11-Deoxycortisol is a corticosteroid hormone in the lamprey David A. Closea,b,1,2, Sang-Seon Yuna,3, Stephen D. McCormickc, Andrew J. Wildbilla, and Weiming Lia,1 aDepartment of Fisheries and Wildlife, Michigan State University, East Lansing, MI 48824; bDepartment of Natural Resources, Confederated Tribes of the Umatilla Indian Reservation, Pendleton, OR 97801; and cUS Geological Survey, Conte Anadromous Fish Research Center, Turner Falls, MA 01376 Edited* by Jan-Åke Gustafsson, Karolinska Institutet, Huddinge, Sweden, and approved June 18, 2010 (received for review December 8, 2009) Corticosteroid hormones are critical for controlling metabolism, assay using the ligand binding domain (LBD) of lamprey CR fused hydromineral balance, and the stress response in vertebrates. with a mammalian GAL4-DBD (6). To better understand the evo- Although corticosteroid hormones have been well characterized in lution of CRs, Stolte et al. (7) performed phylogenetic analysis using most vertebrate groups, the identity of the earliest vertebrate the lamprey CR. Their findings showed that the gene clustered with corticosteroid hormone has remained elusive. Here we provide extant MR genes, suggesting that an MC complex evolved first. evidence that 11-deoxycortisol is the corticosteroid hormone in the Because aldosterone was not found at physiologically relevant levels lamprey, a member of the agnathans that evolved more than 500 in the lamprey (6), receptor sensitivity to aldosterone was suggested million years ago. We used RIA, HPLC, and mass spectrometry to be a by-product of sensitivity to 11-deoxycorticosterone, a postu- analysis to determine that 11-deoxycortisol is the active corticoste- lated ancestral hormone (8). Notably lacking, however, have been roid present in lamprey plasma. We also characterized an 11- direct identification of the circulating corticosteroid, characteriza- deoxycortisol receptor extracted from sea lamprey gill cytosol. The tion of its native receptor, and establishment of biological actions, receptor was highly specific for 11-deoxycortisol and exhibited which are all necessary to define a hormone. Here we provide direct corticosteroid binding characteristics, including DNA binding. Fur- evidence that 11-deoxycortisol is the corticosteroid hormone in the thermore, we observed that 11-deoxycortisol was regulated by lamprey, the closest living relative of the earliest vertebrate, thus the hypothalamus–pituitary axis and responded to acute stress. 11- offering further insight into steroid hormone evolution. Deoxycortisol implants reduced sex steroid concentrations and up- regulated gill Na+,K+-ATPase, an enzyme critical for ion balance. We Results PHYSIOLOGY show here that 11-deoxycortisol functioned as both a glucocorticoid Identification of Circulating Corticosteroids. To determine whether and a mineralocorticoid in the lamprey. Our findings indicate that corticosteroids are present in lamprey, we assumed that the struc- a complex and highly specific corticosteroid signaling pathway ture of a lamprey corticosteroid might be related to modern ver- evolved at least 500 million years ago with the arrival of the earliest tebrate GCs. We isolated putative corticosteroids (Fig. 1A)by vertebrate. screening lamprey plasma with RIAs for cortisol and corticosterone and with a binding protein assay for cortisol after HPLC fraction- evolution | nuclear-receptor | stress response ation. These assays showed no cortisol and corticosterone in the plasma (Fig. 1 B and C), but revealed cross-reactivity in fractions orticosteroid hormones in vertebrates are critical for metab- where 11-deoxycortisol and 11-deoxycorticosterone standards eluted Colism, growth, reproduction, immunity, and ion homeostasis, on HPLC (Fig. 1 B and C). To isolate sufficient quantities of and are an important part of the coping mechanisms involved in steroids for mass spectrometry (MS) analysis, we fractionated the stress responses (1). Corticosteroids primarily act by binding extracted plasma by group partition chromatography (LH-20) to cytosolic receptors, which are then transported to the nucleus, and screened it with 11-deoxycortisol and 11-deoxycorticosterone where they act as positive and negative transcription factors (2). RIAs (Fig. S1). LH-20 fractions were combined and further iso- Their activity leads to the expression or repression of various lated by HPLC fractionation followed by MS analysis. regulatory proteins that counteract the effects of external stres- To confirm the identity of the compounds in the immunoreactive sors, thus maintaining homeostasis (2). fractions, we subjected the analyte ions to tandem MS (MS/MS) The actions of corticosteroids appear to be conserved. Most product ion analyses. The product ion spectra of authentic 11- + vertebrates that have been examined share a stress response that deoxycortisol [M+H] ion at m/z 347 and 11-deoxycorticosterone + includes increased glucocorticoid (GC) hormones that further [M+H] ion at m/z 331, obtained by direct infusion of standards regulate metabolic, endocrine, and immune functions. However, (Fig. 1D, Inset, and E, Inset), matched the fragmented daughter ions active hormones may differ among species. In tetrapod groups, of the plasma samples (Fig. 1 D and E). Taken together, the binding- – there are at least two active GC hormones, either cortisol or assay guided isolation and chromatographic and MS analyses dem- corticosterone, and the mineralocorticoid (MC) that regulates ion onstrated that 11-deoxycortisol and 11-deoxycorticosterone were balance is aldosterone. In contrast, in teleosts, cortisol apparently acts as both GC and MC, whereas aldosterone is not present (3). However, the corticosteroid signaling pathway in the earliest Author contributions: D.A.C., S.-S.Y., and W.L. designed research; D.A.C., S.-S.Y., S.D.M., and A.J.W. performed research; D.A.C., S.-S.Y., S.D.M., and A.J.W. analyzed data; and D.A.C., vertebrate has remained elusive because of lack of information on S.-S.Y., and S.D.M. wrote the paper. the identities of corticosteroids. The authors declare no conflict of interest. Recent work with receptors has shed light on the primitive cor- *This Direct Submission article had a prearranged editor. ticosteroid of vertebrates. A single putative corticosteroid receptor (CR) was identified through PCR-based homology cloning in the Freely available online through the PNAS open access option. 1 sea lamprey, a member of the oldest vertebrate lineages from which To whom correspondence may be addressed. E-mail: d.close@fisheries.ubc.ca or liweim@ msu.edu. jawed vertebrates diverged about 500 million years ago (4, 5). 2 Transactivation studies using cloned lamprey and hagfish CRs, as Present address: Fisheries Centre and Department of Zoology, University of British Co- “ ” lumbia, Vancouver, BC, Canada V6T 1Z4. well as a resurrected ancestral CR that was inferred from known 3Present address: Department of Marine Biotechnology, Kunsan National University, Gun- CRs, resulted in MC receptor (MR)–like “promiscuous binding” to san, Jeonbuk 573-701, Korea. multiple corticosteroids. Aldosterone and 11-deoxycorticosterone This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. had the highest levels of activation in a luciferase reporter gene 1073/pnas.0914026107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.0914026107 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 A Corticosteroid Sex steroid Cholesterol pathway pathway CYP11A1 CYP17 CYP17 Pregnenolone 17-OH-Pregnenolone Dehydroepiandrosterone Androstenediol 3Β-HSD Progesterone 17-OH-Progesterone Adrostenedione Testosterone CYP21 11-Deoxycorticosterone 11-Deoxycortisol Estrone Estradiol CYP11B1 Corticosterone Cortisol CYP11B2 Aldosterone B C 3.0 11-Deoxycortisol 1.0 11-Deoxycorticosterone Cortisol Corticosterone 2.0 AB CBP 0.5 1.0 Cortisol (ng/ fraction) 0.0 0.0 Corticosterone (ng/ fraction) 10 20 30 40 50 60 70 10 20 30 40 50 60 70 Fig. 1. Identification of corticosteroids in Retention time (min) Retention time (min) lamprey plasma. (A) Biosynthetic pathways of corticosteroids and sex steroids. Steroidogenic DE enzymes are indicated on arrow labels. (B) Authentic 311.0 329.0 Authentic 313.1 Screening of HPLC-fractionated plasma extract 100 100 11-deoxycorticosterone 11-deoxycortisol CH2OH CH OH with cortisol RIA and binding protein assay. AB, OC 329.0 2 313.1 100 OC 295.1 100 OH anticortisol antibody; CBP, cortisol binding 331.1 331.1 protein. Arrows indicate where cortisol and 11- 50 347.1 293.0 50 deoxycortisol standards elute. (C) Screening of 311.1 O 269.0 HPLC-fractionated plasma extract with corti- O 347.0 costerone RIA. Arrows indicate where cortico- 0 0 sterone and 11-deoxycorticosterone elute. (D) 50 100 150 200 250 300 350 50 100 150 200 250 300 350 Identification of 11-deoxycortisol by atmo- 269.1293.1 spheric pressure chemical ionization MS (APCI MS-MS) analysis. Fragmentation patterns gen- Plasma sample Plasma sample Relative Abundance Relative Relative Abundance Relative 295.2 erated from the authentic 11-deoxycortisol (inset) matched the plasma sample. (E) Identi- 0 0 fication of 11-deoxycorticosterone by APCI MS- 100 150 200 250 300 350 100 150 200 250 300 350 MS analysis. Fragmentation patterns gener- ated from the authentic 11-deoxycorticoster- m/z m/z one (Inset) matched the plasma sample. present in circulation and thus potential corticosteroids in the sea Kinetic studies of the 11-deoxycortisol binding moiety showed that lamprey. These results were intriguing because