Hormone Biosynthesis, Metabolism, and Mechanism of Action Adrian Quesada Rojas, MD Definitions Hormone: Substance produced in tissue => bloodstream => responsive cells Provides means of communication chemical regulatory and signaling Bloodstream Paracrine: cell to cell (contiguos) Autocrine: same cell Intracrine: same cell (unsecreted) Nomenclature Steroid hormones Sex steroids (Cholesterol derivatives) 3 groups (number of carbon atoms) 21 carbons: pregnane nucleus 19 carbons: androstane 18 carbons: estrane Nomenclature Lipoproteins and Cholesterol All steroid prod organs can synth (acetate) LPP: transport of non polar fat in polar solvent Chylomicrons: chlol 10, TG 90 formed in GI VLDL: more dense IDL: removal of some TG from VLDL interior LDL: end prod VLDL catabolism (50% chol) major carrier of chol in plasma HDL: highest protein content Lipoproteins and Cholesterol Steroidogenesis Reactions: Cleavage of side chain Conv hydroxyl <=> ketones (dehydrogenase) Addition of OH group (hydroxylation) Creation of double bonds (remov of H) Addition of hydrogen (saturation) Enz are dehydrogenases or C P450 oxidases Steroidogenesis Rate limit step: transfer Chol from outer to inner mitochondrial membrane (SSC) Once Pregnenolone: two ways in ovary D5 pathway (3-b hydroxysteroids) => DHEA D4 pathway (3-ketone) => 17a hydroxyP Conversion of pregnenolone to P: 3b hydroxysteroid dehydrogenase D4-5isomerase reaction (3-OH group to ketone + transfer double bond 5-6 to 4-5) Steroidogenesis P hydroxylated at 17 position 17a OH P (precursor of C19 androgens) By peroxide formation at C20, followed by epoxidation of C17, C20 carbons, side chain is split off forming Androstenedione 17 ketone may be reduced to 17b OH to form testosterone (C 19) C 19 steroids => aromatase => C18 phenolic steroid estrogen (E1 and E2) Steroidogenesis Alternative Pregnenolone conv to D5 3b OH C19 steroid (DHA) by 17a OH lation followed by SCC With D4 3 ketone formation, DHA is converted into Androstenedione (C19 steroids) C 19 steroids undergo aromatization (hydroxylation of angular 19 methyl group, followed by oxydation and loss of 19 C as formaldehyde and dehydrogenation) Steroidogenesis Two Cell System FSH receptors on granulosa FSH receptors are induced by FSH LH receptors on theca initially, as follicle grows FSH induces LH receptors on granulosa FSH ind aromatase activity on granulosa Actions regulated by autocrine and paracrine factors Two Cell System Blood Transport of Steroids Most E and T bound to protein carrier (SHBG) 30% is bound to albumin 1% free Hyperthyroidism, pregnancy, E adm SHBG Corticoids, A, P, GH, Insulin SHBG SHBG inversely related to weight Hyperinsulinemia (low SHBG predictor of DM) Body fat distrib (central=> hyperinsulinemia) Blood Transport of Steroids Estrogen Metabolism Ovary => Estradiol / estrone Estriol => periph metab of estrone / E not a secretory prod of ovary conversion to less active form Androgens are precursors of estrogens Adrenal gland (source of A, Androstenedione) Estrogen Metabolism Estrogen Metabolism E2 100-300 mg/day Androstenedione 3 mg/day ( 1% conversion) => 20- 30 % of E1 (produced every day) Progesterone Metabolism NO peripheral conversion Secretion from adrenal and ovaries Preov < 1 mg /day Post ov 20-30 mg /day 10-20% of P is excreted as Pregnanediol Pre ov <1 mg/day, post ov 3-6 mg/d (home ovulation test) Androgen Metabolism Ovary: DHA, Androstenedione, testosterone Adrenal cortex: Gluco, mineralo, sex steroid Sex steroids (less than ovary) DHA (½ adrenal, ¼ ovary, ¼ periph) Androstenedione (½ adrenal, ½ ovary) Testost 0.2-0.3 mg/day 50% periph conv of androstenedione and DHA 25% adrenal 25% ovary Androgen Metabolism Androgens Test binding capacity Androg effects depends on unbound fraction Hirsutism Test => 5a reductase => DHT (princ androg) Androstenedione > Test (women) DHT is derived from Androst and DHA DHT largely metabolized intracell Only 1/10 of levels of Test Not all tissue requires DHT from Test (Wolffian) Cellular Mechanism of Action Two major types of hormone action Tropic hormones (recep at cell memb) Steroids (recep inside the cell) Multiple cell receptors: Intracellular: In the nucleus (transcription activity) G protein: Single polypeptide chain (cell memb) Cellular Mechanism of Action Ion gate channel: Cell surface, mult units (ACh) Intrinsic enz activ: Trans memb recep with intracell component with tyrosine or serine kinase activity Mechanism of action Steroid hormones Includes: Hormone diffusion across cell memb Steroid binding to receptor protein Interaction of Horm-Recep complex with DNA Synthesis of mRNA Transport of mRNA to ribosomes Protein synthesis in cytoplasm Mechanism of action Steroid hormones Mechanism of action Steroid hormones Biological activity Mantained only while H-R complex attached to nuclear site Duration of exposure to hormone is as important as dose Major factor in potency differences among various estrogens is length of time E-R complex occupies nucleus Mechanism of action Steroid hormones Cortisol and P must circulate in large concentrations 2 to receptor complexes short half-lives in nucleus H-R complex after gene activation (processing) Rapid degradation of R unbound to E Much slower degradation of bound R Continuous presence of E is important factor in continuing response Estrogen Receptors ER-a (rapid turnover) ER-b 96% homologous in DNA binding domain and 53% homologous in the hormone binding domain (when compared to ER-a) Respond in comparable manner to same hormones Estrogen Receptors Estrogen Receptors E receptors => nucleocytoplasmic shuttling Recep can diffuse out of nucleus and be transported back in or undergo metabolism If shuttling impaired Receptors are degraded rapidly E antagonists prevent nuclear translocation and thus increase cytoplasmic degradation Estrogen Receptors Prior to binding R is inactive complex that includes a variety of heat shock proteins Activation => dissociation of HSP When H binds R => conformational change Conformational shape determines exact message transmitted to the gene E2, raloxifene, tamoxifen induces a different conformational shape Estrogen Receptors 1. 2. Once activated H receptor activates transcription in partnership with several groups of polypeptides Transcription factors (polymerase enzyme and DNA) Coactivators and corepressors (intracel prot called adaptor proteins and activate or suppress the TAF) Estrogen Receptors Phosphorylation Also stimulated by ligand binding Occur in specific receptor sites Increases potency of molecule to regulate transcription cAMP and prot kinase pathways increase transcriptional activity of E-R Estrogen Receptors Differences ER-a and ER-b ER-b prevalent in brain, CV system, granulosa cells Breast expresses ER-a and ER-b ER-b acts as natural suppressor of ER-a activity on breast tissue Colon contains only ER-b, reduction of colon CA in postmenopausal on HRT may reflect antiproliferative effect of beta receptor Progesterone Receptor Induced by E, decreased by Progestins Two major forms (A and B) A and B are expressed differently in Breast and Endometrial tissues In most cells B positive regulator of Presponsive genes, A inhibits B activity A and B have different molecular functions, affecting different genes Progesterone Receptor Mice lacking of P receptors (A,B) are unable to ovulate (failure to expel a mature oocyte in a fully developed follicle) PR-A protects against uterine and mammary gland hyperplasia induced by PR-B Androgen Receptor 1. 2. 3. Androgens only in one of three ways: T => DHT (Intracrine) T itself (Endocrine) Intracel conversion of T to E (Intracrine) Wolffian duct derivatives (T) Hair follicles, urogenital sinus derivatives (req T => DHT) Androgen Receptor T and DHT binds to same high affinity A-receptor (DHT has greater affinity) Anti-A also bind to same R (20% affinity of T) A-R: two forms B (full length) and A (shorter) Functional differences yet to be determined A and P can cross react for their receptors (pharmacologic concentrations) Progestin can act as an anti-E and anti-A Androgen Receptor Androgen Insensitivity Syndrome Congenital abnormality in A intracel R A-R gene is on X chromosome (Xq11-12) X-linked disorder Deletion of amino acids from steroid binding domain 2 to nucleotide alterations in gene Androgen Receptor Agonists and Antagonists Agonist => stimulates response Antagonist => inhibits actions of an agonist blockage of receptor message Examples: Tamoxifen Mifepristone Histamine receptor antag Agonists and Antagonists Physiologic Antagonists P is not an E antagonist Modifies E action by depleting E receptors P can induce conversion of E2 to E1 Androgens block E actions (unclear mechan) Agonists and Antagonists Two groups of anti-E: Pure anti-E Mixed agonists antagonists Tamoxifen: Similar to clomiphene Competitively inhibits E binding to receptor E affinity for receptor is 100-1000x better than tamoxifen Agonists and Antagonists Agonists and Antagonists Tamoxifen–E receptor binds to DNA Agonistic – Antagonistic actions depends on coactivators present in specific cell types Estrogenic actions: Lowers FSH levels Decreases Cholesterol, LDL Stimulates P receptor synthesis Maintenance of bone, vagina, endometrium Agonists and Antagonists Causes endometrial hyperplasia, polyps and 4x increase in endometrial cancer Mechanism of action: TAF-1 and TAF-2 can both activate transcription Tamoxifen agonistic ability is due to activation of TAF-1 Antagonistic activity is due to competitive inhibition of E-dependent activation of TAF-2 Estrogen Receptors Agonists and Antagonists Response to E and anti-E depends on: Nature of E receptor E response elements and promoters Cell content of protein coactivators Properties of ligand Modulation by growth factors and agents that affect phosphorylation and protein kinases Agonists and Antagonists Tamoxifen reduces risk of recurrent disease in E-R positive and negative breast cancer Besides binding to E-R (competitive inh), Inhibits protein kinase C activity (phosphor) Inhibits calmodulin-dependent cyclic AMP phosphodiesterase (binds to calmodulin) Stim secretion of TGF-b (inh growth CA cell) Agonists and Antagonists Tamoxifen: treatment should be for only 5 years Emergence of tamoxifen-resistant tumors, how? Loss of E receptors Loss of cellular control / loss of E-R expression Variant and mutant E-R Changes in coactivators Differential metabolism Resistance occur when E-R is NOT dominant in growth of CA cells Agonists and Antagonists Selective E Agonist / Antagonist Raloxifene Induce conformational shape change that results in modified action influenced by cellular regulating proteins Anti-E in uterus and breast Agonist in certain tissues (bone, lipids) Antiprogestins Mifepristone Binding => different conformational change => prevents full gene activation Has also some agonistic activity Final response is determined by coact, corepr Affinity to P-R is 5x greater than P In absence of P can produce weak agonistic effect Also works as weak anti-androgens Tropic Hormones Receptor in cell membrane R functions as ion channel or as enzime Can also couple an active agent (messenger) Cyclic AMP IP3 1,2 DG Ca Cyclic GMP Tropic Hormones Cyclic AMP FSH, LH, hCG, TSH and ACTH Ligand => R activates adenylate cyclase Conversion of ATP into cAMP cAMP binds cytoplasm receptor protein and activates protein kinase Catalyze phosphorilation of serine in cell proteins => physiologic events Tropic Hormones Tropic Hormones cAMP is degrated by PDE into 5’-AMP PG’s stim adenylate cyclase and cAMP accum Other tropic hormones Oxytocin Insulin GH do not use Adenylate cyclase PRL hPL Tropic Hormones Calcium messenger system Intracel Ca regulates cAMP, cGMP levels Ca mess system is linked to H-R function by specific enzyme Phospholipase C Catalyzes hydrolysis of phosphatidylinositols (cell membrane phospholipids) PLC activ generates two intracell messengers Tropic Hormones IP3 and DAG Both initiate functions of 2 parts of Ca system First part => Ca activ Prot kinase Sustained cellular responses Second part => Calmodulin (regulator) Acute cell responses Tropic Hormones Tropic Hormones Kinase receptors Cell membrane receptors of insulin, IGF, EGF, platelet-derived GF and fibroblast GF are tyrosine kinases Extracell, transmembrane, cytoplasmic domain Cytoplasmic (autophospholylation) Insulin / IGF (2a, 2b subunits) Tropic Hormones Regulation of tropic hormones Four major components Autocrine and paracrine reg factors GF are produced by local gene expression Modulate activ in cells (nearby or producing cells) Involved in reproductive physiology (activin, inhibin, IGF-I, IGF-II, TGF-b, FGF, EGF) Also cytokines (modulate ovarian steroidogenesis) Heterogeneity of tropic hormones (FSH, hCG) Glycoproteins are not single proteins (isoforms) Regulation of tropic hormones Up and down-regulation of receptors Positive or negative modulation of receptors by homologous hormones Little is known regarding up regulation (PRL, GnRH) Regulation of adenylate cyclase Regulation of tropic hormones Regulation of Adenylate cyclase G protein system: Adenylate cyclase (composition): Receptor Guanyl nucleotide regulatory unit (GTP regul) Catalytic unit (converts ATP to cAMP) Regulation of tropic hormones Regulation of tropic hormones Regulation of tropic hormones Ligand => receptor => nucleotide regulatory unit (GTP uptake) => catalytic enzyme converting ATP to cAMP Coupling of regulatory unit with catalytic unit Enzyme activity is terminated by hydrolysis of GTP to GDP Regulation of tropic hormones G protein system Stimulatory nucleotide regulatory G protein Inhibitory nucleotide regulatory G protein Not limited to cAMP signal, can activate other messenger-generating enzymes Regulation of tropic hormones