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Molecular Endocrinology 19(6):1402–1411
Copyright © 2005 by The Endocrine Society
doi: 10.1210/me.2004-0480
PERSPECTIVE
A Life-Long Search for the Molecular
Pathways of Steroid Hormone Action
Bert W. O’Malley
Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas
77030
The O’Malley laboratory first showed that estrogen
and progesterone act in the nucleus to stimulate
synthesis of specific mRNAs (ovalbumin and avidin),
coding for their respective inducible proteins. The
overall molecular pathway of steroid-receptor-DNAmRNA-protein-function was then established and
provided a coherent foundation for future studies of
the impact of estrogen and progesterone receptors
on endocrine tissue development, adult function, and
in pathologies such as cancer. The lab group went on
to: biochemically demonstrate ligand-induced conformational activation of progesterone and estrogen
receptors, discover the concept of ligand-independent activation of steroid receptors, discover key
steroid receptor coactivator intermediary coactivators for receptor function, and define the role of coactivators/corepressors in selective receptor modulator drug action and in cell homeostasis. This body
of work advanced our molecular understanding of
the critical role of steroid hormones in normal and
abnormal physiology and also generated a base of
scientific knowledge that served to further modern
hormonal therapy and disease management. (Molecular Endocrinology 19: 1402–1411, 2005)
THE BEGINNING
the estradiol binding protein he discovered was a
true receptor, but I found the whole concept to be
fascinating and almost certainly correct as presented by Elwood. It was at this point that I became
interested in studying aspects of the molecular
pathway of steroid hormone action. My first and
memorable mentor for my work on the chicken oviduct was Stan Korenman, but he then left the NIH at
the end of that first year. I stayed at NIH and continued to develop the chick oviduct as a model for
hormone action. Why the oviduct? The oviduct was
chosen because of the work of Roy Hertz in the early
1940s. He was attempting to devise a new bioassay
for estrogenic substances and published weight
changes in the chicken oviduct in response to estrogens. Knowing the protein composition of egg
white and guessing that the proteins were made in
the oviduct, Stan and I employed the chicken oviduct for the early studies on induction of specific
radiolabeled protein synthesis. I remember my
around-the-clock injections of chickens during that
first year. Indeed, we showed that estrogen and
progesterone induced de novo synthesis of the
specific proteins, ovalbumin and avidin, respectively
(1, 2).
M
Y INTEREST in steroid hormones began in
earnest when I entered the National Institutes
of Health (NIH) as a clinical associate in the Endocrine Branch of the National Cancer Institute in
1965, then under the direction of Mort Lipsett. My
plan was to do steroid chemistry for a year and then
to move my efforts toward examining the actions of
these hormones. I remember the discussions at
Branch coffee breaks about the radiolabeling of estradiol and the tissue-binding experiments performed by Jensen in his pioneering studies. At that
time, there was considerable argument among
Jensen and certain other scientists as to whether
Abbreviations: AF, Activation function; AIB1, amplified in
breast cancer 1; ER, estrogen receptor; GR, glucocorticoid
receptor; LBD, ligand binding domain; NR, nuclear receptor;
PR, progesterone receptor; SERM, selective ER modulator;
SRC, steroid receptor coactivator; SRM, selective receptor
modulator; TR, thyroid hormone receptor.
Molecular Endocrinology is published monthly by The
Endocrine Society (http://www.endo-society.org), the
foremost professional society serving the endocrine
community.
1402
O’Malley • Perspective
DEMONSTRATING THAT NUCLEAR RECEPTOR
(NR) LIGANDS REGULATE NUCLEAR RNAs
These protein experiments provided the critical background information to encourage me to attempt to
define the overall pathway and mechanism of action of
estrogen and progesterone. The sum total of my subsequent life’s work has been focused on this goal.
Although it was suspected by some that steroid hormones could induce new protein synthesis, there was
considerable confusion as to the molecular mechanism in the late 1960s and early 1970s. Some biochemists were still wed to the theory that steroids
activated enzymes directly, and many prominent
physiologists believed that steroid hormones exerted
their primary effects at the cell membrane; the latter is
a thought that only recently has been reborn and now
appears to be bearing some fruit. At the time that I
entered the field in the 1960s, studies by other labs
showed that steroids caused an increase in incorporation of radiolabeled precursor nucleotides into trichloroacetic acid-precipitable RNA. A widespread
interpretation that steroids increased RNA synthesis
only by enhancement of radiolabeled nucleotide uptake into cellular pools was set to rest by the sum of
our 1968 study showing that new species of RNA,
measured by RNA-DNA hybridization and RNA composition analyses, were induced in chick oviduct target tissue by steroid hormones, and that their induction was coincident with the synthesis of new specific
proteins (3). With these analyses, carried out by my
first postdoctoral fellow, Bill McGuire, we predicted
that steroid hormones acted on DNA to turn on the
synthesis of genes. This result strongly focused my
thoughts on the nucleus and gene expression, where
they remained for the next 30 yr. I should mention that
we did not forget about the receptor itself during this
time period. In 1970, Merry Sherman and I published
the most complete physical chemical characterization
of a steroid receptor [oviduct PR (progesterone receptor)] to that point in time, and suggested that this
information would soon allow us to trace the receptor
directly to nuclear gene targets (4).
THE MECHANISM OF STEROID HORMONE
ACTION: LIGANDS INDUCE SPECIFIC mRNAs
At this stage, I was almost certain that steroid hormones (and their receptors) acted at the level of DNA
to turn on (and off) genes. Nevertheless, even among
those who favored the nucleus as the primary target of
steroids, transcriptional regulation was not the universally accepted theory. In fact, major labs promoted
both posttranscriptional regulation (5) and translational regulation (6) to explain the increase in protein
synthesis. Thus, it was imperative that we prove that
the specific mRNAs that coded for our oviduct proteins were induced by steroid hormones. Unfortu-
Mol Endocrinol, June 2005, 19(6):1402–1411 1403
nately, the methodology required to prove this was
only in its infancy. I moved to Vanderbilt University in
1969, and for the next decade of my scientific career,
I was very fortunate to have the opportunity to enter
into a critical and invaluable collaboration with Tony
Means. In 1969, our primary goal was to demonstrate
unequivocally that steroid hormones can induce actual
specific mRNAs (i.e. ovalbumin and avidin mRNAs)
that would then code for their respective specific proteins. I also wanted to carry out these experiments in
a living animal because of prior criticisms of cell culture artifacts. We were fortunate to be the first to
publish that estrogen induced ovalbumin mRNA levels
in the chick oviduct (7–9) (Fig. 1). We also next published that progesterone induced avidin mRNA, and
the concept of nuclear action of steroid receptors was
born. In the subsequent year, the Schimke lab substantiated our estrogen induction of ovalbumin mRNA
in the same tissue (10). The overall pathway for steroid
hormone action now could be predicted, i.e. steroid
hormones induce synthesis of proteins by first inducing levels of their specific mRNAs. We proposed that
the hormonal induction of mRNAs for avidin and
ovalbumin was due to new mRNA synthesis because
there was no mRNA produced before hormone administration to the chick oviduct. Therefore, the possibility,
suggested by others, of increasing mRNA (i.e. ovalbumin) posttranscriptionally to levels equivalent to almost 50% of the cellular mRNA by inhibiting the degradation of the mRNAs could be calculated as
mathematically infeasible. Also, translational regulation by the hormones could be ruled against because
there was no mRNA detectable in tissue and no mRNA
on oviduct polysomes in the absence of hormone
stimulation. Most importantly, the test of time proved
these results and our interpretation of transcriptional
regulation to be entirely correct.
Our elucidation of the overall action pathway for
steroid hormones by the demonstrations of estrogen
induction of ovalbumin mRNA and progesterone induction of avidin mRNA in 1972 was exciting indeed.
I realize that some of the readers of this perspective
were likely in grade school at the time and cannot
fathom any possibilities for regulation other than transcriptional. Nevertheless, it was a period of intense
excitement in our lab group, and we could sense the
whole climate changing in our field. In my opinion, it
was our lab’s single most important contribution to the
field of steroid hormone action. I say that because
many have suggested that this series of publications
changed the field of endocrinology, fostering the
emergence of the new fields of hormone action and
molecular endocrinology. Hormone action and transcriptional regulation were now inextricably linked in
all meetings, beginning with the then heavily oversubscribed Gordon Conferences on Hormone Action.
There was a logarithmic explosion of publications on
steroid hormone action and steroid receptors (see the
“About NURSA” section in National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)/Nu-
1404 Mol Endocrinol, June 2005, 19(6):1402–1411
O’Malley • Perspective
Fig. 1. Illustration of our 1972 experiment (see Refs. 8 and 9) showing the effect of DES (diethylstilbestrol) on the rate of
ovalbumin protein synthesis (dashed lines) and levels of ovalbumin mRNA (bars) during 48 h after administration of the hormone
to immature chicks. The coincidence of the curves implies cause and effect and was interpreted as a direct transcriptional
response to a steroid hormone.
clear Receptor Signaling Atlas (NURSA) web site: www.
NURSA.org), which then extended to studies encompassing all other hormones. To add to the excitement,
Jensen had applied his estrogen receptor (ER) antibody to analysis of breast tumors to predict the patients who would benefit from hormonal therapy. The
tree of molecular endocrinology had some transcriptional roots, as can be noted in the early works of
Tomkins, Williams-Ashman, Mueller, Tata, Kenny,
Baxter, Thompson, Gorski, Liao, Wilson, and many
others. However, in the mid-to-late 1970s, this hormone action tree sprouted many more branches as an
influx of a large number of additional outstanding molecular scientists [i.e. Gustafsson (1974), Yamamoto
(1975), Chambon (1981), Evans (1982), Rosenfeld
(1982), Roeder (1993), etc.] expanded the hormone/
receptor/action field. They were drawn by an interest
in transcriptional regulation, and they elevated further
the quality of our field’s experimental science and our
scientific meetings. The NR field is one of the most
innovative, progressive, and productive fields of all of
the medical science subspecialties.
I next moved my lab group to Baylor College of
Medicine in 1973 and continued to generate additional substantiating data for our gene regulation
model. During the next decade, we carried out stud-
ies to purify an mRNA (ovalbumin), to clone the first
cDNAs for ovalbumin and avidin, to determine the
exact number of copies of the mRNA in cells before
and after hormone stimulation, to study the structure of the ovalbumin gene, and to show that the
hormone-receptor complex acted on target cell
chromatin to effect transcription. The pace and level
of competition was impressive among the labs in the
field. Yamamoto published glucocorticoid induction
of radiolabeled mRNA synthesis of a viral gene
(mouse mammary tumor virus) in 1975 (11), substantiating hormone action to be at the level of transcription; since then, the model has been confirmed repeatedly by other labs. Hormone response elements
were next identified by Gustafsson and Yamamoto.
A few years later, we and others were able to accomplish stimulation of gene transcription by a purified PR in a chromatin cell-free transcription system; this system allowed us to directly demonstrate
that the receptor acted via the promoter region to
somehow stabilize general transcription factor complexes on the TATA element (12). Although we saw
some ability for purified receptor to interact with
certain key components of the basic transcriptional
machinery such as transcription factor IIB, we repeatedly observed that there was some powerful
O’Malley • Perspective
magical protein component in nuclear extracts that
greatly enhanced the action of the receptor—and
that our purified receptor did not act efficiently in the
presence of purified TATA transcriptional components unless we added back small aliquots of a
nuclear extract. It was not for another 5 yr that we
were able to isolate and clone one of these elusive
magical components, the protein coactivator SRC
(steroid receptor coactivator)-1 (13). More excitement was to come before then, however.
NRs CAN BE ACTIVATED BY SECOND
MESSENGER PATHWAYS IN THE ABSENCE
OF LIGAND
By 1990, I thought we knew almost everything about
the basic aspects of ligand binding and activation of
steroid receptors. We certainly were in for a surprise.
In the course of doing some control experiments, a
postdoctoral fellow (Larry Denner) in the lab, along
with my close collaborators, Bill Schrader and Nancy
Weigel, made the observation that the avian PR could
be activated by phosphorylation pathways in the absence of ligand. Because the results were initially difficult to believe, we repeated the experiments many
times, but the conclusion was inescapable—-a ligandindependent pathway existed for activating certain receptors. The paper describing this work presented a
concept not previously predicted over the preceding
25 yr of research in the receptor field—and had future
consequences to the biology and pathology of NRs.
As one can imagine, there was considerable skepticism both among reviewers and my colleagues when
we presented these data. We first showed that cellular
phosphorylation pathways could activate a receptor in
the absence of ligand (14). Next, we demonstrated that
dopamine, not by binding to PR but by acting at its
own D1 membrane receptor, could activate PR both in
cultured cells, and in living animals (15, 16). This novel
steroid-independent pathway for activation of a steroid receptor is now known to occur via signaling
cascades from membrane regulatory molecules such
as cAMP, dopamine, growth factors, cytokines, and
possibly other cellular regulators acting at the membrane (16). Ligand-independent activation represents
a prime means by which membrane receptors and
NRs communicate at the level of the genome, and a
mechanism by which the cellular environment can
modulate NR function and transcription. These ligandindependent pathways for receptor function are particularly important in the mechanism of action of neurotransmitters such as dopamine in central nervous
system neurobehavior (16) and of growth factors, as
demonstrated clearly by the Korach lab (17). Moreover, applications are substantiated now in pathologies such as inflammation and in breast/prostate cancers where the ER/androgen receptors have been
shown to be activated by phosphorylation cascades
Mol Endocrinol, June 2005, 19(6):1402–1411 1405
induced by tumor growth factors in the absence of
ligand. Importantly, because approximately 50% of
the orphan receptors of the NR family are thought not
to have primary endogenous physiological ligands, our
discovery of ligand-independent activation uncovered
the mechanism by which this large group of orphan
receptors now are considered to be activated and/or
regulated (18). In this latter case, we solved a conundrum before it arose (19).
After Bill Schrader and Nancy Weigel purified the PR
and demonstrated the first isoform structures of a
steroid receptor (PRa; PRb) (20), Donald McDonnell
showed that the PRa and PRb forms of PR have differing and opposite functional activities. Our early PRa
and PRb work was the harbinger of NR isoforms as a
later developing concept in our field, one that proved
important for retinoic acid receptor, thyroid hormone
receptor (TR), and retinoid X receptor. Unfortunately,
our efforts to clone the first receptor clearly failed,
being led astray by a false immunogenic protein that
contaminated our antibody pool. After the clonings of
a partial cDNA for rat glucocorticoid receptor (GR) by
Gustafsson (21) and Yamamoto, the first full-length
clone of human GR by Evans (22) and Rosenfeld, and
ER by Jensen (23) and Chambon (24) in this issue, Orla
Conneely succeeded in cloning PR. We next went on
to clone the vitamin D receptor and confirmed its role
in human vitamin D-resistant rickets (25). The cloning
of receptors led to another increase in the publication
rate in our field and made important reagents available. The Evans lab was first to identify an orphan
receptor (the ER relateds), but soon many more were
discovered. Although we cloned five of the original
orphans, I chose to maintain my focus on classical
steroid receptors and mechanisms. However, in 1992,
we did publish the prediction that most of the orphan
receptors would turn out to be activated (or inactivated) either by metabolic ligands or by phosphorylation signaling pathways (19). This prediction turned out
to be correct.
LIGANDS DIRECTLY ACTIVATE RECEPTORS FOR
TRANSCRIPTIONAL RESPONSES AND THE CTERMINAL REGION OF RECEPTORS CONTAINS
THE KEY TO LIGAND ACTIVATION
Although we accepted that liganded receptor was the
mediator of the transcriptional response, the precise
mechanistic details of its function were unclear. Next,
we needed to directly couple the events of receptor
binding to DNA with receptor-mediated transcription
and prove that the ligand mediated this series of reactions in a sequential process. Using a cell free transcription system and purified PR in 1990, Milan Bagchi
demonstrated directly that progesterone specifically
set off a chain of distinguishable receptor events leading to heat shock protein dissociation, conversion
from an inactive to an active receptor form, binding to
1406 Mol Endocrinol, June 2005, 19(6):1402–1411
a PR element, and finally, induction of transcription of the
target gene; the RU486 antagonist promoted all events
except conversion to an active receptor form in our transcription system (26). (Only much later, in 1998, did we
use GR protein produced by Gustafsson to carry out an
experiment to show that steroid receptors bind to their
inverted repeat hormone response elements in DNA as
homodimers (27); Chambon showed identical results for
ER that same month.) After Bagchi’s experiment described above, we now understood that the activation of
receptor by ligand was direct, but we didn’t understand
the conformational step of receptor activation by agonist
and inactivation by antagonist in structural terms. In a
further collaboration with Ming and Sophia Tsai in 1992,
in what I believe was one of our more important but
lesser known biochemical studies, my postdoctoral fellow, George Allen, used molecular mapping with proteo-
O’Malley • Perspective
lytic enzymes and monoclonal antibodies produced by
Dean Edwards to predict that the structural allosteric
mechanism of action by agonist/antagonist ligands occurred at the C-terminal tail (now also known as helix 12)
of NRs (28) (Fig. 2). These results led us to postulate that
the C-terminal tail of the receptor flipped over like a lid
and compacted and covered the ligand binding site
when the ligand binding domain (LBD) was occupied by
hormone; the ligand-induced surface predicted was later
shown to be the coregulator interaction region of the
molecule. We were excited, however, when the liganded
crystals of two receptors were published in 1995. Our
structural model in our 1992 paper (Fig. 2) (28) was
proven to be surprisingly accurate in terms of that found
in the first important crystal structures of two agonistbound NR LBDs published simultaneously by Baxter
(TR) and Chambon (retinoic acid receptor).
Fig. 2. A model diagram of receptor structure-function relationships induced by agonist and antagonist ligands and published
in our 1992 paper (see Ref. 28). It shows the receptor in oval shape and illustrates the results of our molecular mapping studies
of receptors using proteolytic enzymes and site-specific antibodies. When the receptor LBD is unoccupied by ligand and inactive,
the C-terminal tail is extended; when agonist binds, the tail flips over and covers the ligand binding site, providing an activation
surface for interaction with coactivators (not discovered at that time); when the LBD is occupied by antagonist, the tail does not
position similarly and assumes more the inactive/unoccupied configuration (preventing coactivator from binding to the activation
surface). In this model, the primary regulatory structural alterations of receptors induced by ligand binding occur via C-terminal
tail positioning. Subsequent later detailed crystallizations and x-ray analyses of NRs confirmed the general accuracy of this model.
hsp, Heat shock protein.
O’Malley • Perspective
Mol Endocrinol, June 2005, 19(6):1402–1411 1407
EVIDENCE FOR THE EXISTENCE OF CELLULAR
COREPRESSORS FOR NRs
THE FIRST SELECTIVE NR COACTIVATOR
IS CLONED
My main thoughts, however, still remained on the
missing molecular link between the receptor and the
general transcription factor machinery at the TATA
box. I remember being encouraged by the experiments of Mark Ptashne, who was studying regulatory
activator molecules in yeast that did not bind to DNA,
but rather, acted via squelchable protein-protein interactions (29). We performed an ER-PR squelching experiment that was published in 1989 and predicted
that ER and PR interact with some common transcription factor(s) and that this may be a general mechanism for regulation by the steroid receptors (30). The
Chambon lab published a more detailed confirmation
of this squelching hypothesis the next year. However,
we were not happy with this approach because
squelching experiments provided no real information
on the specificity or the precise targets of the squelching reaction. I felt that we needed to back up and
rethink this problem in a manner that identified specific
molecules. As a first step toward this goal, Donald
McDonnell showed in yeast that incorporated mammalian ER and PRs were able to interact with a specific
yeast repressor protein (i.e. SSN6) that repressed the
transcriptional activity of an activation [activation function (AF) 1] domain of the steroid receptors when tamoxifen was bound. On the other hand, the SSN6
corepressor dissociated when estradiol was bound,
leading to receptor activation (31).
We then turned our attention to AF2-mediated repression mechanisms (32, 33). In a series of experiments using TR, my postdoctoral fellow, Aria Baniahmad, first demonstrated a silencing activity associated
with a human activation-mutant receptor; the silencing
activity was contained in the C terminus (33, 34). He
next went on to uncover the existence of a corepressor protein for the AF2 of a NR in mammalian cells. I
presented this complete data and our corepressor
theory at the winter Keystone Nuclear Receptor meeting in 1994 (submitted for publication in September
1994 and published in January 1995) (33). To the best
of my knowledge, our studies using mutational analyses of NRs (TR, ER, PR) were the first to claim the
existence of a new class of unknown soluble NR corepressors in animal cells that functioned by proteinprotein interactions and not by competitive interactions with DNA binding sites. Consequently, I guess
Aria’s paper (33) represents the biochemical discovery
of coregulators for steroid receptors, and in it, I believe
we were first to predict a ligand-induced coactivator
exchange with corepressor at the C terminus of a NR
to achieve ligand regulation of gene expression. Later
that year, Rosenfeld and Evans separately cloned the
first cDNAs for corepressors. Now, more than ever, we
were determined to find this elusive coactivator molecule for NRs. We were not alone in our quest,
however.
The search for coactivators was ongoing in more than
one lab, beginning with the early description of receptor-associated protein fragments by David Moore in
yeast assays and cell-free work on a 160-kDa protein
band that bound to activated ER by Myles Brown and
a 140-kDa band studied by Parker. A detailed description of everyone’s activities is beyond the limitations of
this perspective, but it is safe to say that at that time
the cloning of an authentic molecule containing demonstrable coactivation activity was an elusive goal to
all. As a field, we had no specific guidelines as to what
coactivators should do when added back to animal
cells. In collaboration with Ming and Sophia Tsai, my
postdoctoral fellow, Sergio Oñate, identified the first
selective NR coactivator cDNA clone with clear biological activity, SRC-1 (13). In his publication, we established the criteria that subsequently were used to
categorize the extensive number of coactivators discovered later by our lab and many others. The SRC-1
coactivator (and its two related family members)
turned out to be a key to understanding the transcriptional activity of NRs. As we expected, SRC-1 was
found to be a major booster of the power of transcription in the presence of the liganded receptor, but it
also directed histone acetylase activity to the gene.
McDonnell’s lab cloned hRPF1, and we next cloned a
second coactivator, E6-AP; both were found to be
dual function proteins containing an activation domain
and a second domain with proteolytic/ubiquitinylation
activity (35). Other members of the SRC-1 family were
cloned by other labs, and more and more coactivators
began to appear, each with the same interesting structure of a coactivation domain linked to some enzyme
function.
Soon after cloning SRC-1, we made an important
pharmacologic prediction of principle for coactivators,
when Carolyn Smith and I published that the intracellular coactivator/corepressor ratio can determine the
cellular activity of a mixed antagonist/agonist ligandlike tamoxifen (36). This experiment explained in large
part the puzzling mechanism of the tissue-selective
actions of selective ER modulators (SERMs) and contributed to the expansion of selective receptor modulator development in the pharmaceutical field. Later,
we substantiated this explanatory model in a controlled cell-free chromatin transcription system with
purified receptor and purified SRC coactivators (37).
Surprisingly, as we were cloning additional coactivators, other labs found more, and the number expanded logarithmically. There are now approximately
150 identified coactivator proteins that function with
NRs, and most function in a hormone-dependent
manner (see the “About NURSA” section in NIDDK/
NURSA web site: www.NURSA.org). Many of these
coactivators are enzymes that are recruited to NRoccupied promoters to carry out the multiple steps
1408 Mol Endocrinol, June 2005, 19(6):1402–1411
inherent to gene activation and repression; some coactivators are even noncoding RNA molecules (38).
The difficulty in proving suspected coactivators to be
authentic was a continuing problem. Consequently,
after cloning SRC-1, we felt it critical to quickly go on
to achieve in vivo evidence to prove the biological
activity of coactivators [SRC-1 and SRC-3/amplified in
breast cancer 1 (AIB1)] in living animals via a series of
gene knockout experiments performed by a postdoctoral fellow, Jianming Xu. Our newly generated SRC-1
KO mice were shown to have partial resistance to
multiple steroid hormones (39). Next, SRC-3 KO mice
(40) were constructed and proved to have a separate
and distinct phenotype from the SRC-1 KO, thereby
demonstrating the impressive tissue-specific selectivity even among multiple closely related members of
this SRC-1 family of coactivators. With publication of
these initial KO mouse studies, coactivators were accepted generally to be biologically important in the NR
field.
UNSUSPECTED ROLES OF COACTIVATORS IN
TRANSCRIPTION AND CELLULAR SIGNALING
To say the least, we were quite naive at that time as to
the extensive biology of the coactivators. Subsequent
experimental observations showed that steroid receptors not only mediate initiation/reinitiation of transcription but also can alter the nature of the gene product
by regulating the downstream alternative splicing of
pre-mRNAs in a receptor-specific manner (41). Our
initial experiments were based on principles defined
earlier by the Spiegelman lab for PGC-1 (42). We described multiple receptor coactivators that can regulate the alternative RNA splicing process and defined
the specific peptide domains that provide the coactivation and the alternative splicing activities (43). Furthermore, after Zafar Nawaz and I showed that certain
dual function coactivators have ubiquitin ligase (i.e.
E2/E3) activity that ubiquitinylates proteins within the
receptor-coactivator complex, a postdoctoral fellow,
David Lonard, demonstrated that degradation of the
receptor-coactivator complex by the 26S proteasome
was obligatory for efficient continued receptor-mediated transcription (44). This work led to our realization
that the receptor-coactivator complex has a built-in
enzymatic self-destruct code, and that if the gene was
to remain on, new molecules would need to be continually supplied to the site. There are now well over a
dozen dual function coactivators known that contain
completely distinct enzyme activities—and all are
brought to the target gene by the receptors bound to
the hormone response elements located within promoter regions.
In recent years, our laboratory continued to expand
the biology of coactivators by searching for novel
pleiotropic signaling activities of these molecules in
other pathways such as inflammation, growth, and
O’Malley • Perspective
stress (45). Our work showed that coactivators act as
homeostats to sense environmental signals and to
coordinate the signals emanating from membrane receptors with NRs and gene transcription, a concept of
importance to both normal and pathological signaling
by hormones. The coactivators do this by receiving
phosphorylation signals from the environment via
membrane receptors. More importantly, a coactivator
phosphorylation code exists whereby signals from
kinase pathways selectively phosphorylate serine/
threonine residues in the coactivator molecule, and
depending upon the combination of sites phosphorylated, the coactivator is preferentially directed to bind
and activate distinct sets of downstream transcription
factors (46) (Fig. 3). This code represents an elegant
mechanism by which membrane-initiated signaling
pathways can direct limiting quantities of a coactivator
toward their own relevant downstream transcriptional
activators bound to the target promoter DNA. It is the
product of the cellular concentration of coactivator
and the phosphorylation state of coactivator that
drives the potency and the selectivity of coactivators
for target DNA-bound activator proteins (46). In addition to the concentration of coactivators (or corepressors) in the cell, the site-specific phosphorylations on
the coactivator provide a cell-specific input to a coactivator’s activity, and thus to a ligand’s activity.
ROLES OF COACTIVATORS IN PHARMACOLOGY
AND PATHOLOGY
Our understanding of the complex protein interactions
involved in NR functions has been unraveled now to a
point where our current understanding of mechanisms
has provided a major impact in medicine. The early
studies of Baxter and Tomkins on GR mutants verified
their connection to genetic disease predisposition.
The work of Jensen showed the utility of ER analyses
in therapeutic decisions for breast cancer, and Craig
Jordan’s important observations typified tamoxifen as
the primary prototype SERM for breast cancer therapy. Fueled by both basic and clinical research, interest in selective receptor modulators (SRMs) of NRs
because pharmacologic drugs has been explosive.
After the clinical studies on tamoxifen, second generation SERMs have been developed by a number of
pharmaceutical firms and new SRMs for androgens,
glucocorticoids, and progesterone are in early phase
development in biotechnology/pharmaceutical companies. SRMs also have been synthesized recently for
the orphan receptors that are ligand regulatable. The
new information on the molecular mechanisms of action of coactivators also has been rapidly applied to
abnormal physiology. Coactivators for steroid receptors now have been reported to be overexpressed in
many endocrine tumors, after the initial report directed
to AIB1/SRC-3 (47). We found that SRC-3 can cooperate with oncogenes in transformation of normal cells
O’Malley • Perspective
Mol Endocrinol, June 2005, 19(6):1402–1411 1409
Fig. 3. Signaling through the cell from the environment to the genome, can occur by differential phosphorylation of coactivators,
which in turn differentially activate distinct sets of nuclear DNA-binding transcription factors. The figure illustrates our current
hypothesis for the activations of the SRC-3/AIB1 coactivator. In the figure, estrogen, TNF␣, or other stimuli activate distinct kinase
pathways, and differentially phosphorylate SRC-3. The differential phosphorylation selectively directs SRC-3 to form distinct
coactivator complexes with different downstream transcription factors, thereby creating differential gene activations (see Ref. 46).
NF-␬B, Nuclear factor ␬B; CBP, cAMP response element binding protein-binding protein; CoA, coenzyme A; CoA-X and CoA-Y,
any unknown coactivator.
in culture (47), and that our SRC-3 KO mice were
resistant to carcinogenic/oncogenic induction of
mammary cancers (48). In a recent important study
published by Myles Brown (49), overexpression of
AIB1/SRC-3 in transgenic mice was found to result in
spontaneous appearance of breast cancers in older
animals. The totality of available experimental evidence proves this coactivator to be an authentic oncogene. In fact, recent analyses of clinical human
breast tumor samples indicate that AIB1/SRC-3 is
overexpressed along with HER2/neu in a subset of
human breast cancers (50). This cooperative stimulation of SRC-3 expression and phosphorylation leads
to extremely aggressive growth of breast cancers and
is instrumental in predicting those women who will
manifest early resistance to tamoxifen therapy (50).
These results highlight the pathologic importance of
our discoveries of the deleterious synergism between
coactivators (SRC-3) and ligand-independent pathway activators such as HER2/neu, which cause phosphorylation and maximal activation of SRC-3 in the
endocrine cancer cells. Finally, coactivators (and corepressors) are important determinants of normal response to steroid hormones and provide explanations
for variance in tissue-hormone kinetics and sensitivity,
in growth selectivity, and even in phenotypic diversity
among individuals.
In conclusion, I want to emphasize very clearly that
my work over the past 35 yr has been as leader of a
series of small teams of extremely talented faculty
collaborators, postdoctoral fellows, and students. No
modern scientist works without important contributions from others, and the contributions of my trainees
and collaborators cannot be overstated. I am deeply
appreciative of my outstanding scientific colleagues
and my administrative assistants. In no way do I envision myself as other than the spiritual leader of a
talented scientific choir, whose members also became
close and lifelong friends. Although the vast majority of
our hypotheses over the years were correct, they were
wrong in certain instances. I guess the latter can be
expected when one attempts to push the edge of
current knowledge. Importantly, in the field of science
one presents his/her conclusions, and the scientific
body keeps working toward an accurate verification of
the secrets of nature, continually modifying and retooling hypotheses and models until they reach near certainty. To keep things in perspective, I should also
make clear my great admiration for the large and expert group of scientists that work in the field of NRs
and who have made the numerous and immense contributions that have emanated from our field. Their
intelligence, good humor, and friendship have been a
source of both critical feedback and great pleasure to
1410 Mol Endocrinol, June 2005, 19(6):1402–1411
me for the past 35 yr. Most important of all, I am
grateful for the devotion of my wife, Sally, and my four
children (Sally Jr., Bert Jr., Becky, and Erin) and for
their understanding of the considerable demands of
my career in science. All in all, it is these above individuals who have made possible any small successes
that I have achieved in my career.
Acknowledgments
Received November 30, 2004. Accepted December 24,
2004.
Address all correspondence and requests for reprints to:
Bert O’Malley, Professor and Chairman, Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza,
Houston, Texas 77030. E-mail: [email protected].
This work was supported by National Institutes of Health
Grants HD-07857 and HD-08188.
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