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Department of Internal Medicine III, Erasmus University Medical School (S.V.d.G., J.P.S., E.K., T.J.V.), 3000 DR Rotterdam, The Netherlands; Laboratory of Comparative Endocrinology, K. U. Leuven (S.V.d.G., V.M.D., E.R.K.), 3000 Leuven, Belgium; and the Department of Nuclear Medicine, University of Massachusetts Medical Center (J.L.L.), Worcester, Massachusetts 01655
Address all correspondence and requests for reprints to: Theo J. Visser, Ph.D., Department of Internal Medicine III, Erasmus University Medical School, Room Bd 234, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. E-mail: visser{at}inw3.azr.nl
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is well known that thyroid hormone plays an important role in many crucial developmental events in all vertebrate classes. In birds, this hormone is essential for yolk sac retraction, functional maturation of the lungs, pipping (penetration of the air sac), and hatching (15). Decuypere et al. (16, 17) showed that plasma T3 increases dramatically at the moment of pipping, when the embryo switches from allantoic to lung respiration. This is correlated with a decrease in hepatic D3 activity rather than with an increased D1 activity, suggesting that the peak in plasma T3 at the end of incubation is caused by a decrease in its hepatic breakdown (18). Later studies showed that the increase in plasma GH, also observed at the end of incubation, plays an important role in the suppression of hepatic D3 activity (19, 20).
In this study we generated complementary DNA (cDNA) fragments by reverse transcription-PCR (RT-PCR) of polyadenylated RNA from embryonic day 17 (E17) chicken liver using primers based on the conserved amino acid sequences NFGSCTSecP and YIEEAH. Using these RT-PCR products as probes, we isolated two cDNA clones from an E17 chicken liver cDNA library, each of which appears to code for a Sec-containing protein. One clone shows highest sequence homology with mammalian D1 cDNAs and expresses characteristic D1 activity after transfection into COS-1 cells. The other clone, which lacks approximately the first 16 codons, shows high homology with mammalian and amphibian D3 cDNAs. These two cDNA clones were used in the study of D1 and D3 expression in chicken liver during embryonic development.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]deoxy-ATP were purchased from Amersham
(Aylesbury, UK); polyethylene glycol (PEG6000) was obtained from Merck
(Hohenbrunn, Germany); diethylaminoethyl-dextran and Sephadex LH-20
were purchased from Pharmacia. Nonradioactive iodothyronines were
obtained from Henning Berlin R&D (Berlin, Germany),
[3',5'-125I]T4 (
1200 Ci/mmol)
was obtained from Amersham, and
[3,5-125I]T3 (35 Ci/mmol) was
obtained from Mr. R. Thoma (Formula, Berlin, Germany) courtesy of Dr.
G. Decker (Henning Berlin).
[3',5'-125I]rT3 (2000 Ci/mmol)
and [3,5-125I]T3 sulfate
([3,5-125I]T3S) were prepared in our
laboratory as described previously (21, 22).
6-n-Propyl-2-thiouracil (PTU), iodoacetate (IAc), gold
thioglucose (GTG), dithiothreitol (DTT), and chloroquine were obtained
from Sigma Chemical Co. (St. Louis, MO). All other reagents were of the
highest purity commercially available.
Animals
Fertilized chicken eggs from a rapidly growing broiler strain
(Hybro) were purchased from Euribrid (Aarschot, Belgium) and incubated
in a laboratory incubator as described previously (18). The start of
incubation was defined as E1. Animals were killed between day 14 (E14)
and day 20 (E20) of embryonic development, on day 21 just after
hatching (C0), and 1 day after hatching (C1). Of the animals killed on
E20, approximately half were in the nonpipping stage, and the other
half were in the internal pipping stage. Livers were isolated, frozen
in liquid nitrogen, pooled, and stored at -80 C until further
processing. Livers were pooled from seven (E14), five (E15), four
(E16), three (E17), and two (E18–C2) animals. Blood was collected by
cardiac puncture (embryos) or decapitation (chicks). Plasma pools from
two animals (E14 and E15) or individual plasma samples (E16–C2) were
stored at -20 C until hormone analysis. The experimental protocol was
approved by the ethical experimental animal committee of the K. U.
Leuven.
Cloning
Total RNA was isolated from embryonic chicken liver (ECL) by
homogenization of tissue in TRIzol reagent, and polyadenylated RNA was
isolated on oligo(dT)-cellulose. Oligo(dT)-primed cDNA was obtained
using AMV reverse transcriptase. PCR was performed using the upstream
primer 5'-AATTTTGGCAGTTGTACCTGACC-3', the downstream primer
5'-RTGIGCTTCCTCIATGTA-3', and SuperTaq DNA polymerase. The products
were isolated and ligated into pCR-II. Two different clones were
isolated and sequenced, showing that the inserts were homologous to
known deiodinases. An E17 chicken liver cDNA library was constructed in
Lambda ZAP-Express (Stratagene, La Jolla, CA). The library was blotted
on Hybond-N+ and screened with the RT-PCR products labeled
by extension of the PCR primers using Klenow DNA polymerase in the
presence of [-32P]deoxy-ATP. The phagemids carried in
selected positive bacteriophages were excised, generating cDNA clones
in pBK-CMV. cDNA inserts were then sequenced manually and by automatic
sequencing in both directions using the dideoxy method of Sanger
et al. (23).
Transient transfection
The cDNA encoding chicken D1 (ECL1711) was subcloned from
pBK-CMV into pCI-Neo using EcoRI and NotI. Both
clones isolated (ECL1711 and ECL1715) were 5'-truncated based on
alignment with known deiodinases from other vertebrates (2, 3, 5, 6, 8, 9, 10, 12), with coding sequences that presumably lacked the first 5
and 46 nucleotides, respectively. A eukaryotic translation start site
(24) was engineered in ECL1711 at the EcoRI restriction site
by site-directed mutagenesis using the oligonucleotide
5'-GCTAGCCTCGAGAAATGGGCACGAGGTTGA-3' and the MORPH kit
(5Prime
3Prime, Boulder, CO), yielding the mutant ECL1711M. This
mutation, which was confirmed by sequencing, created an ATG start codon
in a Kozak consensus sequence (24), and resulted in a four-amino acid
(MGTR) addition to the N-terminus of the amino acid sequence encoded by
the cDNA (see Fig. 1
). RNA secondary
structure prediction was performed using the MFOLD program provided by
Dr. M. Zuker (Institute for Biomedical Computing, Washington
University, St. Louis, MO) on the Internet
(http:/www.ibc.wustl.edu/zuker) (25).
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Northern analysis
Total tissue RNA (20 µg/lane) was separated on 1% (wt/vol)
formaldehyde-agarose gels and blotted onto Hybond-N membranes. Blots
were hybridized at 60 C with random hexamer-primed
32P-labeled cDNA probes in 6 x SSC (standard saline
citrate), 0.5% SDS, 5 x Denhardt’s solution (1X Denhardt’s
solution = 0.2% Ficoll, 0.2% BSA, and 0.2% polyvinylpyrrolidone),
and denatured salmon sperm DNA (100 µg/ml). Blots were washed twice
for 15 min each time at 55 C with 3 x SSC-0.5% SDS and twice for
15 min each time at 60 C with 1 x SSC-0.5% SDS. Autoradiographs
were scanned using a Hewlett-Packard Scanjet IIcx (Palo Alto, CA), and
signals were quantified using software developed by Dr. R. Docter
(Department of Internal Medicine III, Erasmus University Medical
School, Rotterdam, The Netherlands). Photographs of the ethidium
bromide-stained gels were analyzed similarly, indicating less than 20%
variation in the amount of applied RNA.
Enzyme assays
Liver microsomal fractions were prepared as described previously
(18). Deiodinase activities of native and recombinant enzyme
preparations were determined by measuring the radioiodide released from
either [3',5'-125I]T4 or
[3',5'-125I]rT3 by ORD, or from
[3,5-125I]T3 or
[3,5-125I]T3S by IRD (21, 22). In
short, 0.5–200 µg tissue or lysate protein were incubated in
triplicate for 20–60 min at 37 C with 10 or 100 nM
[125I]substrate in 0.2 ml 0.1 M sodium
phosphate buffer (pH 7.2), 2 mM EDTA, and 10 mM
DTT. Reactions were stopped, and [125I]iodothyronines
were precipitated by successive addition of 0.1 ml 5% BSA and 0.5 ml
10% trichloroacetic acid. Radioiodide was further isolated from the
supernatant on Sephadex LH-20 minicolumns (21, 22). The characteristics
of the deiodinase activity expressed in ECL1711M-transfected COS-1
cells were compared with those determined in E19 chicken liver
microsomes. For the developmental study, D1 and D3 activities were
assayed as described by Darras et al. (18). For D1 activity,
incubation mixtures contained 1 µM
[3',5'-125I]rT3, 50 µg/ml
microsomal protein, and 5 mM DTT. For D3 activity,
incubations contained 10 nM
[3'-125I]T3, 10 or 500 µg/ml
microsomal protein, 1 µM rT3, 0.1
mM PTU, and 50 mM DTT.
Miscellaneous
Plasma T3 and T4
were measured as described by Darras et al. (28, 29). Unless
indicated otherwise, the results presented are taken from
representative experiments and were reproduced in at least two other
experiments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chicken D1
The longest cDNA clone identified after screening with ECL17a
(clone ECL1711) was 1360 bp long, but it lacked a translation start
site (Fig. 1
). Alignment with the reported sequences of other
deiodinases showed that it shared highest sequence identity with D1s
from other species (2, 5, 7, 9), and based on optimal nucleotide
alignment, this clone presumably lacked the first five nucleotides of
the coding sequence. After subcloning of ECL1711 into pCI-Neo, an ATG
start codon in a Kozak consensus sequence (24) was engineered by
site-directed mutagenesis at the EcoRI restriction site,
expanding the coding sequence by four codons (coding for MGTR), which
then is probably two codons longer than the authentic coding sequence.
Figure 2 shows the alignment of the
deduced amino acid sequence of the mutant, ECL1711M, with those of
other D1s, indicating that it has 62% amino acid identity with human
D1 (5), 60% identity with rat D1 (2), and 58% identity with dog D1
(9). ECL1711M also contains an in-frame TGA codon at position 127,
i.e. at the corresponding position as in the other D1s (Fig. 1). UGA usually functions as a translation stop codon, but is
translated as Sec when a Sec insertion sequence (SECIS) element is
present in the 3'-untranslated region (3'UTR) of selenoprotein-coding
messenger RNAs (mRNAs) (30, 31, 32). A putative SECIS element is located
between nucleotides 1217–1303 in ECL1711M (Fig. 1).
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). Rates of iodothyronine deiodination
decreased in the order rT3 >
T3S > T4 >
T3, which is identical with the substrate
preference demonstrated by native chicken D1 in E19 chicken liver
microsomes (Fig. 3).
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). The potencies by which the different
unlabeled iodothyronines inhibited the ORD of
[125I]rT3 decreased in the order
rT3 > T4 >
T3, with IC50 values of approximately
0.3, 2, and more than 10 µM, respectively. The
Km value for rT3 calculated
from Lineweaver-Burk plots was 0.26 µM for both
ECL1711M-expressed enzyme and native chicken D1 (not shown).
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shows that addition of 0.1–100
µM PTU or IAc or 0.01–10 µM GTG resulted
in the dose-dependent and identical inhibition of the ORD of 0.1
µM rT3 by the ECL1711M-encoded
enzyme and native chicken D1 in the presence of 10 mM DTT.
In both cases, the IC50 values were about 30 nM
for GTG and about 2 µM for both PTU and IAc. Taken
together, these data demonstrate that ECL1711 cDNA codes for chicken
D1.
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). Alignment of this sequence with the
reported sequences of other deiodinases showed that ECL1715 shared
sequence identity with D3s from other species (3, 6, 10, 12), and that
the coding sequence presumably lacked the first 46 nucleotides.
Alignment of the deduced amino acid sequence from ECL1715 with the
sequences of other D3s indicated 62% amino acid identity with rat D3
(3), 63% identity with human D3 (6), 72% identity with X.
laevis D3 (12), and 71% identity with R. catesbeiana
D3 (10) (Fig. 7). The homology between
ECL1715 and the other D3 sequences includes the presence of a
Sec-encoding TGA codon at a corresponding position as well as a SECIS
element in the 3'UTR (Fig. 7).
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Ontogeny of D1 and D3 in the chicken
During the last week of embryonic development, important changes
in circulating thyroid hormone were observed. Plasma
T3 levels increased dramatically after E18 until
the moment of internal pipping (E20IP) and decreased somewhat until
hatching (C0; Fig. 8). Plasma
T4 levels gradually increased from E14 until E18,
and remained relatively constant thereafter (Fig. 8). In comparison,
Fig. 9 shows the ontogeny of hepatic D1
activity, catalyzing the ORD of T4 to
T3 and of rT3 to
3,3'-T2, and Fig. 10 shows
the ontogeny of hepatic D3 activity, catalyzing the IRD of
T4 to rT3 and of
T3 to 3,3'-T2. D1 activity gradually
increased from E14 until a maximum was reached 1 day after hatching
(C1). D3 activity increased between E14 and E17 and rapidly decreased
between E18 and E20IP. When the profiles of the mean plasma thyroid
hormone levels (n = 7–24) and mean hepatic iodothyronine
deiodinase activities (n = 4–5) were compared, plasma
T3 was positively correlated with D1 activity
(r = 0.700; P < 0.05) and negatively correlated
with D3 activity (r = -0.767; P < 0.01). Despite
the increase in the plasma levels of the precursor
T4, plasma T3 remained low
between E14 and E17, and this was associated with a doubling of hepatic
D3 activity. Between E18 and E20IP, plasma T4
remained almost constant, but plasma T3 increased
sharply, which was associated with a dramatic decrease in hepatic D3
activity. No correlation was found between plasma
T4 levels and hepatic D3 activity (r =
-0.359; P = NS). However, a strong positive
correlation was found between plasma T4 levels
and hepatic D1 activity (r = 0.833; P < 0.01);
both showed a gradual increase during ontogeny (Figs. 8 and 9).
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and 10).
Densitometric analysis of these blots revealed that D1 mRNA levels
remained relatively constant during embryonic development. In contrast,
D3 mRNA levels showed a modest increase from E14 to E17 and a strong
decrease thereafter to almost undetectable levels on E20IP. The mean D3
mRNA levels (n = 2) and mean D3 activities (n = 4–5) were
highly significantly correlated (r = 0.977; P <
0.001). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IAc) as native chicken D1. The conclusion that ECL1711M is
derived from chicken D1 mRNA is also supported by the finding that
recombinant and native chicken D1 show identical Km values
for rT3 (0.26 µM), which is similar
to values found for rat (33) and human (34) D1. Since the completion of
our study, an expressed sequence tag (EST) cloned from chicken T cells
was entered in the GenBank/EMBL Data Bank (accession no. AA495711) with
an almost identical nucleotide sequence as ECL1711. The deduced protein
is six amino acids shorter at the N-terminus than ECL1711M and has Ala
instead of Thr at position 37 and Leu instead of Met at position
131. The conclusion that ECL1715 is a partial cDNA clone coding for chicken D3 is based on indirect evidence, as expression studies were not possible. The deduced amino acid sequence shows strong homology with rat (62%), human (63%), X. laevis (72%), and R. catesbeiana (71%) D3. The weak homology of ECL1715 with mammalian, amphibian, and fish D2 sequences (30–45%), in contrast to the strong homology among these D2 sequences (68–90%) (4, 7, 11, 14), strongly suggests that ECL1715 does not encode chicken D2. This is supported by the findings of Galton and Hiebert (35) and ourselves (unpublished observations) that D2 activity is not expressed in embryonic chicken liver. Finally, the strong correlation between hepatic D3 activity and ECL1715-hybridizing mRNA expression during embryonic development represents convincing evidence that ECL1715 is a partial cDNA clone coding for chicken D3.
The availability of chicken D1 and D3 cDNA clones enables the detailed investigation of the regulation of these enzymes under different physiological conditions and in different developmental stages. Birds, in general, and chickens, in particular, are excellent models for studying the role of thyroid hormones in development. In contrast to mammals, which experience intrauterine development, the chicken embryo develops in a closed compartment deprived of the influx of maternal endocrine factors. Circulating thyroid hormone in the embryonic chicken represents the remainder of what was deposited in the yolk before laying (36) and, to increasing extents, hormone synthesized by the embryo itself. During chicken embryonic development, important changes occur in circulating thyroid hormone levels. Plasma T3 levels increase dramatically at the moment of pipping, when the embryo perforates the air chamber and switches from allantoic to lung respiration, and remain high until hatching. These data are in accordance with the work of Decuypere et al. (16, 17) and Darras et al. (18). Plasma T4 levels increase more gradually, reaching a maximum at hatching. Whereas plasma T4 was not correlated with hepatic D3 activity, a negative correlation was found between plasma T3 and D3 activity. Similar results by Borges et al. (37) and Darras et al. (18) suggest that the rise in plasma T3 in embryonic chickens at the end of incubation is at least in part caused by a decreased breakdown due to the decrease in hepatic D3 activity. In recent years it has become clear that this decrease in hepatic D3 activity is at least in part caused by the simultaneous increase in plasma GH (19, 20). It is also interesting to point out that hepatic D1 expression is positively correlated with both plasma T3 and T4 during embryonic development. Although it is unlikely that the prohormone T4 directly induces D1 expression, the positive correlation between plasma T3 and D1 activity is a true "chicken and egg" problem, as T3 is not only a product but also a stimulator of D1 expression, at least in rats (38).
These results demonstrate that iodothyronine deiodinases, especially hepatic D3, are acutely regulated during embryonic development. To find out whether this regulation occurs at the transcriptional or the translational level, we decided to investigate D1 and D3 mRNA expression in chicken liver. We did not standardize D1 and D3 mRNA levels relative to actin or GAPDH mRNA levels, because expression of the latter may change significantly during embryonic development. However, it should be stressed that D1 and D3 mRNA levels were determined in the same RNA preparations, indicating completely different ontogenic patterns. Whereas hepatic D1 activity gradually increases during embryonic development from E14 onward, the D1 mRNA level remains relatively constant. As D1 activity and mRNA level are standardized relative to microsomal protein and total RNA levels, respectively, it cannot be firmly concluded that the ratio of D1 activity to mRNA changes and, thus, that regulation takes place at the translational or posttranslational level. In contrast, hepatic D3 activity and D3 mRNA level were very strongly correlated; both showed an increase between E14 and E17, followed by a dramatic decrease. These results strongly suggest that the regulation of hepatic D3 expression during embryonic development occurs predominantly at the pretranslational level. Whether these changes are due to alterations in transcription rate or mRNA stability requires further investigation. Also, the role of GH in this mechanism remains to be elucidated.
In conclusion, 1) ECL1711 and ECL1715 are near-full-length clones for chicken D1 and D3, respectively; 2) both cDNAs contain an in-frame TGA codon and a consensus SECIS element in the 3'UTR, making them members of the Sec-containing iodothyronine deiodinase family; and 3) the regulation of hepatic D3 expression in the chick during embryonic development largely represents a pretranslational mechanism.
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Acknowledgments |
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Footnotes |
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2 These authors contributed equally to this work.
3 Sanders, J. P., S. Van der Geyten, E.
Kaptein, V. M. Darras, E. R. Kühn, J. L. Leonard,
and T. J. Visser, manuscript in preparation.
Received June 2, 1997.
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) levels during embryonic development. Results are presented as the
mean ± SEM of 7–24 plasma samples. NP, Nonpipping;
IP, internal pipping.
