527
Novel approaches for the study of vertebrate steroid
hormone receptors
Satomi Kohno,,1 Yoshinao Katsu,† Taisen Iguchi† and Louis J. Guillette Jr
*Department of Zoology, 223 Bartram Hall, University of Florida, Gainesville, FL 32611, USA; †Okazaki Institute for
Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences and Department
of Basic Biology, School of Life Science, Graduate University for Advanced Studies, Okazaki, Aichi 444-8787, Japan
Synopsis Steroid hormones are essential for the normal function of most organ systems in vertebrates. Reproductive
activities in females and males, such as the differentiation, growth and maintenance of the reproductive system, require
signaling by sex steroid hormones. Although extensively studied in mammals and a few fish and bird species, the
evolution and molecular mechanisms associated with the nuclear steroid hormone receptors are still poorly understood in
amphibians and reptiles. Given our interest in environmental signaling of sex determination as well as a major interest in
environmental contaminants that can mimic steroid hormone signaling, we have established an approach to study the
molecular function (ligand binding and trans-activation) of steroid hormone receptors cloned from reptiles. This
approach involves molecular cloning and sequencing of steroid hormone receptors, phylogenic analysis and in vitro transactivation assays using endogenous or exogenous ligands. Comparing the in vitro trans-activation induced by different
ligands with receptors cloned from different species would develop additional functional relationships (classification)
among steroid hormone receptors. This approach can provide insight into understanding why each species could have
different responses to exogenous ligands. Further, we have developed a novel and less invasive approach to obtaining
mRNA for molecular cloning and sequencing of steroid hormone receptors in reptiles and other non-mammalian species,
using blood cells as a source of genetic material. For example, white blood cells (WBCs) and red blood cells (RBCs) of the
American alligator both express steroid hormone receptors and have adequate amounts of mRNA for molecular cloning.
This approach would allow us to analyze components of endocrine function of steroid hormones without sacrificing
animals. Especially in endangered species, this approach could provide an understanding of endocrine functions, elucidate
the phylogenic relationships of various receptors in vitro, such as the steroid hormone receptors, and determine possible
effects of environmental contaminants in a minimally invasive manner.
Introduction
Steroid hormones are essential for the normal
function of most organ systems in vertebrates.
Estrogens regulate ovarian development and differentiation, induce maturation of the female reproductive tract, and stimulate hepatic vitellogenesis in
reptiles. Androgens are involved in testicular development and differentiation, control of spermatogenesis,
maturation of the male reproductive tract and
differentiation of external genitalia (Norris 1980;
Bentley 1998).
The differentiation, growth, and maintenance of the
reproductive system require endocrine signaling by
sex steroid hormones via their receptors. Two different types of receptors for steroid hormones have
been described: those that belong to the super family
of nuclear receptors (Blumberg and Evans 1998;
Bertrand et al. 2004) and those that function as
membrane-linked (extra-nuclear) receptors (Hammes
and Levin 2007). Two subtypes of receptor proteins
have been described to represent the extra-nuclear
version: G-coupled steroid hormone receptors
(Filardo et al. 2000; Zhu et al. 2003; Revankar et al.
2005; Thomas et al. 2006) and membrane-localized
classical steroid hormone receptors (Razandi et al.
2003; Pedram et al. 2007). Nuclear receptors work as
transcription factors regulating mRNA expression,
and have six functionally distinct domains (Krust
et al. 1986). These domains are labeled the A through
F domains with the C and E domains playing essential
roles of DNA binding and ligand binding, respectively
(Fig. 1). The C domain of a steroid receptor is the
DNA-binding domain, which uses a zinc finger motif
to accomplish binding to target DNA. The E domain
From the symposium ‘‘Reptile Genomics and Evolutionary Genetics’’ presented at the annual meeting of the Society for Integrative and
Comparative Biology, January 2–6, 2008, at San Antonio, Texas.
1
E-mail: [email protected]
Integrative and Comparative Biology, volume 48, number 4, pp. 527–534
doi:10.1093/icb/icn080
Advance Access publication August 2, 2008
ß The Author 2008. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved.
For permissions please email: [email protected].
528
is the ligand-binding domain, which is involved in
binding to steroid hormone and recruiting various
transcriptional cofactors to the steroid receptor so
that transcription can occur (Pratt and Toft 1997).
Extra-nuclear receptors for steroid hormones have
been demonstrated and various receptor types have
been cloned. The rapid reactions to steroid hormones
characterizing these membrane receptors, as well as
membrane-localized classical steroid hormone receptors, appear to be induced by G-protein coupling,
which causes post-translational modifications or
phosphorylation (Razandi et al. 2003; Hammes and
Levin 2007; Watson et al. 2007).
Although, extensively studied in mammals and in a
few fish and bird species, the evolution and molecular
mechanisms associated with the nuclear steroid
hormone receptors have been poorly understood,
especially in amphibians and reptiles (Young et al.
1995). For example, it is well accepted that the estrogenic sex steroid hormones play an important role in
sex determination in many non-mammalian and nonavian vertebrates (Crews 2003; Yao and Capel 2005).
Fig. 1 Comparison of amino-acid sequence for each domain of
vertebrates (A) estrogen receptor (ESR1) and (B) androgen
receptor (AR). The DNA-binding (DBD) and ligand-binding
(LBD) domains of both ESR1 and AR are well conserved among
vertebrate species. Numbers indicate present sequence identity
compared to the human sequence. Bar indicates 100 amino acids.
S. Kohno et al.
In mammals and birds, sex determination is dependent on the genotype, XX:XY or ZW:ZZ, at fertilization. However, in some lizards, and turtles and all
crocodilians studied to date, sex determination is
dependent on incubation temperature during a
narrow time period of embryonic development
(Crews 2003). Generally, lower temperatures produce
males whereas higher temperatures produce females
in turtles. In contrast, lower and very high temperatures produce females and intermediate temperatures produce males in crocodilians (Crews 2003). Sex
determination by environmental signals has been
investigated, and the important role of sex steroid
hormones on sex reversal in reptilian sex determination has been established (Crews 2003).
Given this sensitivity of sex determination
to steroid hormone signaling, contamination of the
global environment with chemicals that can mimic
steroid hormones should be a major concern. It has
been well established that some environmental contaminants can act as agonist or antagonist ligands on
various steroid hormone receptors or they can alter
the synthesis/degradation of endogenous hormones
(Tyler et al. 1998; Guillette and Gunderson 2001;
Milnes et al. 2006). Several studies suggest that various
contaminants can directly act on steroid hormone
receptors as an agonist or antagonist (Rooney and
Guillette 2000; Gray et al. 2002). Indeed, endocrine
alterations which result from developmental exposure
to endocrine disruptive contaminants has been
observed in a number of wildlife species, including
turtles (Willingham et al. 2000) and the American
alligator (Guillette et al. 2000; Guillette et al. 2007).
Exposure to various contaminants with estrogenic
potential during development alters sex determination
and gonadal steroidogenesis in freshwater turtles
(Willingham and Crews 1999; Willingham et al.
2000) and the American alligator (Crain et al. 1997).
Further, recent studies have documented altered
expression of estrogen receptor (ESR2) mRNA in
gonadal tissue from juvenile American alligators
caught in Lake Apopka (FL, USA) (Kohno et al.
2008). This lake is polluted with various endocrine
disruptive contaminants, some of which have been
shown to be estrogenic (Vonier et al. 1996; Guillette
et al. 2002). Male gonadal tissue from animals
obtained at Lake Apopka exhibited elevated expression of ESR2 mRNA when compared to animals of
similar age and size from a reference population
(Kohno et al. 2008). The altered expression and
functioning of ESR2 might be important in explaining
the gonadal alterations induced by contaminants in
the American alligator as previous studies have
suggested that this receptor type is reactive with a
Vertebrate steroid hormone receptors
wide array of exogeneous estrogenic chemicals
(Kuiper et al. 1996). Therefore, functional details of
steroid hormone receptors in wildlife, especially in
reptiles, need to be investigated. Here, we summarize
several novel approaches to studying the molecular
function (ligand binding and trans-activation) of
steroid hormone receptors cloned from reptiles and
fishes.
Two different trans-activation assays
for examining steroid receptors in vitro
Reporter gene assays using luciferase in mammalian
cell lines are commonly used to characterize transactivation by steroid hormone receptors. Generally,
the cells are transfected with plasmids including
cDNA for a steroid hormone receptor. The reporter
plasmids, which have a specific sequence of a hormone response element (HRE) and luciferase reporter
cDNA, are also transfected into the cells (Fig. 2).
Following ligand (steroid hormone or test chemical)
binding to the receptor, the receptor–ligand complex
interacts with the HRE, inducing the transcription of
luciferase cDNA. As a result, the reporter protein,
luciferase, accumulates in the cells and the luminescence signal increases in a dose-dependent manner
(Fig 2). However, these results would be somewhat
limited as they indicate potential trans-activation
using only a canonical HRE. Generally, investigators
529
use a common sequence of HRE for analysis of steroid hormone receptors cloned from any vertebrate,
although it’s rare to find canonical HREs in the
promoter region (O’Lone et al. 2004). The canonical
HREs do not produce problems in the trans-activation assay; however, each steroid hormone receptor
from different species could have differing responses
to the canonical HRE given the variation in aminoacid sequence and folding of each unique receptor.
Therefore, we have added to this approach a new
assay, the modified GAL4 system that does not require
DNA recognition of an HRE. To eliminate this
dependence on the canonical sequence of the HRE,
we have developed a novel reporter–gene assay using a
modified GAL4 system (Promega, Madison, WI) that
is traditionally used in the two-hybrid system for
mammalian cells (Katsu et al. 2006a). The cells are
transfected with the expression construct for a steroid
hormone receptor-GAL4 fusion protein. Following
ligand binding to the receptor, and if it can induce
trans-activation, the receptor-GAL4 fusion protein
interacts with the GAL4 binding site and activates
transcription. As a result, luciferase accumulates in the
cell and a luciferase signal increases with the dose of
the ligand (Fig. 2). The modified GAL4 system, using
red-belly turtle (Pseudemys nelsoni) ESR1, for example, exhibits a 10-fold higher induction with estradiol17 when compared to the HRE-Luciferase System;
however, the sensitivity between the two assay
Fig. 2 Two different systems utilized for trans-activation assays with luciferase for analysis of steroid hormone receptors: a typical
luciferase assay using the HRE (left); a novel luciferase assay using the modified GAL4 system.
530
systems, measured as an EC50 for example, is not
different (Katsu et al. 2008). The two assay systems use
different mammalian cell lines, as HEK 293 or Hep G2
were used for the trans-activation assay with HRE of
ESR1 or AR, whereas CHO-K1 cells were used for the
GAL4 trans-activation assay. This difference in the
fold induction of luciferase induced by the various
tested ligands could be caused by different regulatory
sequences of the luciferase reporter cDNA (HRE
versus GAL4) or by differing cell lines (HEK293 or
Hep G2 cells for HRE; CHO-K1 for GAL4). Moreover,
this new system, as described above, is completely free
of HRE sequence issues. However, one potential issue
with this system relates to the effect of fusing the
steroid hormone receptor and GAL4 protein on transactivation. Although both systems need to recruit
cofactors for trans-activation, recruiting the cofactors
could be a potential problem with the steroid
hormone receptor-GAL4 fusion protein. The transfected plasmids of the luciferase reporter cDNA have
only a limited regulatory region such as the HRE or
the GAL4 binding site to express luciferase and don’t
have AP1 or Sp1 sites. Therefore, it is possible to miss
potential inductions of trans-activation via action
of membrane-localized, classical, steroid-hormone
receptors, or other transcription factors working
with steroid–hormone receptors, such as Sp1 or AP1.
Estrogen receptor (ER ; ESR1)
Phylogenic analysis of ESR1 amino-acid sequences
clearly has shown that crocodilian estrogen receptors
are a sister group of Aves and form an archosaurian
clad (Katsu et al. 2004; Katsu et al. 2006b). Red-belly
turtle (P. nelsoni) is in an immediate out-group of
Archosauria with the most distinct group of squamates (lizards and snakes) when birds and reptiles are
S. Kohno et al.
compared (Katsu et al. 2008). Ten ESR1 sequences
have been cloned from Sauropsida (birds and reptiles);
however, to date, only four ESR1 sequences have
been characterized by trans-activation. The DNAbinding and ligand-binding domains of ESR1 are well
conserved among vertebrate species compared with
amino-acid sequences (Fig. 1A). Specifically, the DNAbinding domain is highly conserved with 94–100%
identity among vertebrates. This conservation of
sequence suggests that ESR1 could regulate similar
target genes among vertebrate species. However, this
needs to be tested and further clarification is possible,
once trans-activation data are available, from assays of
ESR1 action as described above. Similarly, the ligandbinding domain is well conserved, but it does not
show the same very high degree of sequence similarity
as seen in the DNA-binding domain (Fig. 1A). This
indicates that ESR1 could potentially bind to a wide
range of ligands with species specificity.
Trans-activation assays using red-belly turtle
(P. nelsoni) and roach (Rutilus rutilus) ESR1 revealed
that estradiol-17 (E2), ethynylestradiol (EE2), and
diethylstilbestrol (DES) have higher potentials than
estrone (E1) or estriol (E3), whereas DES showed
higher potentials than E2 in mosquitofish (Gambusia
affinis affinis) (Fig. 3). As a unique way of examining
steroid hormone receptor activity, we performed a
cluster analysis using each EC50 in the trans-activation
assay of ESR1 (Fig. 5A). This analysis showed that
mosquitofish ESR1 has more similar characteristics to
red-belly turtle ESR1 than to roach ESR1, as ligands
were classified into two groups by their characteristics
of trans-activation; that is, group of E2, EE2, and DES
and group of E1 and E2 (Fig. 5A). This initial analysis
is small as it used just three species and five ligands,
but is promising in that it could provide important
Fig. 3 The trans-activation assay on (A) red-belly turtle, (B) mosquitofish, and (C) roach estrogen receptor (ESR1). Red-belly and
roach ESR1 revealed that E2, EE2, and DES have higher potentials than does E1 or E3, whereas DES showed higher potentials than did
E2 in mosquitofish ESR1. Charts were modified from publications by Katsu et al. (2007a, 2007b, 2008).
531
Vertebrate steroid hormone receptors
predictive information about species risk to various
environmental contaminants.
Androgen receptor (AR)
A recent phylogenic analysis of AR amino-acid
sequences in reptiles and birds has been presented
(Katsu et al. 2008). Phylogenic relationships for the
ARs, based on molecular sequences, were similar to
those observed for ESR1. There are only six sequences
of full-length AR cloned from Sauropsida in the
database, and only three receptors have been characterized by trans-activation. The DNA-binding and
ligand-binding domains of AR are well conserved
among vertebrates (Fig. 1B). Trans-activation of the
red-belly turtle AR with a variety of endogenous
(DHT, 5-dihydrotestosterone; T, testosterone; 11-KT,
11-ketotestosterone) and pharmaceutical androgens
(MT, 17-methyltestosterone; Tren, trenbolone)
revealed the following order in trans-activation potential: DHT ¼ MT ¼ Tren4T (Figs. 4 and 5B).
Mosquitofish (G. affinis affinis), like other teleost fish
studied to date, have two ARs: type-1 and type-2 (Katsu
et al. 2007a). Trans-activation on both types of
mosquitofish AR revealed a similar pattern; that is,
MT had the highest potential, whereas Tren ¼
11-KT4DHT ¼ T (Figs. 4 and 5B). However, cluster
analysis of ARs using their EC50 for different ligands
revealed a different relationship than expressed by the
phylogeny based on amino-acid sequences (Fig. 5B).
On the other hand, DHT and T showed different
characteristic from MT, Tren and 11-KT on transactivation of AR in the cluster analysis of ligands
(Fig. 5B).
Although more data, both from additional species
and ligands, are needed to obtain better estimates
of classifications, these results indicate that the
functional characteristics of recombinant protein
could be a great source of data to estimate novel
physiological responses that depend on the functions
of cloned proteins. Considering both sequence-based
and functional-characteristics-based clustering or
classification could provide help in understanding
not only endogenous endocrine systems, but also
be used to develop risk assessments for vertebrate
species exposed to a wide array of endocrine-altering
environmental contaminants.
A novel approach for cDNA cloning
and analysis of steroid receptors
Most non-mammalian species, including alligators,
have RBCs with a functional nucleus and organelles
similar to other cells (Kregenow 1977); thus, RBCs
likely regulate mRNA expression as do other type of
cells. Therefore, both WBCs and RBCs are likely to
respond to endogenous/exogenous environmental
factors at the mRNA level. They could be a potential
source of data on environmental perturbation and
have enough mRNA to analyze by current molecular
biological techniques such as cDNA cloning or quantitative real-time PCR. Collection of blood samples
and analysis of plasma are very common techniques,
even for endangered species. If the investigators
saved the blood cells properly after they transfer the
plasma in any non-mammalian species, including
endangered species, a component of the endogenous
endocrine system could be studied. This could
open various directions in research, such as comparing mRNA expression in blood cells with hormone
concentrations in the plasma or comparing alterations
in mRNA expression over bleeds. Indeed, in the
American alligator, stress from constraint increased
the concentrations of corticosterone and glucose
Fig. 4 The trans-activation assay on (A) red-belly turtle, (B) mosquitofish type-1, and (C) type-2 androgen receptor (AR). Charts were
modified from publications by Katsu et al. (2007a, 2007b, 2008).
532
S. Kohno et al.
Fig. 5 Cluster analysis of EC50 on trans-activation assay of (A) estrogen receptor alpha (ESR1) and (B) AR. Cluster was calculated by
Agglomerative Nesting (v1.0.2) in Free Statistics Software (v1.1.22-r6) using the Ward method (Wessa 2008). On both analyses, redbelly turtle ESR1 or ANDR revealed similarities to roach or mosquitofish type-1 more than to other teleosts, respectively. These
relationships were different from a strict phylogeny based on amino-acid sequences. Table of EC50 was modified from the tables in the
publications by Katsu et al. (2007a, 2007b, 2008). E2, estradiol-17; DES, diethylstilbestrol; EE2, ethinyleestradiol.
in plasma. Glucocorticoid receptor (GR) mRNA in
whole blood cells collected from the same blood was
detectable by quantitative real-time PCR, and GR
mRNA was slightly induced at 8 h after stress from
restraint (Kohno and Guillette, unpublished data).
Further, the blood cells expressed enough mRNA
to clone many of the steroid hormone receptors
(e.g., ESR1, GR or AR). These results indicate that it
would be possible to obtain and analyze the transactivation of steroid hormone receptors cloned from
blood cells of endangered species and further allow
determination of whether a given species is at greater
risk to environmental contaminants with agonistic
or antagonistic steroid hormone potential. Such a
study is in its initial stages for the Japanese Giant
Salamander (Andrias japonicus), a national monument of Japan and a highly endangered species (Katsu
et al. 2006a).
Conclusion
Two novel applications for cDNA cloning and transactivation assays could make the functional analysis
of steroid hormone receptors from endangered species possible. Cluster analysis, using ligand-receptor
trans-activities is likely to provide new functional
aspects of receptor-ligand interactions by grouping
similar receptor function and/or ligand potential.
Furthermore, it might be possible to estimate effects
of, and risk to environmental contaminants via
nuclear steroid hormone receptors on many vertebrate
species, including threatened or endangered species.
Funding
This work was supported in part by grants to LJG (UF
Opportunity Fund; NIEHS R21 ES014053-01A1;
HHMI Professor Program) and TI (Core Research
for Evolutionary Science and Technology, Japan
Science and Technology; Grant-in-Aid for Scientific
Research from the Ministry of Education, Science,
Sports and Culture of Japan; grants from the Ministry
of Environment, Japan).
Acknowledgments
We thank Brandon C. Moore, Heather J. Hamlin,
and Thea M. Edward, Department of Zoology,
University of Florida, for their critical reading of
the manuscript.
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