Biochemistry of Corticosteroids: Lipid Metabolism and Stress Response

Biochemistry of Corticosteroids

Corticosteroids are a class of endogenous cholesterol-based steroid hormones with metabolic and electrolyte balance properties.

There are two classes of corticosteroids:

• Glucocorticoids regulate carbohydrate metabolism
• Mineralcorticoids regulate sodium and potassium levels

Glucocorticoids and Rheumatoid Arthritis

In 1948 at Mayo Clinic a patient with rheumatoid arthritis began daily injections of compound E, a synthetic version of a steroid hormone. Within 3 days, the patient was nearly asymptomatic

Glucocorticoids are steroid hormones involved in several physiological processes:

• Carbohydrate metabolism
• Electrolyte balance
• Immune response
• Development
• Growth
• Cardiovascular function
• . Mood and cognitive function
• Reproduction

Glucocorticoids are the pillar in the treatment of inflammatory and autoimmune pathologies

Anatomy of The Adrenal Gland

Capsule Zona glomerulosa - Adrenal gland Zona fasciculata · Medulla · Cortex Zona reticularis Kidney - Adrenal medulla (a) (b)

Adrenal Gland Regions and Steroid Production

Glomerulosa Fasciculata Cortex - Reticularis Medulla Aldosterone Cortisol Androgens Catecholamines - Mineralcorticoids Glucocorticoids Regional differences in steroid production exist because of the distribution of enzymes

Steroidogenesis Pathway

OH Dehydroeplandrosterone Androstenedlol 17B-HSD 17,20-Lyase (CYP17) HO HO* LOH 3B-HSD OH OH HO 5c-Reductase 3B-HSD O DIhydrotestosterone Testosterone HO Pregnenolone 3B-Hydroxysteroid dehydrogenase (3)-HSD) CYP17 CYP19 OH 0 17B-HSD HO Estradiol Progesterone OH 21-Hydroxylase (CYP21A2) 11-Deoxycortisol 26 27 25 24 23 21 22 18 20 11 13 19 C D 16 9 2 10 8 A B 3 5 4 HO 6 Cholesterol branched hydrocarbon chain Corticosterone OH Aldosterone synthase (CYP11B2) HO Aldosterone 0 ZONA GLOMERULOSA ZONA RETICULARIS CYP11B1 OH O Deoxycorticosterone 40 OH 11B-Hydroxylase (CYP11B1) OH O CYP17 HO Cortisol ZONA FASCICULATA 17B-Hydroxysteroid dehydrogenase Androstenedione (17B-HSD) Aromatase (CYP19) 17c-OH-Progesterone OH CYP21A2 HO Estrone Desmolase (CYP11A1) HO 17c-OH-Pregnenolone 3B-HSD 0 CYP17 LOH Steroid nucleus 17 15 7 Cholesterol 170-Hydroxylase (CYP17)

Steroidogenesis from Cholesterol

All steroid hormones are synthesized from cholesterol through an enzymatic process called steroidogenesis Most of the supply of cholesterol comes from plasma low-density lipoproteins (LDLs) derived from dietary cholesterol The first and rate-limiting step is the conversion of cholesterol to pregnenolone by the cholesterol side-chain cleavage enzyme (P450scc) in mitochondria Transcription of the CYP11A1 gene encoding P450scc determines the amount of P450scc enzyme and the net steroidogenic capacity of a cell

Cholesterol 1 P450scc + StAR P450c11₿ 3₿HSD2 P450c21 P450c11AS P450c11AS 18OH Aldo Corticost P450c17 P450c17 3₿HSD2 P450c21 P450c118 17OH-Preg +17OH-Progesterone +11-Deoxycortisol + Cortisol (17OHP) P450c17+ b5 3₿HSD2 P450aro DHEA Androstenedione Estrone 17₿HSD3/5 17₿HSD3/5 17₿HSD1 * 3BHSD2. Testosterone P450aro Androstenediol Estradiol - 5aRed2 DHT P450c11AS Pregnenolone Pregesterone -+ DOC ->Corticosterone

Steroidogenesis - P450scc Enzyme

P450scc catalyzes the conversion of cholesterol (C27) to pregnolone (C21) in 3 monooxygenase reactions:

1. 22-hydroxylation of cholesterol
2. 20-hydroxylation of 22(R)-hydroxycholesterol
3. Oxidative scission of the C20-22 bond of 20(R),22(R)-dihydroxycholesterol (the side-chain cleavage event)

Resulting in the production of pregnenolone and isocaproic aldehyde These three reactions occur on a single active site of P450scc that is in contact with the IMM. Each reaction requires 2 electrons from NADPH, that are transferred from NADPH to P450scc via two electron transfer proteins: ferredoxin reductase and ferredoxin. The 3 proteins together constitute the cholesterol side- chain cleavage complex

21 22 24 26 20 23 25 18 12 17 11 13 16 C D 19 15 1 9 2 10 8 A B 3 5 7 4 6 HO Cholesterol (C27) P450scc (CYP11A) Cholesterol desmolase CH3 I CEO HO Pregnenolone (C21) 27 14

Steroid Acute Regulatory Protein (StAR)

21 24 22 26 20 18 25 Cholesterol 12 I 17 11 27 13 19 16 14 9 Cholesterol 1 2 H H 5 7 3 .6 StAR HO- Zona fasciculata Glucocorticoids CYP17A1 Zona reticularis Androgens CYP11A1 Pregnenolone 17-OH-pregnenolone DHEA HSD3B2 Progesterone 17-OH-progesterone Androstenedione CYP21A2 Deoxycorticosterone 11-Deoxycortisol OH 21 Testosterone CYP11B2 CYP11B1 0 19 18 HO OH Corticosterone Cortisol 12 17 19 16 14 9 15 1 10 8 2 3 .. 5 7 Cortisol Aldosterone 6 0 & Zona glomerulosa Mineralocorticoids StAR triggers the flow of cholesterol from the outer mitochondrial membrane (OMM) to P450scc on the IMM (rate-limiting step) StAR mediates the acute steroidogenic response and the rapid production of adrenal steroids CYP17A1 Cholesterol 10 8 15 4 23 13 CYP11B2 20

Regulation of Steroidogenesis

Steroidogenic cells store very little steroid. A rapid steroidogenic response requires rapid synthesis of new steroid ACTH (Adrenocorticotropic hormone) promotes steroidogenesis in three time frames:

1. Promotes adrenal growth (weeks to months)
2. Promote the transcription of genes encoding various steroidogenic enzymes via cAMP (days)
3. Rapidly stimulates StAR gene transcription to increase the flow of cholesterol (minutes)

ACTH Adenylyl cyclase x B ATP . CAMP ACTH receptor Induction of steroidogenic genes PKA P HSD3B CYP21 CYP11B2 StAR CREB CYP11A1 Nucleus Cholesterol StAR Pregnenolone Progesterone 11-Deoxy corticosterone Corticosterone HSD3B CYP21 CYP11B2 CYP11A1 CYP17 17a-Hydroxy pregnenolone 17a-Hydroxy progesterone 11-Deoxy cortisol Cortisol Mitochondria HSD3B CYP21 CYP11B2 Choong S et al BBA 2011

Cortisol Biosynthesis

Plasma Cytoplasm Acetate /De novo synthesis Receptor-mediated Lysosomes Cholesteryl ester storage endocytosis Low-density lipoprotein LDL Cholesterol 90€ Mitochondrion Rate-limiting step mediated by StAR Cortisol Cortisol 11B-Hydroxylase (CYP11B1) 11-Deoxycortisol Pregnenolone Pregnenolone 3B-Hydroxysteroid dehydrogenase 11-Deoxycortisol Progesterone 21-Hydroxylase (CYP21) 17a-Hydroxylase (CYP17) 17a-Hydroxyprogesterone Endoplasmic reticulum Zona Fasciculata Cholesterol Cholesterol side- chain cleavage enzyme (CYP11A)

The HPA Axis and GC Production

• Adrenal GC production is regulated by the hypothalamic-pituitary-adrenal (HPA) axis
• Under basal GCs are released from the adrenal glands in the bloodstream in a circadian rhythm and stress increases the activity of the HPA axis
• When the HPA-axis is stimulated, CRH and AVP are released from the PVN that induce release of ACTH from the pituary gland
• Circulating GC ultimately turns off the HPA neuroendocrine activity and restores a steady state via negative feedback

Circadian cues OH 0 JOH CRH ACTH Stress Hypothalamus AVP A Cortisol (humans) Cholesterol · IL-1 H TLR2 TLR4 · IL-6 A · TNF HO Cain D.W. et al Nat Rev Immunology 2017 Immune system CRH corticotropin-releasing hormone AVP arginine vasopressin PVN hypothalamic paraventricular nucleus HO Pituitary gland Adrenal gland Steroidogenesis H

Circadian Regulation of GC Synthesis and Secretion

Circulating GC levels are higher during the activity period and peak levels are linked to the beginning of the activity period Circadian rhythms of GC levels are generated by multiple regulatory mechanisms:

1. SCN regulation through the HPA- axis and the autonomic nervous system
2. The local clock machinery in the adrenal gland

GC synchronize the circadian timing system

PVN .. CRH, AVP K ACTH ANS Pituitary Neural input SCN HPA axis activity Adrenal gland Adrenocortical GC-producing cells Adrenal peripheral clock CLOCK BMAL1 -E StAR V Daily GC rhythm Adrenal clock-dependent mechanism Rhythmic GC biosynthesis: StAR Adrenal sensitivity to ACTH Choong S et al BBA 2011

REPORT: Resetting of Circadian Time in Peripheral Tissues by Glucocorticoid Signaling

Aurélio Balsalobre1, Steven A. Brown1, Lysiane Marcacci1, François Tronche2, Christoph Kellendonk2,*, Holger M. Reichardt ... + See all authors and affiliations Science 29 Sep 2000: Vol. 289, Issue 5488, pp. 2344-2347 DOI: 10.1126/science.289.5488.2344 A EtOH Dex Y 0 1 1 hours Per1 Per2 - TBP C Y 0 8 12 16 20 28 hours Per2 Per3 Rev-erba - TBP B Y 0 4 8 12 16 20 24 28 32 36 40 44 hours Per1 Per2 Per3 --- --- -- Cry1 DBP Rev-erba - TBP

Mechanism of Action of Glucocorticoids

GC Biological Availability

In the bloodstream GCs are bound to and transported by plasma proteins which keep them inactive Corticosteroid-binding globulin (CBG) is the main GC-binding protein in the plasma, with about 80-90% of the GCs bound to it Free GCs diffuse through the cell membrane, however the bioavailability is regulated by the balance between active (cortisol) and inactive (cortisone) forms 11ß-hydroxysteroid dehydrogenase 1 (11ß- HSD1): cortisone cortisol OH OH H H H H H human 113-HSD1 Cortisone OH € 118-HSD2 H H H H H H 11-dehydrocorticosterone OH HO OH Cortisol OH HO 11-HSD2 cortisol cortisone mouse Corticosterone Timmermans S et al Frontiers in Immunology 2019

Nuclear Receptors

a Human nuclear receptors Endocrine ERa RARa MR PR ERß RARß AR THR VDR RARY GR THRß DAX1 COUPTF1 LRH1 TLX Orphan SHP COUPTF2 EAR2 PNR TR2 TR4 NOR1 GCNF REVERBa REVERBß NURR1 NUR77 HNF4a HNF4Y CAR RORa Adopted ERRa PPARa RXRa ROR ERRØ PPAR8 RXRß RORY ERRY PPARY RXRY LXRa SF1 PXR FXR LXRØ Primary structure Tertiary structure DNA binding AF2 Ligand AF1 Zn |Zn Transactivation Ligand binding, transactivation, dimerization, repression Dhiman VK et al Nat Rev Genetics 2018 Glass CK et al Nat Rev Immunology 2005

Nuclear Receptor Transcriptional Regulation

Baba Non-genomic actions Genomic actions - Ligand dependent Ligand independent Gene Gene Gene Gene Gene activation depression repression transrepression repression Perissi V et al Nat Rev Mol Cell Biol 2005

Nuclear Receptor Transcriptional Regulation: Histone Modifiers

Histone arginine methyltransferase ATP-dependent chromatin remodelling Histone acetyltransferase RNA and RNA processing Mediator CBP/p300 complex TIP60 complex P/CAF complex GCN5/ TRAPP complex PGC1 SRA NUMAC complex CARMTI p160S TRAP/DRIP/ARC complex SWI/SNF complexes Activation TAFS IIF (IIJ TBP ATA Pol 1| Nuclear receptors Mediator/SRB Repression ---- NCOR/SMRT CtBP complex RIP140 LCOR HDAC3 GPS2 TBLR1 TBL1 NURD/Mi2 complex Ligand-dependent corepressors TAB2 IR10 KAISO KIAA0677TRIP5 Sin3 complex Sin3 complex KAPI SWI/SNF ATP-dependent chromatin remodelling Ski Histone deacetylase Perissi V et al Nat Rev Mol Cell Biol 2005 c IIA) IIB SWI/SNF complexes