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Department of Nutritional Sciences, University of Connecticut, Storrs, CT 06269-4017
4To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • zinc • thyroid hormone • retinoic acid • growth hormone • diethylenetriaminepenta-acetic acid • rat pituitary tumor cells
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,
Mangelsdorf et al. 1995
). Although some differences
exist between these receptors, they share a fundamental mechanism of
action. Binding of the appropriate hormone/nutrient/ligand to the
receptor leads to a change in the interaction of the receptor with
specific recognition sequences known as hormone response elements in
target genes. This differential interaction is communicated to the
basal transcriptional apparatus, leading to a change in the rate at
which the gene is transcribed. An important feature of these receptors
is a highly conserved DNA binding region, which is crucial for
recognition of and binding to the hormone-response elements
(Evans 1988
). The DNA binding region contains nine
invariant cysteine residues, eight of which are thought to chelate two
atoms of zinc. It has been proposed that zinc is necessary for the
folding of the receptors into a conformation appropriate for DNA
binding (Berg 1990
, Evans 1988
). This
would predict a requirement for zinc for the functioning of these
nuclear receptors and their ligands. The evidence to back up this
sequence-based prediction is variable and probably best for the
glucocorticoid receptor. Bacterially expressed glucocorticoid receptors
have been shown to contain zinc (Freedman et al. 1988
).
Glucocorticoid receptors in which crucial cysteine residues have been
altered are inactive (Severne et al. 1988
). For the
thyroid hormone receptor, removal of zinc in vitro prevents receptor
binding to DNA (Miyamoto et al. 1991
).
Numerous
proteins, notably enzymes and transcription factors, bind zinc and are
thought to be dependent upon zinc for function (Vallee and Falchuk 1993
). The consequences of zinc deficiency are well
known in both humans and experimental animals and include failure of
growth and development (Brandao-Neto et al. 1995
,
Prasad 1991
). The linkage between this physiological
picture of deficiency and loss of function of specific
zinc-dependent proteins has been hard to establish. Since ligands
acting through the nuclear receptor pathway are also important
regulators of growth and development, one possibility is that zinc
deficiency causes loss of the mineral from nuclear receptors and
therefore loss of function of a number of hormones and nutrients.
We sought to test this hypothesis using a well-characterized cell
culture model for thyroid hormone action. GH3, rat pituitary tumor
cells, express growth hormone (GH)5
and this process is
regulated by the active thyroid hormone, triiodothyronine (T3) at the
transcriptional level (Samuels et al. 1988
). Much of the
early work on thyroid hormone receptors and mechanism of action
utilized this or closely related cell lines. Thus the GH gene promoter
and its regulation, particularly regarding T3, are well described
(Brent et al. 1991
, Force and Spindler 1994
). We incubated GH3 cells with the membrane-impermeable
chelator, diethylenetriaminepenta-acetic acid (DTPA) to determine
its effects on the T3-signaling pathway (Chattopadhyay and Freake 1998
). In the absence of T3, DTPA had little effect on
GH mRNA levels. However, when cells were treated with T3, addition of
DTPA led to a surprising but consistent enhancement of GH expression.
The effect was half-maximal at about 50 µmol/L of DTPA, where a
3–5-fold increase in GH mRNA levels was observed. Coincubation of
cells with equimolar concentrations of zinc prevented the effect of
DTPA though equivalent concentrations of other cations were
ineffective. These results are the opposite from what might be
predicted from the model outlined above.
In the studies reported here we have sought to extend these
observations. We have taken advantage of the fact that the GH promoter
is also regulated by all-trans and 9-cis retinoic
acid (Davis et al. 1994
, Garcia-Villarba et al. 1993
, Morita et al. 1989
, Sugawara et al. 1994
) to test its response to these agents in the presence of
DTPA. In addition, we have used a second chelator and performed time
course and reversibility experiments to further probe the mechanisms
whereby chelation of zinc enhances thyroid hormone action.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell culture.
GH3 cells were obtained from the American Type Culture Collection
(ATCC, Rockville, MD). They were grown in serial monolayer culture in
Ham’s F-10 nutrient mixture supplemented with horse serum (15%),
newborn calf serum (2.5%) and antibiotics (penicillin 5 x 104 IU/L, streptomycin 50 mg/L) in a humidified atmosphere
of 95% air-5% CO2 at 37°C. Forty-eight hours prior
to experimentation, the media was replaced with Ham’s F-10 nutrient
mixture supplemented with 10% newborn calf serum, depleted of
endogenous hormones by incubation with an ion-exchange resin
(Samuels et al. 1979
). The concentration of zinc in
Ham’s F10 medium is 0.1 µmol/L. Thus most of the zinc within the
system comes from the calf serum, which, at the dilution used, will
give a final concentration in the media of about 5 µmol/L. The cells
were treated with combinations of T3, all-trans retinoic
acid or 9-cis retinoic acid, DTPA and zinc as described
in the individual experiments. After the time periods shown (usually
48 h, except for the time-course and reversibility
experiments), cells were lysed and RNA was extracted. For the
time-course studies, after the depletion period, T3 (10 nmol/L) was
added to all cells 48 h prior to RNA extraction. DTPA (50
µmol/L) was added either simultaneously, as in the standard protocol,
or at various times thereafter such that the cells were exposed to
chelator for the final 12 to 48 h, depending on treatment group.
In a further experiment, cells were exposed to T3 for an additional
48 h, prior to the addition of any chelator, to ensure that any
delay observed in the effects of DTPA was not due to a lag in the
action of T3. To test the ability of zinc to reverse the effects of
DTPA, cells were treated with both T3 and DTPA for 48 h to induce
GH mRNA expression. The medium was replaced and the incubation
continued with T3 and DTPA, in the presence or absence of 40 µmol/L
of zinc. Cells were lysed and RNA extracted at various time points in
the subsequent 6 to 48 h.
RNA extraction and northern analysis.
Total RNA was extracted from each 25 cm2 flask by the
modified Peppel/Baglioni method (Salvatori et al. 1992
)
with slight modifications as reported earlier (Chattopadhyay and Freake 1998
). The RNA was size-separated in 1% agarose-6%
formaldehyde denaturing gels and transferred onto nitrocellulose
membranes (MSI NitroPure, 0.45 µm, Westborough, MA). After UV
cross-linking and baking, the membranes were hybridized separately
with radiolabeled GH, metallothionein-1 (MT-1) and RPL32 cDNA probes. A
750 bp rGH insert (Seeburg et al. 1977
) was isolated
from pGEM3Z using Pst 1. Mouse MT-1 was a kind gift from Dr. Richard
Palmiter, University of Washington, Seattle. RPL32 was provided by Dr.
Mary McGrane, University of Connecticut. [32P]-labeled
cDNA probes of high specific activity were synthesized using a random
primer labeling kit (Life Technologies, Rockville, MD). Hybridization
was carried out overnight at 42° C, as previously described
(Chattopadhyay and Freake 1998
). After posthybridization
washes, the membranes were autoradiographed at -80°C with an
intensifying screen, and the resulting images were quantified using
Molecular Analyst software for the GS-670 densitometer (Bio-Rad,
Hercules, CA). The mRNA levels are expressed as the ratio between
densitometric signals measured for GH and RPL32.
Data analysis.
Each observation is derived from an independent RNA preparation from an individual flask of cells. Each treatment employed two to three replicates, and each experiment was repeated two to four times. ANOVA, followed by Scheffe’s test, was used to determine significant differences between treatment groups (Statview 4.01; Abacus Concepts, Berkeley, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Time-course experiments were performed to discover the period of
exposure to the chelator required for this stimulation to be observed.
Treatment with DTPA for the final 12 h had no effect on GH mRNA
levels (Fig. 2
). A small increase in GH mRNA expression may have occurred after 24-h
exposure (P = 0.1), though significant increases were
only found at 48 h, at which point a 3.5-fold induction was seen.
This delay was not shortened when cells were pretreated with T3 for
48 h prior to the beginning of the experiment, to ensure maximal
T3 effects throughout the time course (Fig. 2)
.
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, it was of interest to determine whether subsequent addition
of zinc could reverse the effects of the chelator. Therefore, cells
were pretreated with DTPA plus T3 for 48 h to induce GH mRNA.
These cells had 14-fold higher levels of GH mRNA in comparison to those
not exposed to chelator or T3 (2.79 ± 0.15 vs. 0.18 ± 0.02
GH mRNA/RPL32 mRNA). The incubation was then continued in fresh medium
with DTPA and T3, with or without 40 µmol/L of zinc, and GH mRNA
levels assessed from 6 to 48 h later. In cells not given
additional zinc, GH mRNA continued to accumulate, such that after
48 h a further 1.2-fold increase was observed (P
< 0.01, Fig. 3
). In the presence of zinc, GH expression did not change significantly
over the first 24 h. GH mRNA levels then dropped back such that
after 48 h they were 53% of 0 time cells or 25% of cells
incubated for the same length of time in the absence of zinc. Thus, the
effects of DTPA were reversed by subsequent addition of zinc.
|
). A significant increase (P < 0.01) was not
observed until 200 µmol/L of EDTA was used and this concentration was
still less effective than 50 µmol/L of DTPA. In common with DTPA,
EDTA had no effects on GH mRNA in the absence of T3.
|
). Addition of DTPA further enhanced the all-trans retinoic
acid effect by about 50% (P < 0.01). Simultaneous
addition of zinc resulted in intermediate GH mRNA levels, which were
not significantly different from cells treated with retinoic acid
alone. Treatment with both T3 and all-trans retinoic acid
resulted in greater levels of GH mRNA than when either was used
independently (P < 0.001). This stimulation was
further enhanced by DTPA (70%, P < 0.001), but not
when additional zinc was added.
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).DTPA increased expression in the presence of 9-cis retinoic
acid (P < 0.005), though not if exogenous zinc was
added simultaneously. Addition of both T3 and 9-cis retinoic
acid stimulated GH mRNA expression, in comparison to either one by
itself (P < 0.005). This effect was amplified about
100% by DTPA (P < 0.001), but not in the presence of
additional zinc.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Previously, we showed that the
DTPA stimulation of T3-induced GH mRNA was uniquely and specifically
reversed by zinc out of seven divalent cations tested
(Chattopadhyay and Freake 1998
). These agents,
therefore, appear to be acting through their chelating ability, rather
than in some nonspecific manner.
It is important to note that the effects of DTPA on the cell and on the
GH expression pathway are reversible. Following induction of GH mRNA by
DTPA, subsequent addition of zinc reduces these mRNA levels. This
argues against a toxic and generalized effect of zinc removal on the
cells. There are two further aspects of the specificity of these
effects that are worth noting. The first is that all mRNA results have
been standardized to the level of RPL32 in the same samples. Thus, this
does not represent a generalized effect of DTPA on gene expression.
Secondly, in the absence of T3 (or retinoic acid), chelation by DTPA or
EDTA has very minimal effects on GH mRNA levels (Chattopadhyay and Freake 1998
, Fig. 4
). Removal of zinc appears to be
impacting, or interacting with, the nuclear receptor signaling pathway.
More recently it has been shown that in addition to T3, both
all-trans retinoic acid and 9-cis retinoic acid
enhance GH gene expression (Davis et al. 1994
,
Garcia-Villarba et al. 1993
, Morita et al. 1989
, Sugawara et al. 1994
). Consistent with
these authors, we found that both retinoids had independent effects on
GH mRNA which were additive to those of T3. DTPA amplifies the
inductions by all-trans retinoic acid and 9-cis
retinoic acid, both in the absence and presence of T3, but not when
additional zinc was added. Similar effects were also observed with
dexamethasone (Chattopadhyay and Freake 1998
). Thus, it
appears that the stimulatory effect of zinc chelation on GH gene
expression is not confined to the T3 signal transduction pathway.
All-trans retinoic acid operates through the retinoic acid
receptor (Mangelsdorf et al. 1995
). Its steroisomer,
9-cis retinoic acid can bind to both retinoic acid receptors
and retinoid X receptors. In the present study, very similar effects
were observed with both stereoisomers. The possibility of isomerization
was not investigated, and it is unclear through which receptor these
retinoids are operating in this particular instance. However, Davis et al. (1994)
used a retinoid X receptor-specific agonist to
demonstrate that this receptor is capable of mediating the
9-cis retinoic acid effect on GH gene expression. Thus, it
appears likely that the functions of a number of nuclear receptors in
this model system are positively affected in conditions of zinc
restriction.
While a clear understanding of the mechanism whereby zinc removal
enhances signaling through the thyroid and retinoid signaling pathways
in GH3 cells is not available at this time, some possibilities can be
excluded. There are reports that zinc inhibits T3 binding to its
nuclear receptors (Lu et al. 1990
, Surks et al. 1989
). However, other studies found no effect of zinc on T3
binding to receptors (Lin and Cheng 1991
,
Miyamoto et al. 1991
, Zhu et al. 1994
).
In GH3 cells, using a whole cell binding assay, we found no effect of
either zinc or DTPA on receptor binding of T3 (Chattopadhyay and Freake 1998
). Therefore, metal-induced inhibition of
hormone binding to receptor does not appear to be responsible for the
stimulation by DTPA of hormonal induction of the GH gene in GH3 cells.
It also appears unlikely that zinc inhibits nuclear receptor binding to
DNA. Zinc has been shown to be required for the function of a number of
"zinc-finger" transcription factors, such as TFIIIA and Sp1
(Westin and Schaffner 1988
). Zinc has also been
demonstrated to be required for proper folding of bacterially expressed
human thyroid hormone receptor and its binding to target DNA
(Miyamoto et al. 1991
). It has also been shown for the
glucocorticoid receptor that preventing zinc binding, by mutating
cysteine residues in the DNA binding region of the receptor, results in
an inhibition of receptor/response element binding and target gene
transactivation (Severne et al. 1988
). It is hard to
imagine, given our existing understanding about the nuclear hormone
receptors, that zinc removal, which is likely to dissemble
zinc-coordinated structures, would also potentiate their DNA
binding.
Is also seems unlikely that these effects are mediated by
metallothioneins (MT). MT appear to be capable of removing zinc from or
donating it to other proteins (Maret et al. 1997
,
Zeng et al. 1991
), making it possible that changing MT
levels could influence the availability of zinc for these receptors.
However, MT-1 mRNA levels in GH3 cells were below detection limits,
unless the cells were stressed with high levels of zinc (data not
shown). This is consistent with a previous report documenting low MT
gene expression in rat brain (Choudhuri et al. 1993
).
Incubation with additional zinc, the circumstance under which MT
appears to be expressed, led to only minor reductions of GH mRNA
(Chattopadhyay and Freake 1998
). Most effects on GH mRNA
were seen when the already low levels of zinc in the media were further
reduced by chelation. MT mRNA could not be detected under either of
these conditions, making it unlikely that substantial changes in MT
protein were occurring.
A prolonged period of time is required for DTPA effects on GH mRNA levels to be observed. About 24 h also appears to be required before those effects can be reversed by addition of zinc. It seems unlikely that such lengthy times would be required for the chelator to deplete intracellular zinc levels, and so the delay is more likely attributable to zinc-sensitive biological processes. For example, removal of zinc with DTPA might lead to inhibition of transcription of a gene with a zinc-sensitive promoter. The protein product of this gene would play an inhibitory role in the T3 and retinoic acid stimulation of GH gene expression. Alternatively, zinc chelation might increase production of a gene product whose transcription is repressed by zinc. This product would then play a stimulatory role in the induction of GH expression by T3 and RA.
In last few years, the role of coregulators in silencing and
potentiating T3 and retinoic acid induction of expression of target
genes has widened our understanding about the molecular mechanism of
action of nuclear receptors (Horwitz et al. 1996
).
Corepressors bind with unliganded receptors and inhibit activity of the
transcriptional apparatus. Binding of ligand causes dissociation of
corepressor and recruitment of coactivator, which stimulates
transcription. It is not known whether the amount or activity of any of
these coregulators is in any way dependent on zinc, but they are good
candidates for further investigation of the mechanisms underlying the
effects of DTPA.
There are mammalian proteins whose synthesis is known to be sensitive
to zinc (Falchuk 1993
). Apart from MT, which are
increased by zinc, a chromatin protein, possibly a histone variant, is
induced by zinc deficiency (Falchuk 1993
). This
observation is important because there are reports that chromatin
structural proteins affect transcriptional regulation (Allfrey 1977
). Histone acetylation influences T3 and retinoic
acid-induced GH mRNA levels in GH4C1 cells, a related strain of rat
pituitary tumor cells (Garcia-Villalba et al. 1997
).
Future studies should address the possibility that zinc deficiency as
caused by DTPA alters the content or acetylation status of chromatin
proteins thereby influencing the expression of the GH gene.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 This work was supported by a USDA-NRI grant (94–37200-1084) to HCF.
3 Chattopadhyay contributed equally to this work and should be considered joint first author. Current address: Department of Biochemistry & Molecular Biology. Mount Sinai School of Medicine.
5 Abbreviations used: DTPA, diethylenetriaminepenta-acetic acid; GH, growth hormone; MT, metallothionein; T3, triiodo-L-thyronine.
Manuscript received August 12, 1999. Initial review completed September 7, 1999. Revision accepted October 19, 1999.
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