Steroids and the Scientist

Jan-Åke Gustafsson

Department of Medical Nutrition, Karolinska Institutet, Novum, SE-141 86 Huddinge, Sweden

Address all correspondence and requests for reprints to: Jan-Åke Gustafsson, Department of Medical Nutrition, Karolinska Institutet, Novum, SE-141 86 Huddinge, Sweden.

ABSTRACT

Our interest in nuclear receptors (NRs) originated from early studies on hepatic steroid metabolism. We discovered a new hypothalamo-pituitary-liver axis, imprinted neonatally by androgens and operating through sexually differentiated GH secretory patterns. Male and female patterns have opposite effects on sexually differentiated hepatic genes, explaining sexually dimorphic liver patterns. To further understand steroid action, we purified the glucocorticoid receptor (GR) leading to our discovery of the NR three-domain structure, with separable DNA binding domain and ligand binding domains and a third domain now known to have transcriptional regulatory properties. Knowledge of this domain structure has been immensely important for deciphering NR actions. Using this first purified NR, we collaborated with Keith Yamamoto and first demonstrated specific NR binding to DNA. This also was the first demonstration of a mammalian transcription factor, a breakthrough that led to discovery of NR response elements. In further collaboration with Yamamoto, we cloned the first NR cDNA sequences, leading to cloning of the superfamily of NR genes. With Yamamoto and Kaptein, we determined the first three-dimensional NR structure, that of DNA binding domain. Later work on orphan receptors resulted in the first discovery of: 1) endogenous ligands for an orphan receptor (fatty acids as activators of peroxisomal proliferator-activated receptor {alpha}); 2) liver X receptor ß (OR-1) and its role in central nervous system cholesterol homeostasis; and 3) estrogen receptor ß, leading to a paradigm shift in understanding of estrogen signaling, of importance in endocrinology, immunology, and oncology and to development of estrogen receptor ß agonists for treatment of autoimmune diseases, prostate disease, depression, and ovulatory dysfunction.

OVERTURE: INTESTINAL MICROBIAL METABOLISM AND ENTEROHEPATIC CIRCULATION OF STEROID HORMONES

THIS REVIEW IS written in honor of a beloved mentor, the late Sune Bergstrom, whose support in my early career made all the difference to me. Although Bergstrom is very well known for his prostaglandin research, most people do not remember that he is also the father of the bile acid and cholesterol metabolism field in Sweden (1). Our interest in endocrinology started with steroid hormone metabolism. Developments in gas chromatography-mass spectrometry toward the end of the 1960s offered new possibilities for identification of steroid hormone metabolites in body fluids and excreta. In my thesis work from that period of time, we identified quite a large number of novel steroid hormone metabolites in bile, urine, and feces from germfree and conventional rats as part of an effort to investigate the role of the intestinal microflora in the enterohepatic circulation of steroid hormones. I have a vivid memory of myself whistling joyfully, injecting samples onto the gas chromatograph when suddenly, Sune Bergstrom was over my shoulder inquiring whether "the work went better with all the music." I still do not know whether my off key noise was disturbing him or whether he was just surprised that a Swede would be expressing feelings so openly. In any case, we found many examples of microbe-catalyzed steroid dehydrations, dehydroxylations, and oxidoreductions. This work revealed the existence of an extensive metabolic interplay between liver and gastrointestinal tract connected via the enterohepatic circulation where steroidal compounds could be recirculated about ten times before finally being excreted in urine and/or feces. Accordingly, a substantial fraction of steroid metabolites in urine was found to be of mixed microbial/hepatic origin, a finding that at the time was surprising to most investigators in the field. These studies were published in a series of papers between 1968 and 1970 (2, 3).

DISCOVERY OF IMPRINTING OF HEPATIC METABOLISM AND THE SEXUALLY DIFFERENTIATED HYPOTHALAMO-PITUITARY-LIVER AXIS

Upon identification of steroids in urine and feces, it became apparent that there were striking differences between male and female rats in the profile of excreted steroid metabolites (4). In an effort to understand the mechanisms behind these sex differences, we widened our experimental arsenal to encompass not only in vivo gas chromatography-mass spectrometry techniques but also in vitro studies of steroid metabolism in subcellular fractions of rat liver. Our observations indeed showed the existence of a very remarkable sexual dimorphism in hepatic metabolism of steroid hormones in rat liver. Interestingly, we found that these sex differences resulted from neonatal imprinting by aromatized androgens (5), in much the same way as sexual behavior is imprinted. Our further investigations showed that the sexually differentiated liver metabolism in rats arose as a consequence of neonatal programming of the hypothalamo-pituitary axis, or, more precisely, of the GH secretory pattern (6). At this time, male and female rodents were shown to have very different GH secretory patterns (7), with males displaying a peak/trough pattern with peaks every 3–4 h, and females with a relatively stable GH secretory pattern. The female pattern of liver enzymes could be mimicked experimentally by the use of osmotic minipumps loaded with GH (8). These early studies demonstrated the existence of a novel hypothalamo-pituitary-liver axis that controls hepatic steroid hormone metabolism. Furthermore, they showed that GH may elicit completely opposite responses in the liver depending upon how it is presented to the liver. Finally, our results showed that the action of sex hormones in the liver in many instances occurs indirectly via the hypothalamo-pituitary axis.

DISCOVERY OF NOVEL, SEXUALLY DIMORPHIC CYTOCHROME P-450 ISOFORMS IN RAT LIVER AND THE GH REGULATORY MECHANISMS BEHIND THEIR EXPRESSION

One of the extremely interesting sex differences was in the in vivo metabolism of corticosterone in rats. The absolutely predominant corticosterone metabolite in female rats is 15ß-hydroxy-allo-tetrahydrocorticosterone (3{alpha}, 11ß, 15ß, 21-tetrahydroxy-5{alpha}-pregnan-20-one), which is excreted as a sulfate. This metabolite is completely absent from male rats, which prefer to metabolize corticosterone to highly polar 16{alpha}-hydroxylated compounds. The female-specific corticosterone metabolite is generated by an unusual female-specific cytochrome P-450-dependent hepatic 15ß-hydroxylase that specifically uses 21-sulfurylated corticosterone derivatives as substrates. In 1988, we cloned this P-450 (named P-450 2C12) and showed (9) that 2C12 is under strict control by the female-specific GH secretory pattern. Interestingly, the male-specific GH secretory pattern instead induces expression of the male-specific cytochrome P-450 2C11, which is a 16{alpha}-hydroxylase that we and others cloned in 1988 (10). There followed several years of intensive mechanistic studies aimed at unraveling signal transduction pathways turned on by GH and resulting in control of the sex differentiated P-450 isoforms in rodent liver. Importantly, GH regulation of sexually differentiated genes also occurs in the human liver and, in collaboration with others, estrogen regulation of lipoprotein receptors were found to be mediated—at least partially—via GH and the hypothalamo-pituitary-liver axis (11). Attempts have been made to apply these findings in medicine in the form of GH treatment of certain forms of disturbances of lipid homeostasis (12).

NUCLEAR RECEPTORS (NRs) BC (BEFORE CLONING)—A SOMEWHAT UNDEREVALUATED ERA; PURIFICATION OF THE FIRST NR, THE GLUCOCORTICOID RECEPTOR (GR), TO HOMOGENEITY

In parallel with our research on steroid metabolism in the liver and its regulation, we developed a keen interest in mechanisms of steroid hormone action. Our particular feeling at the time was that metabolites of steroid hormones were underestimated as ligands for steroid hormone receptors and we started to undertake a major effort to purify the rat liver GR to homogeneity to analyze the nature of its copurified endogenous ligand. It turned out to be difficult to identify the endogenous GR ligand with this approach. However, our success in the first purification of an NR (the GR) to homogeneity provided a reagent that was critical for the field for the solution of a number of issues and in doing so led us to discover the modular nature of NRs that has been critical to deciphering differential receptor functions (13). This was indeed a tour de force and took several years to complete.

It should be emphasized that, at this point in time (1970s), the overall structure of steroid receptors was unknown and the subject of much confusion and speculation. The use of sucrose gradients, the preferred sizing method of the time revealed as many as six different size forms, and the abundance of these varied in different tissues. These observations led to suggestions that the physiological functions of glucocorticoids were mediated by multiple GRs, or else that the basic GR was very small with multiple associated proteins. Purification of the receptor led to a clarification of these issues. The varied sizes were in some cases due to differences in association of these receptors with heat shock proteins, but in other cases was due to proteolysis of the large 90-kDa single polypeptide chain receptor that we purified. The observation that the receptor domains were susceptible to proteolytic cleavage turned out to be key to discovering the receptor’s domain structure. We demonstrated that GR had three domains, a notion that was confirmed by others many years later, after the cloning of the full-length GR. In view of the importance of the three-domain concept for the development of the steroid receptor field, our key findings with reference to this issue are detailed below.

DISCOVERY OF DOMAIN STRUCTURE OF NRs

The modular nature of NRs that characterizes the entire family dictates numerous aspects of their properties, from regulation of transcription in different cellular environments, to combinatorial interactions with proteins and diverse DNA elements. These domains are the amino terminal fragment (NTD), the DNA binding domain (DBD), and the ligand binding domain (LBD).

The breakthrough discovery of the modular nature of GR was a set of experiments in our laboratory initiated through cleavage of these domains of the GR from the full-length receptor with proteases followed by examination of their properties (14, 15, 16, 17). The domain structure of the GR was reminiscent of the domain structure of prokaryotic transcription factors.

1. NTD
The NR NTD interacts with coactivators and corepressors and thus is a critical domain, separate from the DBD and LBD. Differential use of this domain vs. the LBD or in collaboration with the LBD results in enormous combinatorial possibilities for receptor function and helps to explain activities of NR modulators such as SERMS [selective estrogen receptor (ER) modulators] (see Three-Dimensional Structure of the Agonist/Antagonist ...). We first identified this domain as separable from the LBD and DBD, by demonstrating a proteolytic fragment of the receptor that could react with antibodies to the GR and that was separable from the DBD and LBD (17).

2. DBD
a. Discovery. We first discovered the DBD as a proteolytic fragment that binds DNA (15).

b. Specific DNA Binding. In collaboration with Keith Yamamoto, we subsequently showed the specificity of the GR DBD function by first demonstrating (for any NR or any mammalian transcription factor) that purified GR bound to specific DNA sequences, in mouse mammary tumor virus (MMTV) DNA (18, 19, 20). This collaboration also led to the discovery that the specific DNA sites behave as response elements that confer hormone responsiveness onto downstream genes (21). This represented a paradigm shift in understanding signaling in general, and contributed, for example, to solidify the concept of enhancers.

These important results on specific DNA binding of GR came from an intense and very productive collaboration between Stockholm and San Francisco that started in 1979 and lasted till the middle of the 1980s. For many years, on a monthly—or sometimes weekly—basis, we organized an "air bridge" of freshly prepared, homogeneous rat liver GR for the MMTV DNA binding studies at University of California-San Francisco.

The reason for the success of this heretofore impossible task was the combination of access to homogeneous steroid receptor, together with access to unlimited quantities of a glucocorticoid-sensitive gene.

3. LBD
The LBD is separable from the DBD and amino terminal fragment. We first determined the major function of the LBD (for any NR) by demonstrating binding of the cognate ligand to a proteolytic fragment of GR (15) that was separable from the DBD.

CLONING OF RECEPTOR GENE SEQUENCES

A major leap forward was our first cloning of any NR gene sequences, in this case a fragment of the rat GR cDNA (22, 23, 24). This was done in collaboration with the Yamamoto laboratory. We had produced a panel of polyclonal and monoclonal anti-GR antibodies that our laboratory had developed against purified rat liver GR. These antibodies were key for demonstrating in a cDNA expression library clones that contained and expressed receptor gene sequences. These clones provided for the first time probes to detect receptor mRNAs that could be used for quantification in various tissues, including GR mutants in glucocorticoid-resistant cell lines. More importantly, this achievement demonstrated that receptor gene sequences could be cloned, a forerunner for cloning of the entire superfamily.

THREE-DIMENSIONAL STRUCTURE OF A RECEPTOR DOMAIN
During the latter half of the 1980s, we expressed large quantities of recombinant GR DBD and sent this to Kaptein’s nuclear magnetic resonance spectroscopy laboratory in Utrecht, the Netherlands, for three-dimensional structural studies. The Yamamoto lab was also at this time furnishing the Kaptein lab with recombinant GR DBD and these interactions resulted in a three-way collaboration where the first three-dimensional structure of an NR domain, the GR DBD, was determined by solution nuclear magnetic resonance spectroscopy (25). This landmark discovery also defined the structure of the now classical two-zinc-finger motif shared by all NRs, where two zinc atoms are coordinated by conserved cysteine residues. The DBD was found to consist of a globular body from which the finger regions extend. Indeed, the GR DBD structure turned out to be applicable to all thus far defined NR DBD structures, which differ only slightly from one another. Finally, this discovery set the stage for obtaining the numerous receptor three-dimensional structures that have provided great insight into the mechanisms of receptor action.

THREE-DIMENSIONAL STRUCTURE OF THE AGONIST/ANTAGONIST BOUND ER{alpha} LBD AND DISCOVERY OF THE HELIX 12 SWITCH

Before the x-ray structure determination of an antagonist bound to an NR LBD, investigators had concluded that the antagonist must perturb the folding of the LBD helix 12 into the body of the receptor, but they did not know how this occurred. In collaboration with the biotechnology company KaroBio AB (Stockholm, Sweden), and the laboratories of Rod Hubbard and Geoff Greene, we determined the first x-ray crystal structure of an NR LBD bound to an antagonist, the ER{alpha} LBD complexed with raloxifene (26). This demonstrated the antagonist perturbation of helix 12 that is general for NRs, and how it occurs. A side group of the ligand actually binds to helix 12 and holds it in an alternative position that precludes formation of a coactivator binding surface that is critical for certain receptor functions. This discovery also helped to solidify understanding of the NR modular domain function including actions of SERMs because the ligand selectively perturbs the LBD, and not the N-terminal domain. This discovery has also been useful for design of pharmaceuticals.

FIRST DISCOVERY OF A LIGAND FOR AN ORPHAN NR (PEROXISOMAL PROLIFERATOR-ACTIVATED RECEPTOR {alpha})

Before the cloning era, we had many very specific ligands (the steroid hormones, vitamin D, retinoids, peroxisome proliferators, pregnenolone 16{alpha}-carbonitrile) but very few specific receptors. After cloning of NRs, the problem was the opposite. Cross-hybridization led to identification of numerous NRs for which no ligand could be identified. These were called orphan receptors. In an effort to identify ligands for these receptors, we began analyzing extracts from blood and tissues and discovered that one of these orphan receptors, peroxisomal proliferator-activated receptor {alpha}, was activated by fatty acids (27). This finding led to the now important concept that NRs bind a great spectrum of small molecules that occur naturally, especially those involved in nutrition. This discovery also expanded the field of regulation of lipid homeostasis by NRs, with consequent impact on pharmaceutical design and understanding molecular mechanisms involved in adipose tissue differentiation.

DISCOVERY OF LIVER X RECEPTOR (LXR) ß

Understanding the role of novel ligands and NRs in lipid metabolism was further influenced by our cloning of LXRß (OR-1) (28) and delineation of its actions as separate from LXR{alpha}. LXRß is an oxysterol-activated NR that regulates cholesterol homeostasis. We demonstrated through knockout experiments that LXR{alpha} are necessary for normal brain function; deletion of the LXR{alpha} genes leads to severe neurodegeneration and massive cholesterol accumulation in parts of the brain, a finding of potential interest in view of the alleged association between neurodegenerative disease and disturbed cholesterol homeostasis (29).

DISCOVERY OF ERß

Although by 1995 many NRs had been cloned on the basis of the homology of their DBDs it was, nonetheless, an enormous surprise when we cloned the second ER, ERß, from a prostate cDNA library (30). The discovery of ERß changed our concepts of estrogen signaling and opened a new chapter in estrogen research and in design of estrogenic pharmaceuticals. ERß commonly counteracts ER{alpha} action, explaining previously puzzling findings where estrogens gave contrasting effects. But most fortuitously, ERß is not expressed in the adult endometrium or pituitary and this means that selective ERß agonists will be free from two of the most unwanted side effects of estradiol use, i.e. chemical castration in males and endometrial cancer in females.

With ERß –/– mice, we found that ERß is necessary for normal functioning in a variety of tissues including the immune system, the prostate where ERß is antiproliferative, the ovary, and the brain (31, 32, 33, 34). We also found that ERß is the preferred receptor for phytoestrogens (35), and this probably explains the reputed beneficial effects of phytoestrogens in the body. The notion that selective ERß agonists could be used to treat diseases was enthusiastically pursued by drug companies, and this has resulted in development of ERß agonists for treatment of autoimmune diseases (rheumatoid arthritis and inflammatory bowel disease), benign prostatic hyperplasia, prostate cancer, infertility, and depression, respectively (Table 1Go). These drugs are in different phases of development; the most advanced are in phase II clinical trials.


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Table 1. Potential Pharmaceuticals of ERß Agonists (Although Only ~9 yr Have Elapsed Since I First Presented the Discovery of ERß at a Keystone Meeting, an Astounding Number of Pharmaceutical Possibilities Based on Targeting ERß Have Emerged)

 
THE ERß-3ßAdiol PATHWAY

As we studied ERß and its functions in the prostate I came back full circle to my earliest studies on steroids and their metabolism in the liver. In those early days, we found that 3{alpha}Adiol was a very good substrate for liver cytochromes P450, but its isomer 3ßAdiol was not (36). Sometime later, we identified a specific cytochrome P450 in the prostate that selectively catalyzed hydroxylation of 3ßAdiol (37, 38). The meaning of this specific metabolism was a great intellectual challenge for us until we began to study ERß in the prostate. Suddenly, all the pieces fell into place. 3ßAdiol was the estrogenic metabolite of 5{alpha}-dihydrotestosterone and was part of a clever feedback mechanism whereby androgens promote growth and differentiation of the prostate via the androgen receptor and inhibit growth via ERß (39). The implication of this pathway for pharmacological intervention in prostatic disease is profound. We predicted that use of 5{alpha}-reductase inhibitors, by eliminating the ERß ligand, 3ßAdiol, would result in worse prostate cancer, and this turned out to be the case when the results of the Prostate Cancer Prevention Trial were released (40). We suggest that if ERß agonists are given along with 5{alpha}-reductase inhibitors the patient will benefit from loss of 5{alpha}-dihydrotestosterone and from the prodifferentiative effects of ERß. This would result in a lower incidence of prostate cancer and the cancers should be more highly differentiated, i.e. less malignant. Thus, steroid hormone metabolites seem to have come in from the cold to again assume important roles in endocrinology!

FOOTNOTES

Abbreviations: DBD, DNA binding domain; ER, estrogen receptor; GR, glucocorticoid receptor; LBD, ligand binding domain; LXR, liver X receptor; NR, nuclear receptor; NTD, amino terminal fragment; SERM, selective ER modulator.

Received for publication November 29, 2004. Accepted for publication January 6, 2005.

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