A brief history of adrenal research

Molecular and Cellular Endocrinology 371 (2013) 5–14

Contents lists available at SciVerse ScienceDirect

Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce

Review

A brief history of adrenal research Steroidogenesis – The soul of the adrenal Walter L. Miller University of California, San Francisco, San Francisco, USA

a r t i c l e

i n f o

Article history: Available online 30 October 2012 Keywords: Adrenal cortex Adrenal history Adrenal hyperplasia Brown-Sequard Steroid hormones Steroidogenic enzymes

a b s t r a c t The adrenal is a small gland that escaped anatomic notice until the 16th century, and whose essential role in physiology was not established until the mid 19th century. Early studies were confounded by failure to distinguish the effects of the cortex from those of the medulla, but advances in steroid chemistry permitted the isolation, characterization and synthesis of many steroids by the mid 20th century. Knowledge of steroid structures, radiolabeled steroid conversions, and the identification of accumulated urinary steroids in diseases of steroidogenesis permitted a generally correct description of the steroidogenic pathways, but one confounded by the failure to distinguish species-specific differences. The advent of cloning technologies and molecular genetics rapidly corrected and clarified the understanding of steroidogenic processes. Our laboratory in San Francisco was one of several contributing to this effort, focusing on human steroidogenic enzymes, the genetic disorders in their biosynthesis and the transcriptional and post-translational mechanisms regulating enzyme activity. Ó 2012 Walter L. Miller. Published by Elsevier Ireland Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5. 6. 7.

Introduction to the Keith Parker Memorial Lecture . Anatomists: discovery of the adrenal . . . . . . . . . . . . Physiologists: adrenal function . . . . . . . . . . . . . . . . . Chemists: adrenal hormones . . . . . . . . . . . . . . . . . . . Clinical investigators: adrenal hyperplasias . . . . . . . Steroidogenic enzymes: proteins, cDNAs and genes Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . .

5 5 6 8 10 10 11 12 12

1. Introduction to the Keith Parker Memorial Lecture

2. Anatomists: discovery of the adrenal

It was indeed an honor to give the second Keith Parker Memorial Lecture at the biennial Adrenal Cortex Conference. Keith was a friend and a creative, ground-breaking scientist who was taken from us far too soon. We all miss him deeply. Following his discovery of the transcription factor SF1 (steroidogenic factor 1), much of Keith’s career concerned adrenal development in rodent systems, while mine has concerned human steroidogenesis, but we were both focussed on the adrenal. Below I shall recount some of the history of adrenal research, then mention some contributions from my lab and suggest some future directions.

Whereas it is self-evident that the adrenal has always been with us, early anatomists apparently failed to note its presence. In the context of describing animal sacrifices, Leviticus 3:4 and 4:9 both refer to ‘‘. . .the two kidneys, and the fat that is on them, which is by the flanks. . .’’ (King James translation) and in a different context Claudius Galen (ca. 130–201) only described ‘loose flesh’ atop the left kidney (Leoutsakos and Leoutsakos, 2008). Thus it seems that the ancients could not distinguish the adrenals from the peri-nephric fat, and anyone who has collected ungulate adrenals at an abattoir will confirm that the differentiation of the adrenal from adjacent fat and lymph nodes is not trivial. In 1563 Bartolomeo Eustachius (1520(?)–1574), who was Professor of Anatomy at the Collegio della Sapienza in Rome and a challenger

E-mail address: [email protected]

0303-7207/$ - see front matter Ó 2012 Walter L. Miller. Published by Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mce.2012.10.023

6

W.L. Miller / Molecular and Cellular Endocrinology 371 (2013) 5–14

3. Physiologists: adrenal function

Fig. 1. Plate 47 from Eustacchio’s ‘Tabulae Anatomicae’, as reproduced by GM Lancisi in 1714 as ‘Tabulae Anatomicae’. Image captured on line.

of Galenic authority, published his tome ‘Opuscola Anatomica’. This work, which correctly described the anatomy of the inner ear and the eustachian tube, referred to the adrenals as ‘glandulae renibus incumbentes’ in the description of the kidneys. However, because Eustacchio feared the Roman Catholic inquisition, most of the accompanying anatomic engravings (completed in 1552 by Pier Matteo Pini) were not published, but were sequestered in the Papal library until Clement XI gave them to GM Lancisi, who published them in 1714 as ‘Tabulae Anatomicae’ (Carmichael, 1989; Mezzogiorno and Mezzogiorno, 1999). Eustaccio’s plate 47 shows the adrenal anatomy with remarkable accuracy (Fig. 1), but throughout the 150 years that the church hid Eustacchio’s work, most anatomists questioned his claim to have discovered a suprarenal organ. In 1716, the Academy of Sciences in Bordeaux conducted an essay competition to determine the function of the adrenals, but none of the entries was deemed worthy of the prize, and the Academy then redirected its attention more productively to wine. The Danish anatomist Caspar Bartholin, better known for Bartholin’s glands, described the adrenals as hollow organs filled with ‘black bile’, probably a poetic description of the adrenal medulla undergoing post-mortem autolysis. Finally, in 1805, Georges Cuvier (1769–1832) distinguished the cortex and medulla, but offered no functional insights (Cuvier, 1805).

The linkage of earlier anatomy with clinical observation in the mid 19th century began the modern era of adrenal research. Thomas Addison (1793–1860) first described tuberculosis of the adrenal in 1849 (Addison, 1849) and then wrote his famous, detailed monograph ‘‘On the Constitutional and Local Effects of Disease of the Suprarenal Capsule’’ in 1855 (Pearce, 2004). In studies of (autopsied) patients with anemia, he found bilateral adrenal pathological changes that appeared to be independent of the anemia (Fig. 2). His clinical description is classic: ‘‘The discoloration pervades the whole surface of the body, but is commonly most strongly manifested on the face, neck, superior extremities, penis, scrotom, and in the flexures of the axillae and around the navel ... The leading and characteristic features of the morbid state to which I would direct your attention are, anaemia, general languor and debility, remarkable feebleness of the heart’s action, irritability of the stomach, and a peculiar change of the colour in the skin, occurring in connection with a diseased condition of the suprarenal capsules.’’ Addison was a brilliant observer who also described the first cases of what we now know to be pernicious anemia (vitamin B12 deficiency) and adrenoleukodystrophy, which is more commonly associated with Paul Schilder (1886–1940). But Addison did not understand how adrenal disease led to the symptoms he described so clearly. Addison’s work influenced Charles-Edouard Brown-Sequard (1817–1894), arguably the most brilliant and intriguing character in 19th century medical science (Fig. 3A). Born to an American sea captain father and French mother on the (then British) island of Mauritius in the Indian Ocean, Brown-Sequard was educated in Paris and at various times practiced or held Professorships in London, Boston, New York, Philadelphia, Richmond, Glasgow, Dublin and Paris, was elected to the UK’s Royal Society (1860), the US National Academy of Sciences (1868), and France’s Academie des Sciences (1886), and published over 500 papers. He reportedly crossed the Atlantic Ocean 60 times, spending a total of 6 years at sea (Rengachary et al., 2008). He is best known today as one of the fathers of modern neurology, including discovery of the decussation of sensory fibers in the spinal cord and his description of spinal hemiparaplegia (Brown-Sequard Syndrome). Brown-Sequard was the first to go beyond mere observation and bring experimentation and quantitation to medical research. In 1856 he demonstrated that adrenalectomy (but not a sham operation) was lethal, and conjectured that the adrenals secreted the

Fig. 2. Reproduction of a figure of a (deceased) patient, from Thomas Addison’s monograph ‘‘On the Constitutional and Local Effects of Disease of the Suprarenal Capsule’’. Image captured on line.

W.L. Miller / Molecular and Cellular Endocrinology 371 (2013) 5–14

7

therapeutics. A tireless collector of facts, if his reasoning power had equaled his power of observation he might have done for physiology what Newton did for physics.’’ (Bowditch, 1902). But as the first to prove that a gland was essential for life and the first to attempt hormonal replacement therapy, he is clearly the father of endocrinology. Soon thereafter, in 1865 Luigi de Crecchio, Professor of Legal Medicine at the University of Naples (Obituary, 1895), described the autopsy of Giuseppe Marzo, the first report of a case of classic, non-salt-losing congenital adrenal hyperplasia (CAH). Alfred M. Bongiovanni (who discovered 3b-hydroxysteroid dehydrogenase deficiency and 11b-hydroxylase deficiency) provides a partial translation (Bongiovanni and Root, 1963) of de Creccio’s lengthy manuscript (de Creccio, 1865), and Maria New has reproduced a drawing of the Marzo cadaver, along with interesting speculations about potential earlier cases (Fig. 4) (New, 2011). De Creccio comments that ‘‘. . . it is sometimes extremely difficult or even impossible to determine sex during life’’ and Bongiovanni says ‘‘He described the cadaver of a ‘man’ having a penis with first degree hypospadias and without externally visible or palpable gonads. Dissection revealed a vagina, uterus, fallopian tubes and ovaries within the abdomen. In addition, the adrenal glands were described as very much enlarged’’. Bongiovanni tells us that de Creccio investigated Marzo’s life. ‘‘It was established that Giuseppe Marzo transacted all his affairs of life, including sexual intercourse, as a man, despite the fact that he was declared a female at birth and was finally designated a male, once and for all at the age of 4 years.’’ Concerning his demise, Bongiovanni says ‘‘It is perhaps

Fig. 3. Charles-Edouard Brown-Sequard. (A) Signed portrait. (B) Heading of the paper in Lancet 2, 1889. (C) Nineteenth century advertisement for ‘organic extract’. Images captured on line.

life-sustaining factors we now call hormones (Brown-Sequard, 1856). Reviled for being an unapologetic vivisectionist, he was also occasionally his own experimental subject: in 1889 he reported decreased fatigue, improved strength (‘tested with a dynamometer’) and improved projection of his urinary stream after injecting himself with filtered aqueous extracts of dog testes (Fig. 3B) (Brown-Sequard, 1889). The results of his ‘organotherapy’ were a placebo effect that stimulated both quackery and further research (Fig. 3C). In a grudging memoir for the US National Academy of Sciences, Bowditch wrote ‘‘His work upon the so-called ‘Elixir of life’, which was received with so much incredulity by most physicians, gave an important stimulus to the study of the internal secretion of glands, a branch of physiology which gives promise of leading to important advances in

Fig. 4. Cadaver of Giuseppe Marzo, from de Crecchio, 1865. Image courtesy of Dr. Maria New.

8

W.L. Miller / Molecular and Cellular Endocrinology 371 (2013) 5–14

significant that he often suffered from emesis and diarrhea and that he died after such an episode, during which he ‘was reduced, in a few days, to a state of extreme weakness and exhaustion’. Could this have been an addisonian crisis?’’ (Bongiovanni and Root, 1963). Unfortunately, de Creccio went no further with CAH. Adrenal zonation was described soon thereafter (Arnold, 1866; Gottschau, 1883), but its significance was unknown. In 1901 Jokichi Takamine, an expatriate Japanese chemist working in New York, isolated and patented the first hormone, epinephrine (Yamashima, 2003). Takamine and many who followed believed he had identified the active principle of the suprarenal gland (Takamine, 1901), so that much subsequent physiologic work examining the effects of adrenalectomy on carbohydrate metabolism failed to differentiate between the effects of the hormones of the cortex from those of the medulla. In 1912 Ernest Glynn, a pathologist in Liverpool correctly concluded ‘‘The adrenal cortex and medulla have a different development and different functions; the former is especially connected with growth and sex characters, the latter with blood pressure’’ and ‘‘ In pseudo-hermaphroditism the sex abnormalities are mainly congenital, and the adrenal lesions, if any, are bilateral hyperplasia or cortical rests.’’ (Glynn, 1912). Nevertheless, confusion persisted; when Walter Cannon described the ‘fight-or-flight’ response, he emphasized the sympathetic nervous system and catecholamines, but was not aware that cortical hormones also participated (Cannon, 1915). In their Nobel-prize winning work (along with Bernardo Houssay), Carl and Gerty Cori described glycogenolysis (the Cori cycle), which is driven by catecholamines, and noted in 1927 that adrenalectomy decreased hepatic glycogen, a glucocorticoid effect, but did not then know the hormonal difference (Cori and Cori, 1927). Baumann and Kurland (1927) documented that adrenalectomy (in cats) resulted in hyponatremia and hyperkalemia and Rogoff and Stewart (1928) showed that adrenalectomized dogs could be kept alive with adrenal cortical extracts. Definitive evidence for an adrenocortical hormone appeared in 1930 when Swingle & Pfiffner and Hartman & Brownell reported the use of lipoidal extraction procedures to prepare adrenal extracts that could sustain the life of adrenalectomized animals and would relieve the symptoms of Addison disease (Swingle and Pfiffner, 1930, 1931; Swingle et al. 1933; Hartman and Brownell, 1930; Hartman et al. 1930). Hartmann termed the extract ‘cortin’ but the Swingle–Pfiffner preparation was first to be used to save an adrenally insufficient, moribund patient (Rowntree et al., 1930). Noting that adrenalectomized animals become hyponatremic, Loeb showed that oral saline administration alone extended the life of a patient with Addison’s disease (Loeb, 1933), but the basis of this effect remained unclear. Although Cushing (1932) described the pituitary tumors (‘pituitary basophilism’) that cause what we now call Cushing disease, he apparently failed to note the role of the adrenal. Adrenal feedback on the pituitary was reported several years later, but it was apparent that the pituitary did not control all adrenal function, because adrenalectomy killed animals more rapidly than hypophysectomy (Ingle and Kendall, 1937). An antiinflamatory effect of adrenal extracts was noted in 1940, but was thought to be secondary to a reduction in capillary permeability (Menkin, 1940). Selye, the father of research concerning the stress response first noted that ‘toxic substances’ elicited hyperplasia of the adrenal cortex and involution of the thymus – the discovery of glucocorticoid action (Selye, 1936). He integrated these and other findings into the conceptualization of the HPA axis, coined the terms ‘mineralocorticoid’ and ‘glucocorticoid’, and emphasized that both were needed for survival (Selye, 1946). The next step thus had to be the chemical identification of the responsible factor(s).

4. Chemists: adrenal hormones The general organic chemistry of polycyclic compounds received much attention in the early 20th century. Heinrich Wieland and Adolph Windaus received the 1927 and 1928 Nobel prizes in chemistry for their work on bile acids and the structure of cholesterol, but their structures were incorrect, featuring fourring structures with two 5-carbon rings (Wieland, 1928; Windaus, 1928); the correct cyclopentanophenanthrene structure was then determined by Rosenheim and King in 1932 (reviewed in Rosenheim and King, 1934). Leopold Ruzicka and Adolf Butenandt (a student of Windaus) were awarded the 1939 Nobel prize in chemistry for isolating estrone, androsterone and progesterone. Ruzicka delivered his Nobel lecture after World War II (Ruzicka, 1945), however, because the German pacifist Carl von Ossietzky had received the 1935 Nobel Peace prize while in a Nazi concentration camp, Butenandt, who had joined the Nazi party in 1936 (party member no. 3716562), declined the prize. He was listed officially as a Nobel laureate in 1949 and received his diploma and medal, but did not receive the money or deliver a Nobel lecture. Butenandt directed the Keiser Wilhelm Institute for Biochemistry from 1937 to 1945, directing biochemical research for the war effort, some of which was ethically dubious at best (Trunk, 2006). With Karlson he later isolated and determined the structure of the insect steroid hormone, ecdysone (Butenandt and Karlson, 1954). Thus, by the 1930s, the stage was set for studies of adrenal steroids. At the Mayo Clinic, Edward Kendall had already gained fame for having isolated thyroxine (Kendall, 1915), although he was never able to determine its structure or synthesize it. In the early 1930s he turned his efforts to purifying the hormone of the adrenal cortex (like most investigators, he thought there was only one) (Kendall et al., 1934). As related by Ingle, for 5 years the Kendall laboratory processed 900 lb of bovine adrenals each week, preparing an extract (‘cortin’) used to treat the Addisonian patients who failed treatment with a high sodium, low potassium diet (Ingle, 1975). By 1935, four groups were isolating adrenal compounds: Kendall at the Mayo Clinic, Pfiffner who had moved from Princeton to Columbia University, Cartland and Kuizenga working at Upjohn, Inc. (Cartland and Kuizenga, 1936) and Tadeus Reichstein in Zurich. Much as Kendall had first gained fame for isolating thyroxin(e), Reichstein had achieved international prominence for the isolation, structural characterization and synthesis of vitamin C (Reichstein and Grüssner, 1934), a procedure still known as the Reichstein process. Reichstein only first turned his attention to steroids in 1934, but was the superior chemist. Wintersteiner and Pfiffner were first to isolate ‘compound E’ (cortisone) (Wintersteiner and Pfiffner, 1936), quickly followed by Reichstein, but both groups thought the compound was biologically inactive. Kendall purified the same compound as a diketone and found androgenic activity (Mason et al., 1936); Reichstein had made a similar observations concerning androgenic ketone derivatives of adrenal compounds (Reichstein 1936a,b). These observations indicated that the adrenocortical hormones were steroids. Working in intense competition with one another, the four groups quickly isolated, purified and determined chemical structures for 29 different steroids, including cortisone (compound E), corticosterone (compound B), 11-dehydrocorticosterone (compound A) (Steiger and Reichstein, 1937) and eventually cortisol (compound F) (Fig. 5). These were designated by different and inconsistent letters in each laboratory; some of this alphabet soup terminology still persists, but is clearly antiquated. All of these initially isolated compounds were 11-oxy steroids that Selye termed ‘glucocorticoids’, but the 11-desoxy steroid(s) responsible for mineralocorticoid activity remained unidentified. Kendall described an ‘amorphous fraction’ from adrenals

W.L. Miller / Molecular and Cellular Endocrinology 371 (2013) 5–14

9

Fig. 5. Steroids illustrated in Reichstein’s Nobel lecture (1950).

that affected electrolyte balance, later called ‘electrocortin’, but it was not until 1952 that Reichstein turned his attention to this steroid. In collaboration with Reichstein, Simpson and Tait developed a sensitive bioassay permitting the crystallization of 21 mg of electrocortin from 500 kg of bovine adrenals (Simpson et al., 1953) and the subsequent determination of its structure (Simpson et al., 1954): aldosterone had been discovered (Williams and Williams, 2003). Chemistry without biology is boring. The physiologists and chemists of the 1930s knew from their bioassays that adrenal steroids were important, but it took clinical investigation to show that steroids had incredible potential as therapeutic agents. Philip Hench, a rheumatologist at the Mayo Clinic, first noted symptomatic remission of chronic rheumatoid arthritis in seven patients who had hepatitis and jaundice (Hench 1933). He expanded his clinical experience to 31 cases, noting that the degree of relief was correlated with the degree of jaundice (liver failure) rather than its cause, that there was a contemporaneous improvement in allergies, and that the benefits persisted long after remission of the liver disease, indicating that the salutary effects were not mediated by metabolites of bilirubin. Hench postulated the effect was due to an unknown innate ‘substance X’ (Hench, 1938), and it is now clear that all these effects were secondary to decreased hepatic clearance of cortisol. Hench observed a similar remission of arthritis in pregnant patients, fostering the hypothesis that the responsible factor was a ‘bisexual hormone’ (Hench, 1950). Hench and Kendall began to collaborate around 1935, and by 1941 had

concluded that ‘compound E’ (cortisone) was the substance X that was inducing remission of arthritis, but World War II delayed further work. As related by Ingle, the US Army believed that the German Luftwaffe was importing large quantities of beef adrenals from Argentina and using adrenal extracts to permit its pilots to resist stress and fly to great altitudes (Ingle, 1975) (this was related to Butenandt’s work in wartime Germany on oxygen consumption in stress). There was no truth to the rumor, but the Office of Scientific Research and Development gave high priority to ‘compound A’, and with the help of information from Reichstein, Merck and Co. prepared 100 g of this steroid. Clinical tests with Addison’s disease were negative, but Kendall pushed for more work on cortisone, which was more difficult to prepare. Lewis Sarett at Merck developed the first large-scale production of cortisone, generating 9 g by 1948 at a cost of $14 million. There was no clinical indication for its use, but before ‘pulling the plug’ on the project, Merck distributed its precious cortisone to several investigators; via Kendall, Hench obtained several grams, and began clinical studies. With Charles Slocomb, Hench treated a 29 year-old, wheelchairbound arthritic woman; after receiving 100 mg daily for 4 days, she was able to walk out of the hospital. After treating another 15 patients with similar dazzling results, they announced their results (Hench et al., 1949), and, although they cautioned that excessive doses would cause Cushing syndrome (Sprague et al., 1950), the world lauded the ‘miracle cure’ and Kendall, Hench and Reichstein received the only Nobel Prize for work with adrenal hormonal steroids in 1950 (Fig. 6).

10

W.L. Miller / Molecular and Cellular Endocrinology 371 (2013) 5–14

Fig. 6. The 1950 Nobel laureates. Image captured on line.

5. Clinical investigators: adrenal hyperplasias The effectiveness of cortisone in Addison’s disease and its increased availability quickly permitted its trial in other clinical settings. Wilkins was first to report its successful use in CAH (Wilkins et al., 1950, 1951); Bartter published similar results as an abstract (Bartter et al., 1950) and then as a full paper (Bartter et al., 1951). This opened a vigorous era of clinical investigation of the pathways of steroidogenesis in a variety of inherited adrenal and gonadal disorders. In 1950 it was not known that CAH was an enzymatic defect, and the pathways of steroidogenesis that are so familiar today were also unknown. Knowledge of the structures of many steroids, mainly from the work of Reichstein, suggested precursor/product relationships, so that attention was quickly directed toward the pathways of steroidogenesis. The identification of steroidogenic defects was necessarily indirect, and was limited by the contemporary understanding of genetics. Based on studies in yeast, Beadle and Tatum, who received the Nobel Prize in 1958, had established the principle of ‘one gene, one enzyme’ (Beadle and Tatum, 1941), which was widely believed to mean ‘one enzyme, one enzyme activity’; this seemed to be confirmed by the subsequent demonstration that genes and polypeptides are colinear (Yanofsky et al., 1964). Thus the conventional wisdom was that if A was the precursor of B, there must be a unique A ? B-ase; accordingly, enzymes were named for their presumably unique activities. Thus clinical investigators looked for patients who lacked a biologically active steroid end product such as cortisol, and overproduced precursor steroids, evidenced by the increased excretion of their urinary metabolites. Normal animal adrenals and occasional affected human tissues were also incubated with radiolabeled precursor steroids permitting an enzymatic activity to be demonstrated more directly. Because it was thought that the human steroidogenic pathways were the same those in cattle, several errors were promulgated (such as the notion that 17OHprogesterone is a precursor of sex steroid synthesis by conversion to androstenedione), that were not corrected until human materials could be studied in the subsequent era of molecular biology. With the above approaches Joseph Jailer, who tragically died at age 46 (Obituary, 1960), and Bongiovanni separately and painstakingly demonstrated that CAH was caused by a lesion in steroid 21-hydroxylation (Jailer et al., 1955; Bongiovanni, 1958; Franz et al., 1960). Discovery of other steroidogenic defects soon followed. The use of urinary metabolites sometimes obscured the

question of what was made by the adrenal as opposed to what were the (hepatic) degradation products, but by simple paper chromatography Bongiovanni identified ‘Reichstein’s compound S’ (11-deoxycortisol) in the serum of a patient with a hypertensive form of CAH, permitting the incisive and correct conclusion that ‘‘These findings suggest an essentially complete deficiency of adrenal ‘11-hydroxylase’ in the hypertensive form of congenital adrenal hyperplasia.’’ (Eberlein and Bongiovanni, 1956). The study of urinary steroids permitted the discovery of 3b-HSD deficiency (Bongiovanni and Kellenbenz, 1962) and of 17-hydroxylase deficiency (Biglieri et al., 1966). However, there were also some notable missteps. The seemingly logical notion that a patient’s failure to convert A to B meant a defect in an A ? B-ase led to the long-held view that congenital lipoid adrenal hyperplasia represented ‘‘20,22-desmolase deficiency’’ (Prader and Gurtner, 1955), whereas it is actually a defect in a cholesterol transporter, the steroidogenic acute regulatory protein (StAR) (Lin et al., 1995; Bose et al., 1996). Similarly, the view that each enzymatic activity had to be catalyzed by separate enzymes failed with the initial report of so-called ‘‘17,20 desmolase deficiency’’ (Zachmann et al., 1972), which is now known to be a syndrome that may be caused by mutations in several different proteins, including the 17a-hydroxylase/17,20 lyase enzyme and its co-factors (Miller, 2012).

6. Steroidogenic enzymes: proteins, cDNAs and genes An understanding of steroidogenic processes required identification of the responsible steroidogenic enzymes. The discovery of cytochrome P450 enzymes was central to the understanding of steroidogenesis (for reviews and personal reflections, see Cooper, 1973; Estabrook, 2003; Omura, 2011). Early studies noted that steroid 21-hydroxylation by bovine adrenal microsomes was inhibited by carbon monoxide and reversible by light, but the responsible protein was not identified (Ryan and Engel, 1956, 1957). Others identified a ‘‘carbon monoxide binding pigment’’ in liver microsomes (Klingenberg, 1958; Garfinkel, 1958), and Omura and Sato provided key evidence that this hepatic system involved a hemoprotein they called ‘‘cytochrome P450’’ (Omura and Sato, 1962). Procedures for its spectral quantitation (Omura and Sato, 1964a,b) led to a major breakthrough: the discovery that steroid 21-hydroxylation was P450-mediated (Estabrook et al., 1963;

W.L. Miller / Molecular and Cellular Endocrinology 371 (2013) 5–14

Cooper et al., 1963, 1965). While it took several years to determine that the P450 moiety actually was the catalytic component, as opposed to a generic co-factor (a la cytochrome b5 or cytochrome c), the discovery that P450 was associated with 21-hydroxylation initiated the isolation of several steroidogenic enzymes (for review, see Miller, 1988). Because it was then known that the cholesterol side-chain cleavage reaction occurred in mitochondria (Halkerston et al., 1961), and because other steroidogenic reactions were associated with P450 enzymes, Simpson and Boyd hypothesized and then demonstrated that this reaction was also catalyzed by a P450 enzyme (Simpson and Boyd, 1966, 1967). The work of Peter Hall was also notable: in addition to isolating P450scc (CYP11A1) (Shikita and Hall, 1973a,b), he isolated P450c17 (CYP17A1) and rigorously demonstrated that this one enzyme catalyzed both 17hydroxylase and 17,20 lyase activities (Nakajin and Hall, 1981; Nakajin et al., 1981, 1984). This was the first demonstration that distinct steroidogenic enzymatic activities could be catalyzed by a single enzyme. Protein isolation quickly gave way to molecular cloning. This vastly more powerful technology permitted determination of the precise nature of each steroidogenic enzyme, and its regulated synthesis according to cell type, development, and hormonal stimulation. Many laboratories, working with different species and different enzymes contributed to this effort (reviewed in Miller, 1988 and in Miller and Auchus, 2011); a selected summary of some prominent early adrenal contributions follows. The first steroidogenic enzyme cDNAs to be cloned were those for bovine P450scc (CYP11A1) (Morohashi et al., 1984), P450c21 (CYP21A2) (White et al., 1984; John et al., 1986; Yoshioka et al., 1986) and P450c17 (CYP17A1) (Zuber et al., 1986), quickly followed by those for human P450scc (Chung et al., 1986b), P450c21 (White et al., 1986), and P450c17 (Chung et al., 1987). Gene sequences for human P450scc (Morohashi et al., 1987a), bovine (Chung et al., 1985, 1986a) and human (Higashi et al., 1986; White et al., 1986) P450c21, and human P450c17 (Picado-Leonard and Miller, 1987) soon followed. The cloning of P450c11b (CYP11B1) (Morohashi et al., 1987b; Chua et al., 1987) and P450c11AS (CYP11B2) (Ogishima et al., 1991; Curnow et al., 1991), showed that different 11-hydroxylases participate in the synthesis of cortisol and aldosterone, and that the single P450c11AS enzyme catalyzed all three reactions (11-hydroxylation, 18-hydroxylation and 18-methyl oxidation) needed to convert DOC to aldosterone. Bovine and human adrenodoxin (Okamura et al., 1985; Picado-Leonard et al., 1988), adrenodoxin reductase (Hanukoglu et al., 1987; Solish et al., 1988) and P450 oxidoreductase (Yamano et al., 1989) were also cloned in the late 1980s, followed in the 1990s by 3bHSD (Lorence et al., 1990a,b; Lachance et al., 1990; Rhéaume et al., 1991), and the two forms of 11bHSD (Agarwal et al., 1989; Tannin et al., 1991; Albiston et al., 1994). Finally, the discovery of the steroidogenic acute regulatory protein (StAR), first as spots on 2-dimensional gels (Pon et al., 1986) and then by its cloning (Clark et al., 1994; Sugawara et al., 1995), solved the mystery of congenital lipoid adrenal hyperplasia (Lin et al., 1995; Bose et al., 1996) and elucidated the different mechanisms regulating the acute and chronic adrenal responses to ACTH (Stocco and Clark, 1996). Thus there were substantially fewer enzymes than had been envisioned from earlier physiologic studies: the three reactions needed to convert cholesterol to pregnenolone were all catalyzed by P450scc; the three reactions needed to convert DOC to aldosterone were all catalyzed by P450c11AS; both 17a-hydroxylation and 17,20 lyase activity were catalyzed by P450c17; the 21hydroxylation of both progesterone and 17OHP were catalyzed by P450c21; the 3b-hydroxysteroid dehydrogenase and D5–D4 isomerase reactions with all the D5 substrates (pregnenolone, 17OH-preg, DHEA) were catalyzed by 3bHSD2. The expression of cloned cDNAs, first in transfected cells and subsequently in yeast

11

and bacterial systems permitted the detailed enzymatic analysis of these enzymes and an understanding of important species-specific differences in their activities. This is perhaps best illustrated by P450c17, where there are major species differences in the 17,20 lyase reaction: the rodent (rat, mouse, hamster, guinea pig) enzyme uses the D4 substrate (17OHP); the human enzyme strongly prefers the D5 substrate (17OH-preg) (Auchus et al., 1998) and the pig, frog and trout enzymes can utilize either substrate (reviewed in Flück et al., 2003). An understanding such seeming details has permitted understanding the steroidal patterns seen in disorders of human steroidogenesis and is central to the design of effective inhibitor strategies. 7. Future directions While the above tour through the adrenal cortex might suggest to the young investigator that everything of interest in the adrenal has been done, that is far from the truth. As illustrated by the outstanding science presented at the 15th Adrenal Cortex Conference, the following areas, in no particular order, require further investigation. (1) The precise molecular itinerary of a cholesterol molecule as it enters the mitochondrion and the mechanism of StAR’s action remain unclear. (2) Crystallographic structures are becoming available for some steroidogenic P450 enzymes, but these often represent modified enzymes from non-human species. Much remains to be done to understand the structural biology of the enzymes and how they interact with their redox partner proteins. (3) New, enzyme-specific inhibitor drugs are needed for treatment of steroid excess states and for ablation of steroidogenesis in steroid-dependent malignancies. (4) The enzymes of the newlydiscovered ‘backdoor pathway’ of steroidogenesis (Wilson et al., 2003; Auchus, 2004) remain poorly characterized (Flück et al., 2011), and nothing is known about the regulation of this pathway in the adrenal or elsewhere. (5) It seems likely that there are disorders of steroidogenesis that remain to be discovered (partial deficiencies of ferredoxinn and ferredoxin reductase?). (6) Much remains to be learned about adrenal embryology, about the role of the fetal adrenal cortex and about its mechanism(s) of perinatal involution. (7) The relationships between adrenal development and adrenal malignancy, and their use to target adrenal cancers, requires substantial further investigation. (8) The mechanism(s) triggering adrenarche, the biological significance of this primate phenomenon and their potential relevance to ovarian

Fig. 7. The author (left) with Dr. Synthia Mellon, at the International Congress of Endocrinology, Quebec, Canada, 1984. ÓW.L. Miller.

12

W.L. Miller / Molecular and Cellular Endocrinology 371 (2013) 5–14

hyperandrogenism remain unclear. Thus the adrenal will continue to be of central importance to biology and medicine well into the foreseeable future.

Acknowledgements The author wishes to acknowledge the dozens of fellows, students and collaborators with whom he has studied steroidogenesis since the early 1980s. One, Prof. Synthia H. Mellon, deserves special mention (Fig. 7). It was reading Sindy’s 1978 Ph.D. thesis (with Seymour Lieberman, who had studied with Reichstein) that catalyzed my departure from pituitary polypeptides and into the world of steroidogenesis, and launched both a successful career and a loving marriage.

References Addison, T., 1849. Chronic suprarenal insufficiency, usually due to tuberculosis of suprarenal capsule. Lond. Med. Gaz. 43, 517–518. Agarwal, A.K., Monder, C., Eckstein, B., White, P.C., 1989. Cloning and expression of rat cDNA encoding corticosteroid 11b-dehydrogenase. J. Biol. Chem. 264, 18939–18943. Albiston, A.L., Obeyesekere, V.R., Smith, R.E., Krozowski, Z.S., 1994. Cloning and tissue distribution of the human 11b-hydroxysteroid dehydrogenase Type II enzyme. Mol. Cell. Endocrinol. 105, R11–R17. Arnold, J., 1866. Ein Beitrag zu der feineren Structur und dem Chemismus der Nebennieren. Virchows Arch. 35, 64–107. Auchus, R.J., 2004. The backdoor pathway to dihydrotestosterone. Trends Endocrinol. Metab. 15, 432–438. Auchus, R.J., Lee, T.C., Miller, W.L., 1998. Cytochrome b5 augments the 17,20 lyase activity of human. P450c17 without direct electron transfer. J. Biol. Chem. 273, 3158–3165. Bartter, F.C., Forbes, A.O., Leaf, A., 1950. Congenital adrenal hyperplasia associated with the adrenogenital syndrome: an attempt to correct its disordered hormonal pattern. J. Clin. Invest. 29, 797. Bartter, F.C., Albright, F., Forbes, A.P., Leaf, A., Dempsey, E., Carroll, E., 1951. The effects of adrenocorticotropic hormone and cortisone in the adrenogenital syndrome associated with congenital adrenal hyperplasia: an attempt to explain and correct its disordered hormonal pattern. J. Clin. Invest. 30, 237–351. Baumann, E.J., Kurland, S., 1927. Changes in the inorganic constituents of blood in suprarenalectomized cats and rabbits. J. Biol. Chem. 71, 281–302. Beadle, G.W., Tatum, E.L., 1941. Genetic control of biochemical reactions in Neurospera. Proc. Natl. Acad. Sci. 27, 499–506. Biglieri, E.G., Herron, M.A., Brust, N., 1966. 17a-Hydroxylation deficiency in man. J. Clin. Invest. 45, 1945–1954. Bowditch, HP., 1902. Memoir of Charles Edouard Brown-Sequard, 1817–1894 (Read before the National Academy of Sciences, April 1897). National Academy of Sciences Biographical Memoirs, vol. IV. Published by the Academy, Washington DC, Judd & Detweiler Printers, 1902. pp. 93–98. Bongiovanni, A.M., 1958. In vitro hydroxylation of steroids by whole adrenal homogenates of beef, normal man, and patients with the adrenogenital syndrome. J. Clin. Invest. 37, 1342–1347. Bongiovanni, A.M., Kellenbenz, G., 1962. The adrenogenital syndrome with deficiency of 3b-hydroxysteroid dehydrogenase. J. Clin. Invest. 41, 2086–2092. Bongiovanni, A.M, Root, A.W., 1963. The adrenogenital syndrome. New. Engl. J. Med. 268 (1283–1289; 1342–1351; 1391–1399 (in three parts)). Bose, H.S., Sugawara, T., Strauss III, J.F., Miller, W.L., 1996. The pathophysiology and genetics of congenital lipoid adrenal hyperplasia. New. Engl. J. Med. 335, 1870– 1878. Brown-Sequard, C.E., 1856. Recherches experimentales sur la physiolgie el la pathologie des capsules surrenales. Arch. Generales de Med. 5 (08), 385–401. Brown-Sequard, C.E., 1889. Note on the effects produced on man by subcutaneous injections of a liquid obtained from the testicles of animals. Lancet 2, 105–107. Butenandt, A., Karlson, P., 1954. Uber die Isolierung eines Metamotphose-Hormons der Insekten in kristallisierter Form. Z Naturforsch (b) 9, 389–391. Cannon, W.B., 1915. Bodily Changes in Pain, Hunger, Fear and Rage: An Account of Researches into the Function of Emotional Excitement. Reprinted by Harper Torchbooks, New York, 1963. Carmichael, S.W., 1989. A history of the adrenal medulla. Rev. Neurosci. 2, 83–100. Cartland, G.F., Kuizenga, M.H., 1936. The preparation of extracts containing the adrenal cotical hormone. J. Biol. Chem. 116, 57–64. Chua, S.C., Szabo, P., Vitek, A., Grzeschik, K.-H., John, M., White, P.C., 1987. Cloning of cDNA encoding steroid 11b-hydroxylase, P450c11. Proc. Natl. Acad. Sci. USA 84, 7193–7197. Chung, B.C., Matteson, K.J., Miller, W.L., 1985. Cloning and characterization of the bovine gene for steroid 2l-hydroxylase (P-450c2l). DNA 4, 211–219. Chung, B.C., Matteson, K.J., Miller, W.L., 1986a. Structure of a bovine gene for P450c21 (steroid 21-hydroxylase) defines a novel cytochrome P450 gene family. Proc. Natl. Acad. Sci. USA 83, 4243–4247.

Chung, B.C., Matteson, K.J., Voutilainen, R., Mohandas, T.K., Miller, W.L., 1986b. Human cholesterol side-chain cleavage enzyme, P450scc: cDNA cloning, assignment of the gene to chromosome 15, and expression in the placenta. Proc. Natl. Acad. Sci. USA 83, 8962–8966. Chung, B.C., Picado-Leonard, J., Haniu, M., Bienkowski, M., Hall, P.F., Shivley, J.E., Miller, W.L., 1987. Cytochrome P450c17 (steroid 17a-hydroxylase/17,20 lyase): cloning of human adrenal and testis cDNAs indicates the same gene is expressed in both tissues. Proc. Natl. Acad. Sci. USA 84, 407–411. Clark, B.J., Wells, J., King, S.R., Stocco, D.M., 1994. The purification, cloning and expression of a novel luteinizing hormone-induced mitochondrial protein in MA-10 mouse Leydig tumor cells. Characterization of the steroidogenic acute regulatory protein (StAR). J. Biol. Chem. 269, 28314–28322. Cooper, D.Y., 1973. Discovery of the function of the heme protein P-450: a systematic approach to scientific research. Life Sci. 13, 1151–1161. Cooper, D.Y., Estabrook, R.W., Rosenthal, O., 1963. The stoichiometry of C21 hydroxylation of steroids by adrenocortical microsomes. J. Biol. Chem. 238, 1320–1323. Cooper, D.Y., Levin, S., Narasimhulu, S., Rosenthal, O., Estabrook, R.W., 1965. Photochemical action spectrum of the terminal oxidase of mixed function oxidase systems. Science 145, 400–402. Cori, C.F., Cori, G.T., 1927. The fate of sugar in the animal body. VII. The carbohydrate metabolism of adrenalectomized rats and mice. J. Biol. Chem. 74, 473–493. Curnow, K.M., Tusie-Luna, M., Pascoe, L., Natarajan, R., Gu, J., Nadler, J.L., White, P.C., 1991. The product of the CYP11B2 gene is required for aldosterone biosynthesis in the human adrenal cortex. Mol. Endocrinol. 5, 1513–1522. Cushing, H., 1932. The basophil adenomas of the pituitary body and their clinical manifestations. Bull. Johns Hopkins Hosp. 50, 137–195. Cuvier, G., 1805. Lecons d’anatomie comparee. 5, 240. Baudouin, Paris. de Creccio, L., 1865. Sopra un caso di apparenze virili in una donna. Morgagni 7, 151–183. Eberlein, W.R., Bongiovanni, A.M., 1956. Plasma and urinary corticosteroids in the hypertensive form of congenital adrenal hyperplasia. J. Biol. Chem. 223, 85–94. Estabrook, R.W., 2003. A passion for P450s (remembrances of the early history of research on cytochrome P450). Drug Metab. Dispos. 31, 1461–1473. Estabrook, R.W., Cooper, D.Y., Rosenthal, O., 1963. The light reversible carbon monoxide inhibition of the steroid C21-hydroxylase system of the adrenal cortex. Biochem. Z. 338, 741–755. Flück, C.E., Meyer-Böni, M., Pandey, A.V., Kempna, P., Miller, W.L., Schoenle, E.J., Biason-Lauber, A., 2011. Why boys will be boys. Two pathways of fetal testicular androgen biosynthesis are needed for male sexual differentiation. Am. J. Hum. Genet. 89, 201–218. Flück, C.E., Miller, W.L., Auchus, R.J., 2003. The 17,20 lyase activity of cytochrome P450c17 from human fetal testis favors the D5 steroidogenic pathway. J. Clin. Endocrinol. Metab. 88, 3762–3766. Franz, A.G., Holub, D.A., Jailer, J.W., 1960. Further evidence of a relative lack of C-21 hydroxylation in congenital adrenal hyperplasia. J. Clin. Invest. 39, 904–908. Garfinkel, S.D., 1958. Studies on pig liver microsomes. I. Enzymic and pigment composition of different microsomal fractions. Arch. Biochem. Biophys. 77, 493–509. Glynn, E.E., 1912. The adrenal cortex, its rests and tumours; its relation to other ductless glands, an especially to sex. Quart. J. Med. 5 (2), 157–192. Gottschau, M., 1883. Structur und embryonale entwickelung der nebennieren bei saugethieren. Arch. f. Anat. u. Entwickelungdgeschich. I, 412–458. Halkerston, I.D.K., Eichhorn, J., Hechter, O., 1961. A requirement for reduced triphosphopyridine nucleotide for cholesterol side-chain cleavage by mitochondrial fractions of bovine adrenal cortex. J. Biol. Chem. 236, 374. Hanukoglu, I., Gutfinger, T., Haniu, M., Shively, J.E., 1987. Isolation of a cDNA for adrenodoxin reductase (ferredoxin-NADP+reductase) implications for mitochondrial cytochrome P-450 systems. Eur. J. Biochem. 169, 449–455. Hartman, F.A., Brownell, K.A., 1930. The hormone of the adrenal cortex. Science 72, 76. Hartman, F.A., Brownell, K.A., Hartman, W.E., 1930. A further study of the hormone of the adrenal cortex. Am. J. Physiol. 95, 670–680. Hench, P.S., 1933. Analgesia accompanying hepatitis and jaundice in cases of chronic arthritis, fibrositis and sciatic pain. Proc. Staff Meet. Mayo Clin. 8, 430–437. Hench, P.S., 1938. Effect of spontaneous jaundice on rheumatoid arthritis. Attempts to reproduce the phenomenon. Brit. Med. J. 20, 394–398. Hench, P.S., Kendall, E.C., Slocumb, C.H., Polley, H.F., 1949. The effect of a hormone of the adrenal cortex (17-hydroxy-11-dehydrocorticosterone [compound E]) and of the pituitary adrenocorticotropic hormone on rheumatoid arthritis. Proc. Staff Meet. Mayo Clin. 24, 181–197. Hench, P.S., 1950. The reversibility of certain rheumatic and non-rheumatic conditions by the use of cortisone or of the pituitary adrenocorticotropic hormone. Nobel Lect. (on line). Higashi, Y., Yoshioka, H., Yamane, M., Gotoh, O., Fujii-Kuriyama, Y., 1986. Complete nucleotide sequence of two steroid 21-hydroxylase genes tandemly arranged in human chromosome: a pseudogene and genuine gene. Proc. Natl. Acad. Sci. USA 83, 2841–2845. Ingle, D.J., 1975. Biographical memoir of Edward C. Kendall, vol. 47. National Academy of Sciences, Washington, DC. Ingle, D.J., Kendall, E.C., 1937. Atrophy of the adrenal cortex of the rat produced by the administration of large amounts of cortin. Science 86, 245–246. Jailer, J.W., Gould, J.J., Vande Wiele, R., Lieberman, S., 1955. 17aHydroxyprogesterone and 21-desoxyhydrocortisone; their metabolism and possible role in congenital adrenal virilism. J. Clin. Invest. 34, 1639–1646.

W.L. Miller / Molecular and Cellular Endocrinology 371 (2013) 5–14 John, M.E., Okamura, T., Dee, A., Adler, B., John, M.C., White, P.C., Simpson, E.R., Waterman, M.R., 1986. Bovine steroid 21-hydroxylase: regulation of biosynthesis. Biochemistry 25, 2846–2853. Kendall, E.C., 1915. The isolation in crystalline form of the compound containing iodin, which occurs in the thyroid. JAMA lxiv (25), 2042–2043. Kendall, E.C., Mason, H.L., McKenzie, B.F., Myers, C.S., Koelsche, G.A., 1934. Isolation in crystalline form of the hormone essential to life from the supranetal cortex: its chemical nature and physiologic properties. Trans. Assoc. Am. Physicians 48, 147–152. Klingenberg, M., 1958. Pigments of rat liver microsomes. Arch. Biochem. Biophys. 75, 376–386. Lachance, Y., Luu-The, V., Labrie, C., Simard, J., Dumont, M., de Launoit, Y., Guerin, S., Leblanc, G., Labrie, F., 1990. Characterization of human 3b-hydroxysteroid dehydrogenase/D5 ? D4-isomerase gene and its expression in mammalian cells. J. Biol. Chem. 265, 20469–20475. Leoutsakos, B., Leoutsakos, A., 2008. The adrenal glands: a brief historical perspective. Hormones 7, 334–336. Lin, D., Sugawara, T., Strauss III, J.F., Clark, B.J., Stocco, D.M., Saenger, P., Rogol, A., Miller, W.L., 1995. Role of steroidogenic acute regulatory protein in adrenal and gonadal steroidogenesis. Science 267, 1828–1831. Loeb, R.F., 1933. Effect of sodium chloride in treatment of a patient with Addison’s disease. Proc. Soc. Exp. Biol. 30, 808–812. Lorence, M.C., Murry, B.A., Trant, J.M., Mason, J.I., 1990a. Human 3b-hydroxysteroid dehydrogenase/D5 ? D4 isomerase from placenta: expression in nonsteroidogenic cells of a protein that catalyzes the dehydrogenation/ isomerization of C21 and C19 steroids. Endocrinology 126, 2493–2498. Lorence, M.C., Corbin, C.J., Kamimura, N., Mahendroo, M.S., Mason, J.I., 1990b. Structural analysis of the gene encoding human 3b-hydroxysteroid dehydrogenase/D5D4 isomerase. Mol. Endocrinol. 4, 1850–1855. Mason, H.L., Meyers, C.S., Kendall, E.C., 1936. Chemical studies of the suprarenal cortex. II. The identification of a substance which possesses the qualitative action of cortin; its conversion into a diketone closely related to androstenedione. J. Biol. Chem. 116, 267–276. Menkin, V., 1940. Effect of adrenal cortex extract on capillary permeability. Am. J. Physiol. 129, 691–697. Mezzogiorno, A., Mezzogiorno, V., 1999. Bartolomeo Eustachio: a pioneer in morphological studies of the kidney. Am. J. Nephrol. 19, 193–198. Miller, W.L., 1988. Molecular biology of steroid hormone synthesis. Endocr. Rev. 9, 295–318. Miller, W.L., 2012. The syndrome of 17,20 lyase deficiency. J. Clin. Endocrinol. Metab. 97, 59–67. Miller, W.L., Auchus, R.J., 2011. The molecular biology, biochemistry and physiology of human steroidogenesis and its disorders. Endocr. Rev. 32, 81–151. Morohashi, K., Fujii-Kuriyama, Y., Okada, Y., Sogawa, K., Hirose, T., Inayama, S., Omura, T., 1984. Molecular cloning and nucleotide sequence of cDNA for mRNA of mitochondrial P450(scc) of bovine adrenal cortex. Proc. Natl. Acad. Sci. USA 81, 4647–4651. Morohashi, K., Sogawa, K., Omura, T., Fujii-Kuriyama, Y., 1987a. Gene structure of human cytochrome P-450(scc), cholesterol desmolase. J. Biochem. 101, 879– 887. Morohashi, K., Yoshioka, H., Gotoh, O., Okada, Y., Yamamoto, K., Miyata, T., Sogawa, K., Fujii-Kuriyama, Y., Omura, T., 1987b. Molecular cloning and nucleotide sequence of DNA of mitochondrial P-450(11) of bovine adrenal cortex. J. Biochem. 102, 559–568. Nakajin, S., Shively, J.E., Yuan, P., Hall, P.F., 1981. Microsomal cytochrome P450 from neonatal pig testis: two enzymatic activities (17a-hydroxylase and C17,20lyase) associated with one protein. Biochemistry 20, 4037–4042. Nakajin, S., Hall, P.F., 1981. Microsomal cytochrome P450 from neonatal pig testis. Purification and properties of a C21 steroid side-chain cleavage system (17ahydroxylase-C17,20 lyase). J. Biol. Chem. 256, 3871–3876. Nakajin, S., Shinoda, M., Haniu, M., Shively, J.E., Hall, P.F., 1984. C21 steroid sidechain cleavage enzyme from porcine adrenal microsomes. Purification and characterization of the 17a-hydroxylase/C17,20 lyase cytochrome P450. J. Biol. Chem. 259, 3971–3976. New, M.I., 2011. Ancient history of congenital adrenal hyperplasia. Endocr. Dev. 20, 202–211. Obituary, 1895. Brit. Med. J., p. 54. Obituary, 1960. Brit. Med. J., 871. Ogishima, T., Shibata, H., Shimada, H., Mitami, F., Suzuki, H., Saruta, T., Ishimura, Y., 1991. Aldosterone synthase cytochrome P450 expressed in the adrenals of patients with primary aldosteronism. J. Biol. Chem. 266, 10731–10734. Okamura, T., John, M.E., Zuber, M.X., Simpson, E.R., Waterman, M.R., 1985. Molecular cloning and amino acid sequence of the precursor form of bovine adrenodoxin: evidence for a previously unidentified COOH-terminal peptide. Proc. Natl. Acad. Sci. USA 82, 5705–5709. Omura, T., Sato, R., 1962. A new cytochrome in liver microsomes. J. Biol. Chem. 237, PC1375–PC1376. Omura, T., Sato, R., 1964a. The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. Biol. Chem. 239, 2370– 2378. Omura, T., Sato, R., 1964b. The carbon monoxide-binding pigment of liver microsomes. II. Solubilization, purification, and properties. J. Biol. Chem. 239, 2379–2385. Omura, T., 2011. Recollection of the early years of the research on cytochrome P450. Proc. Jpn. Acad. Ser. B 87, 617–640. Pearce, J.M.S., 2004. Thomas Addison 1793–1860. J. Royal. Soc. Med. 97, 297–300.

13

Picado-Leonard, J., Miller, W.L., 1987. Cloning and sequence of the human gene encoding P450c17 (steroid 17a-hydroxylase/17,20 lyase): similarity to the gene for P450c21. DNA 6, 439–448. Picado-Leonard, J., Voutilainen, R., Kao, L., Chung, B., Strauss, J.F. III, Miller, W.L., 1988. Human adrenodoxin: cloning of three cDNAs and cycloheximide enhancement in JEG-3 cells. J. Biol. Chem. 263, 3240–3244 (corrected, p 11016). Pon, L.A., Hartigan, J.A., Orme-Johnson, N.R., 1986. Acute ACTH regulation of adrenal corticosteroid biosynthesis: rapid accumulation of a phosphoprotein. J. Biol. Chem. 261, 13309–13316. Prader, A., Gurtner, H.P., 1955. Das Syndrom des Pseudohermaphroditismus masculinus bei kongenitaler Nebennierenrindenhyperplasie ohne Androgenuberproduktion (adrenaler Pseudohermaphroditismus masculinus). Helv. Paed. Acta 10, 397–412. Reichstein, T., Grüssner, A., 1934. Eine ergiebige Synthese der L-Ascorbinsäure (CVitamin). Helv. Chim. Acta 17, 311–328. Reichstein, T., 1936a. ‘‘Adrenosteron’’. Über die Bestandteile der Nebennierenrinde II (vorläufige Mitteilung). Helv. Chim. Acta 19, 223–225. Reichstein, T., 1936b. Über die Bestandteile der Nebennierenrinde IV. Helv. Chim. Acta 19, 402–412. Rengachary, S.S., Colen, C., Guthikonda, M., 2008. Charles-Edouard Brown-Sequard: an eccentric genius. Neurosurgery 62, 954–964. Rhéaume, E., Lachance, Y., Zhao, H.L., Breton, N., Dumont, M., de Launoit, Y., Trudel, C., Luu-The, V., Simard, J., Labrie, F., 1991. Structure and expression of a new complementary DNA encoding the almost exclusive 3b-hydroxysteroid dehydrogenase/D5D4-isomerase in human adrenals and gonads. Mol. Endocrinol. 5, 1147–1157. Rogoff, J.M., Stewart, G.N., 1928. Studies on adrenal insufficiency in dogs: V. The influence of adrenal extracts on the survival period of adrenalectomized dogs. Am. J. Physiol. 84, 660–674. Rosenheim, O., King, H., 1934. The chemistry of sterols, bile acids and other cyclic constituents of natural fats and oils. Annu. Rev. Biochem. 3, 87–110. Rowntree, L.G., Greene, C.H., Swingle, W.W., Pfiffner, J.J., 1930. The treatment of patients with Addison’s Disease with the ‘‘cortical hormone’’ of Swingle and Pfiffner. Science 72, 482–483. Ruzicka, L., 1945. Multimembered rings, higher terpene compounds and male sex hormones. Nobel Lect. (on line). Ryan, K.J., Engel, L.L., 1956. Steroid 21-hydroxylation by adrenal microsomes and reduced triphosphopyridine nucleotide. J. Am. Chem. Soc. 78, 2654–2655. Ryan, K.J., Engel, L.L., 1957. Hydroxylation of steroids at carbon 21. J. Biol. Chem. 225, 103–114. Selye, H., 1936. A syndrome produced by diverse nocuous agents. Nature 138, 32. Selye, H., 1946. The general adaptation syndrome and the diseases of adaptation. J. Clin. Endocrinol. 6, 117–230. Shikita, M., Hall, P.F., 1973a. Cytochrome P-450 from bovine adrenocortical mitochondria: an enzyme for the side chain cleavage of cholesterol. I. Purification and properties. J. Biol. Chem. 248, 5596–5604. Shikita, M., Hall, P.F., 1973b. Cytochrome P-450 from bovine adrenocortical mitochondria: an enzyme for the side chain cleavage of cholesterol. II. Subunit structure. J. Biol. Chem. 248, 5605–5609. Simpson, E.R., Boyd, G.S., 1966. The cholesterol side-chain cleavage system of the adrenal cortex: a mixed-function oxidase. Biochem. Biophys. Res. Commun. 24, 10–17. Simpson, E.R., Boyd, G.S., 1967. The cholesterol side-chain cleavage system of bovine adrenal cortex. Eur. J. Biochem. 2, 275–285. Simpson, S.A., Tait, J.F., Wettstein, A., Neher, R., von Euw, J., Reichstein, T., 1953. Isolierung eines neuen kristallisierten Hormons aus Nebennerien mit besonders hoher Wirksamkeit auf den Mineralsoffwechsel. Experientia 9, 333–335. Simpson, S.A., Tait, J.F., Wettstein, A., Neher, R., Euw, J.V., Schindler, O., Reichstein, O., Reichstein, T., 1954. Aldosteronisolierung und Eigenschaften über Bestandteile de Nebennierenrinde und verwandte Stoffe. Helv. Chim. Acta 37, 1163–1200. Solish, S.B., Picado-Leonard, J., Morel, Y., Kuhn, R.W., Mohandas, T.K., Hanukoglu, I., Miller, W.L., 1988. Human adrenodoxin reductase: two mRNAs encoded by a single gene on chromosome 17cen ? q25 are expressed in steroidogenic tissues. Proc. Natl. Acad. Sci. USA 85, 7104–7108. Sprague, R.G., Power, M.H., Mason, H.L., Albert, A., Mathieson, D.R., Hench, P.S., Kendall, E.C., Slocumb, C.H., Polley, H.F., 1950. Observations on the physiologic effects of 17-hydroxy-11-dehydrocorticosterone (cortisone) and adrenocorticotropic hormone (ACTH) in man. Arch. Int. Med. 85, 199–258. Steiger, M., Reichstein, T., 1937. Desoxy-cortico-steron (21-oxyprogesterone) aus D5–3 -xoy-atio-cholensaure. Helv. Chim. Acta 20, 1164–1179. Stocco, D.M., Clark, B.J., 1996. Regulation of the acute production of steroids in steroidogenic cells. Endocr. Rev. 17, 221–244. Sugawara, T., Holt, J.A., Driscoll, D., Strauss III, J.F., Lin, D., Miller, W.L., Patterson, D., Clancy, K.P., Hart, I.M., Clark, B.J., Stocco, D.M., 1995. Human steroidogenic acute regulatory protein (StAR): Functional activity in COS cells, tissue-expression, mapping of the structural gene to 8p11.2 and an expressed pseudogene to chromosome 13. Proc. Natl. Acad. Sci. USA 92, 4778–4782. Swingle, W.W., Pfiffner, J.J., 1930. The survival of comatose adrenalectomized cats with an extract of the suprarenal coryex. Science 72, 75–76. Swingle, W.W., Pfiffner, J.J., 1931. Studies on the adrenal cortex: IV. Further observations on the preparation and chemical properties of the cortical hormone. Am. J. Physiol., 98, 144–152. Swingle, W.W., Pfiffner, J.J., Vars, H.M., Bott, P.A., Perkins, W.M., 1933. The function of the adrenal cortical hormone and the cause of death from adrenal insufficiency. Science 77, 58–64.

14

W.L. Miller / Molecular and Cellular Endocrinology 371 (2013) 5–14

Takamine, J., 1901. The isolation of the active principle of the suprarenal gland. Cambridge University Press, Great Britain. Tannin, G.M., Agarwal, A.K., Monder, C., New, M.I., White, P.C., 1991. The human gene for 11b-hydroxysteroid dehydrogenase. Structure, tissue distribution, and chromosomal localization. J. Biol. Chem. 266, 16653–16658. Trunk, A., 2006. Biochemistry in wartime: the life and lessons of Adolf Butenandt, 1936–1946. Minerva 44, 285–306. White, P.C., New, M.I., DuPont, B., 1984. Cloning and expression of cDNA encoding a bovine adrenal cytochrome P-450 specific for steroid 21-hydroxylation. Proc. Natl. Acad. Sci. USA 81, 1986–1990. White, P.C., New, M.I., Dupont, B., 1986. Structure of the human steroid 21hydroxylase genes. Proc. Natl. Acad. Sci. USA 83, 5111–5115. Wieland, HO., 1928. The chemistry of the bile acids. Nobel Lect. (on line). Wilkins, L., Lewis, R.A., Klein, R., Rosenberg, E., 1950. The suppression of androgen secretion by cortisone in a case of congenital adrenal hyperplasia. Bull. Johns Hopkins Hosp. 86, 249–252. Wilkins, L., Lewis, R.A., Klein, R., Gardner, L.I., Crigler, J.F., Rosemberg, E., Migeon, C.J., 1951. Treatment of congenital adrenal hyperplasia with cortisone. J. Clin. Endocrinol. 11, 1–25. Williams, J.S., Williams, G.H., 2003. 50th Anniversery of aldosterone. J. Clin. Endocrinol. Metab. 88, 2364–2372. Wilson, J.D., Auchus, R.J., Leihy, M.W., Guryev, O.L., Estabrook, R.W., Osborn, S.M., Shaw, G., Renfree, M.B., 2003. 5a-Androstane-3a,17b-diol is formed in tammar wallaby pouch young testes by a pathway involving 5a-pregnane-3a,17a-diol20-one as a key intermediate. Endocrinology 144, 575–580.

Windaus, A., 1928. Constitution of sterols and their connection with other substances occurring in nature. Nobel Lect. (on line). Wintersteiner, O., Pfiffner, J.J., 1936. Chemical studies of the adrenal cortex. III. Isolation of two new physiologically inactive compounds. J. Biol. Chem. 116, 291–305. Yamano, S., Aoyama, T., McBride, O.W., Hardwick, J.P., Gelboin, H.V., Gonzalez, F.J., 1989. Human NADPH-P450 oxidoreductase: complementary DNA cloning, sequence, vaccinia virus-mediated expression, and localization of the CYPOR gene to chromosome 7. Mol. Pharmacol. 35, 83–88. Yamashima, T., 2003. Jokichi Takamine (1854–1922), the samurai chemist, and his work on adrenalin. J. Med. Biog. 11, 95–102. Yanofsky, C., Carlton, B.C., Guest, J.R., Helinski, D.R., Henning, U., 1964. The colinearity of gene structure and protein structure. Proc. Natl. Acad. Sci. 51, 266–272. Yoshioka, H., Morohasi, K., Sogawa, K., Yamane, M., Kominami, S., Takemori, S., Okada, Y., Omura, T., Fujii-Kuriyama, Y., 1986. Structural analysis of cloned cDNA for mRNA of microsomal cytochrome P-450 (C21) which catalyzes steroid 21-hydroxylation in bovine adrenal cortex. J. Biol. Chem. 261, 4106–4109. Zachmann, M., Vollmin, J.A., Hamilton, W., Prader, A., 1972. Steroid 17,20 desmolase deficiency: a new cause of male pseudohermaphroditism. Clin. Endocrinol. 1, 369–385. Zuber, M.X., John, M.E., Okamura, T., Simpson, E.R., Waterman, M.R., 1986. Bovine adrenal cytochrome P45017ct: regulation of gene expression by ACTH and elucidation of primary sequence. J. Biol. Chem. 261, 2475–2482.