expression Regulation; D006903:Hydroxymethylglutaryl CoA Reductases / Q000187:drug effects / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D007985:Leydig Cells / Q000378:metabolism; D008074:Lipoproteins / Q000378:metabolism; D007986:Luteinizing Hormone / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D013256:Steroids / Q000096:biosynthesis* / Q000737:chemistry; D051782:Sterol Regulatory Element Binding Protein 2 / Q000235:genetics / Q000378:metabolism*
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008074:Lipoproteins; D008565:Membrane Proteins; C102196:SREBP cleavage-activating protein; D013256:Steroids; D051782:Sterol Regulatory Element Binding Protein 2; D007986:Luteinizing Hormone; D002784:Cholesterol; D000242:Cyclic AMP; D006903:Hydroxymethylglutaryl CoA Reductases
SCAP/SREBP; cAMP; cholesterol; phosphodiesterase; steroidogenesis