Differential regulation of IGFBP-2 and IGFBP-5 gene expression
by vitamin A status in Japanese quail
Zhengwei
Fu,
Tadashi
Noguchi, and
Hisanori
Kato
Laboratory of Nutritional Biochemistry, Department of Applied
Biological Chemistry, Graduate School of Agricultural and Life
Sciences, University of Tokyo, Tokyo 113 - 8657, Japan
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ABSTRACT |
To investigate the
involvement of the insulin-like growth factor (IGF) system in vitamin A
(VA)-supported growth, we examined the effects of VA status on IGF
binding protein (IGFBP)-2 and -5 gene expression in Japanese quail. VA
deficiency caused a reduction in IGFBP-2 mRNA only in lung, without
effect in other tissues. However, the expression of IGFBP-5 mRNA was
more sensitive to the change of VA status. IGFBP-5 mRNA levels were
significantly reduced by VA depletion in a tissue-specific manner,
which preceded the decrease in body weight. A single injection of
retinoic acid or retinol to VA-deficient quail did not affect the
levels of IGFBP-2 mRNA, but it rapidly induced the expression of
IGFBP-5 mRNAs in some tissues. These results are the first to show that gene expression of some IGFBPs in vivo are under the control of VA
status and suggest a possible involvement of the IGF system in
mediating the physiological actions of VA in the growth of Japanese quail.
insulin-like growth factor binding proteins-2 and -5; vitamin A
deficiency; vitamin A repletion
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INTRODUCTION |
VITAMIN A (VA), a
class of compounds including retinol, retinoic acid (RA), and other
derivatives, is essential for normal growth, vision, and reproduction,
embryonic and fetal development, the maintenance of numerous tissues,
and overall survival in many species. The indispensability of VA has
become clear by VA-deficient diets being fed to animals. In embryonic
development, maternal VA deficiency during gestation leads to embryonic
malformations including cleft palate and abnormalities of the eyes,
heart, circulatory system, and urogenital tract (38). In
postnatal life, VA deprivation causes many abnormalities including
retarded growth, widespread substitution of keratinizing squamous
epithelium, atrophy of several glandular organs, eye lesions, and
testis degeneration (36, 39). The lesions can be reversed
by VA administration, confirming the importance of this vitamin as an
essential factor in every aspect of life. However, the mechanisms by
which VA exerts its effects in most of these aspects are not well
understood. For example, the precise roles of VA during growth are
unclear despite extensive research. It can be postulated that VA might
exert its effects by influencing a number of biochemical and
physiological factors that control growth and cell differentiation.
Insulin-like growth factors (IGFs) play critical roles in
proliferation, differentiation, and transformation in a variety of
vertebrate tissues (33, 37). It has been thought that
IGF-I produced in the liver is secreted into the circulation and acts on target tissues in an endocrine manner (4, 5). Besides its endocrine effects, IGF-I is also produced in most extrahepatic tissues and can function as an autocrine and/or paracrine growth stimulator (4, 5). Thus it can be thought that IGFs may mediate, at least partly, the action of VA during growth. In fact, we
found in our preceding study (12) that VA deficiency
reduces the levels of serum IGF-I concentration and of IGF-I gene
expression in many tissues of Japanese quail (Coturnix coturnix
japonica), accompanied by growth delay, supporting the
aforementioned hypothesis regarding the physiological role of IGF-I in
mediating VA-supported growth.
The actions of IGFs are modulated by IGF-binding proteins (IGFBPs).
IGFBPs bind specifically and with high affinity to IGFs and have been
suggested to act as modulators by either enhancing or inhibiting the
activity and bioavailability of IGFs (15). Therefore, to
clearly understand the physiological role of IGF-I in the
growth-supportive action of VA, it is indispensable to examine the
effect of VA on the synthesis of IGFBPs. To date, six distinct IGFBPs,
designated IGFBP-1 through IGFBP-6, have been identified in mammals
(15, 31). Of these, only two, IGFBP-2 (28)
and IGFBP-5 (1), have so far been characterized at the molecular level in chickens. In the present study, we examined the
effects of VA deficiency on the expression of IGFBP-2 and IGFBP-5
mRNAs in different tissues of Japanese quail. We also examined the
effects of VA repletion on VA-deficient quail in terms of the
expression of these genes.
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MATERIALS AND METHODS |
Animals and experimental design.
All-trans retinol palmitate and all-trans RA were
purchased from Sigma (St. Louis, MO). Two different diets, a
VA-deficient diet (
VA) and a
VA diet supplemented with 14,000 IU/kg
of all-trans retinol palmitate (+VA), were prepared as
described previously (7, 9). In the
VA diet, other
nutrients aside from retinol were sufficient to support normal growth.
These two diets were prepared weekly and stored at
20°C until use.
In experiment 1, 1-day-old male Japanese quail were randomly
divided into two groups (control and VA-deficient groups). They were
housed in a brooder, maintained under continuous illumination for 3 days, and then subjected to a 14:10-h light-dark cycle. The
VA-deficient group was fed the
VA diet, whereas the control group (VA
sufficient) was maintained on the +VA diet. Diets and water were
available at all times throughout the experiment. Five birds from each
group were randomly selected as samples at 14 and 21 days of age. Each
bird was weighed, killed by decapitation, and bled. Their tissues,
including brain, liver, heart, lung, kidney, and testis, were quickly
dissected, immediately frozen in liquid nitrogen, and then stored at
80°C for analysis of the expression of IGFBP-2 and IGFBP-5 mRNAs.
In addition, serum was collected and used for measuring serum retinol concentrations.
In experiment 2, 1-day-old quail were fed the +VA diet or
the
VA diet for 21 days. Then five quails each were injected
intramuscularly with 0.1 mg of all-trans RA (Sigma) or 0.1 mg of all-trans retinol palmitate (Sigma) or an equal amount
of vehicle. All-trans RA and all-trans retinol
palmitate were dissolved in ethanol (10 mg/ml) and diluted five times
with rapeseed oil before injection. The quail were killed 4 h
after the treatment, and the tissues were then dissected and frozen as
described in experiment 1.
Measurement of VA concentrations in serum and liver.
The degree of VA deficiency was assessed by means of HPLC, measuring
concentrations of retinol in serum and retinyl palmitate in liver as
described previously (7, 8).
Measurement of IGFBP-2 and -5 mRNAs: cloning of quail IGFBP-2 and
-5 cDNA.
Complementary DNA of quail IGFBP-5 mRNA was obtained by RT-PCR.
Three micrograms of total RNA extracted from quail testis were reverse
transcribed into first-strand cDNA with a First Strand Synthesis kit
(GIBCO-BRL, Rockville, MD). Sequences of the oligonucleotide primer
pairs used for PCR amplification of cDNA products of IGFBP-5 were
5'-TGCGAGCTGGTGAAGGAGCC-3' (sense) and 5'-TCACTCCACGTTGCTGCTGTC-3' (antisense), corresponding to bp 473-492 and bp 1136-1156 of
the chick IGFBP-5 cDNA sequence (1), respectively. The PCR
was carried out using AmpliTaq Gold polymerase (Perkin-Elmer,
Branchburg, NJ) in a Perkin-Elmer GeneAmp PCR system 2400. The PCR
product of IGFBP-5 was subcloned into the pCRII vector (Invitrogen,
Carlsbad, CA) by use of a TA Cloning kit.
Plasmid DNA was purified using a Wizard Preps DNA purification system
(Promega, Madison, WI). Sequence analysis was performed by means of a
DSQ-1000 automated fluorescent DNA sequencer (Shimadzu, Tokyo, Japan)
with the use of a thermo sequenase fluorescent-labeled primer
cycle-sequencing kit with 7-deaza-dGTP (Amersham Pharmacia Biotech,
Buckinghamshire, UK), according to the manufacturer's protocols.
Sequence data were analyzed using the GENETYX-MAC (version 10) program
and databases. The nucleotide sequence was compared with that of
chicken IGFBP-5, showing 98% identity with the corresponding region
of the chicken IGFBP-5. Its deduced amino acid sequence also manifested
high homology with that of the chicken sequence. This quail cDNA has
been deposited in GenBank and assigned accession no. AF293839.
A 311-bp PCR fragment of quail IGFBP-2 (GenBank accession no. AF260701)
(10), containing positions 1-311, was
subcloned into the pGEM-T Easy vector (Promega).
RNase protection assay.
To generate the antisense IGFBP-2 and IGFBP-5 cRNA probes, the
IGFBP-2 and IGFBP-5 plasmids obtained above were linearized using
NcoI or XbaI restriction endonuclease,
respectively, and transcribed by SP6 RNA polymerase in the presence of
a [
-32P]UTP, as described previously
(21). Total RNA was extracted from the brain, liver,
heart, lung, kidney, and testis according to the manufacturer's
protocol for TRIzol reagent (GIBCO-BRL). Sample quality and quantity
were assessed by measuring the optical density of each sample at 260 and 280 nm. Sample quality was also checked by ethidium bromide
staining of denatured agarose gels, and the intensity of 18S/28S
ribosomal RNA bands was analyzed using KODAK 1D Image Analysis (Eastman
Kodak, New Haven, CT). Equal amounts of total RNA (20 or 40 µg) from
each sample were then used to determine the expression of IGFBP-2 and
IGFBP-5 mRNAs by means of an RNase protection assay, as described
previously (11, 16). Briefly, total RNA was hybridized
with 200,000 cpm of each probe overnight and then digested with RNase A
and RNase T1. RNase A/T1 were inactivated by proteinase K solution (RNA grade, GIBCO-BRL). Protected mRNA was directly precipitated by adding
isopropanol at the same volume and 4 µl of tRNA (5 mg/ml). Therefore,
all of the processes, from total RNA to electrophoresis, could be
finished in the same tube. To compare equivalent loading of RNA
samples, the intensity of 28S/18S rRNA was used to normalize the
expression of IGFBP-2 and IGFBP-5 mRNAs among tissues, and quail
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (150 bp) was used as
an internal control in the same tissue.
Data analysis.
Hybridized blots were imaged and analyzed using BAS-2000 (FujiPhoto
Film, Tokyo, Japan). All values for mRNA levels are given as a relative
value in an experiment. All data, including body weight, serum and
hepatic VA levels, and the levels of IGFBP-2 and IGFBP-5 mRNAs, were
expressed as means ± SE, and the statistical significance was
determined using a one-way ANOVA test (32). Post hoc
analysis was performed using Tukey's multiple comparisons test
(32).
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RESULTS |
Tissue distribution of IGFBP-2 and -5 mRNAs.
To investigate how IGFBP-2 and -5 genes are expressed among tissues, we
performed an RNase protection assay using 32P-labeled
IGFBP-2 and IGFBP-5 probes and total RNA extracted from various tissues
of 21-day-old quail of the control group. As shown in Fig.
1, both IGFBP-2 and IGFBP-5 mRNAs were
present in all of the tissues examined, but the distribution patterns
were clearly distinct between these two genes. Because both
-actin
and GAPDH mRNA levels differed widely among these tissues (Fig.
1A), we measured the intensity of 28S/18S rRNA to normalize
the expression of IGFBP-2 and IGFBP-5 mRNAs among these tissues (Fig.
1B). As a consequence, IGFBP-2 mRNA levels were relatively
high in brain and testis, intermediate in liver and heart, very low in
lung and kidney. In contrast, IGFBP-5 mRNA levels appeared to be
relatively abundant in testis and kidney compared with those in the
other tissues examined.

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Fig. 1.
Tissue distribution of insulin-like growth factor binding protein
(IGFBP)-2 and IGFBP-5 mRNAs determined by RNase protection assay. Total
RNA (40 µg) isolated from different tissues of Japanese quail
(Coturnix coturnix japonica) at 3 wk of age was hybridized
to IGFBP-2 and IGFBP-5 cRNA probes. A: typical results of
the assay for IGFBP-2 and IGFBP-5, -actin, and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are shown, and
responsive 28S/18S ribosomal RNA bands are also presented. Data were
normalized by the intensity of 28S/18S rRNAs. B:
quantitative representations of multiple results are shown, expressed
as values relative to the minimums. Data are means ± SE;
n = 5 samples.
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Effect of VA deficiency on gene expression of IGFBP-2 and -5.
To assess the degree of VA depletion in the VA-deficient group, serum
retinol and hepatic retinyl palmitate levels were determined at 14 and
21 days of age by means of HPLC (Fig. 2).
Feeding on the
VA diet for 14 days greatly reduced serum retinol
concentrations by ~80% and hepatic retinyl palmitate concentrations
by ~90%, which were in agreement with our previous reports (7,
8). At 21 days of age, both the serum retinol and hepatic
retinyl palmitate levels in the VA-deficient group were below
detectable levels. The detection limits of plasma retinol and hepatic
palmitate were 0.10 µg/dl and 0.02 µg/g, respectively.

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Fig. 2.
Depletion of vitamin A (VA) concentrations in the sera
and livers of quail fed VA-free or VA-supplemented diets. Day-old male
quail were fed the VA-deficient diet ( VA) or VA supplemented with
14,000 IU/kg diet of all-trans retinol palmitate (+VA). Sera
and livers were collected at 14 and 21 days of age, and VA levels were
analyzed using HPLC. The initial VA levels in the serum and liver of
newly hatched quail (day 0) were also measured to assess the
degree of VA deficiency. Data represent means ± SE of 5 samples.
ND, not detected.
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The growth of the quail was significantly retarded by VA deficiency
(P < 0.05; Fig. 3). In
contrast to the rapid body weight gain in the control group (+VA) from
14 to 21 days of age, a small increase in body weight was
observed in the VA-deficient group during the same period.

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Fig. 3.
Influence of VA deficiency on growth. Day-old male quail
were fed the VA or VA supplemented with 14,000 IU/kg diet of
all-trans retinol palmitate (+VA). Data represent means ± SE; n = 5 samples. Means not having the same letters
at the same day of age indicate significant differences
(P < 0.05).
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We then examined how VA deficiency influences the expression of IGFBP-2
and IGFBP-5 mRNAs in various tissues. Levels of IGFBP-2 and IGFBP-5
mRNAs were measured by the RNase protection assay in many tissues on
days 14 and 21, when the quail were under
different degrees of VA deficiency. As illustrated in Fig.
4, IGFBP-2 mRNA levels in the brain,
liver, and heart were not affected by the VA deficiency, regardless of
the degree of vitamin depletion. The expression of IGFBP-2 mRNA in the
kidney of quail fed the
VA diet for 21 days also did not differ from
that in the control quail. However, the expression of IGFBP-2 mRNA in
the lung was significantly altered (P < 0.05). IGFBP-2
mRNA levels increased slightly in the lung of the control group from
days 14 to 21, whereas the levels of IGFBP-2 mRNA
in the VA-deficient group decreased significantly during the same
period. Feeding the
VA diet for 14 days reduced the lung IGFBP-2 mRNA
levels by 26% and for 21 days by about sevenfold compared with those
in the control group (Fig. 4).

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Fig. 4.
Effects of VA deficiency on the expression of IGFBP-2 mRNA. Day-old
male quail were maintained as described in the legend of Fig. 2. At 14 and 21 days of age, 5 birds from each treatment group were killed by
decapitation. Different tissues were dissected and processed for
assaying expression of IGFBP-2 mRNA. Left: representative
results obtained by RNase protection assay from the selected tissues
are shown. The level of GAPDH mRNA was used as an internal control in
the same tissue to normalize the intensities of IGFBP-2 mRNA.
Right: quantitative representations of multiple results are
presented, expressed as values relative to the abundance in the
control. Data represent means ± SE; n = 5 samples. Means not bearing the same letter are significantly different
(P < 0.05).
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Compared with the expression of the IGFBP-2 gene, the expression of
IGFBP-5 mRNA was much more sensitive to the change of VA status. The
changes in the levels of IGFBP-5 mRNA caused by VA depletion were very
different among the tissues examined. Feeding the
VA diet for 14 days
reduced the mRNA levels of IGFBP-5 by 45, 40, and 65% in liver
(P < 0.01), heart (P < 0.01), and
lung (P < 0.01), respectively, compared with those in
the control group (Fig. 5). Further
feeding on the
VA diet for 7 days caused a further decrease in
IGFBP-5 mRNA levels in liver (25% of control), but no further decrease
was observed in heart and lung (Fig. 5). In contrast, expression of the
IGFBP-5 gene in brain, testis, and kidney was unaffected by the VA
deficiency (Fig. 5). In addition, the tissue IGFBP-5 mRNA levels did
not change significantly in the control group with age (from days
14 to 21).

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Fig. 5.
Effects of VA deficiency on the expression of IGFBP-5 mRNA.
Management of quail and sampling and analysis of the expression of
IGFBP-5 mRNAs were carried out as described in the legends of Figs. 2
and 4. Left: representative results obtained by RNase
protection assay from the selected tissues are shown. Right:
quantitative representations of multiple results are presented,
expressed as values relative to the abundance in the control. Data
represent means ± SE; n = 5 sample. Means not
bearing the same letter are significantly different (P < 0.01).
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Effect of repletion of vitamin A on expression of IGFBP-2 and
-5 genes.
Next, we examined whether the decrease in the mRNA levels of IGFBP-2
and -5 caused by VA deficiency would be restored by an injection of VA.
Figure 6 shows the effects of treatment
with retinol or RA on the expression of IGFBP-2 and IGFBP-5 genes in the tissues of the VA-deficient quail. An intramuscular injection of
either retinol or RA (0.1 mg/bird) did not alter the levels of IGFBP-2
mRNA in all tissues examined including the lung, where the expression
of IGFBP-2 mRNA was significantly reduced by the VA deficiency (Fig. 6,
left).

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Fig. 6.
Injection of retinol or retinoic acid (RA) to the VA-deficient
quail show different regulation of the gene expression of IGFBP-2 and
IGFBP-5. Quail were depleted of VA by being fed a VA-free diet for 21 days. They were killed 4 h after treatment with 0.1 mg/bird of
all-trans retinol palmitate (+VA) or all-trans RA
(+RA), and their tissues were processed to measure the expression of
IGFBP-2 and IGFBP-5 mRNAs by RNase protection assay. Top:
typical results of each IGFBP-2 or IGFBP-5 mRNA are shown;
bottom: the quantitative representation of multiple results
are presented expressed as values relative to the abundance in the
vehicle treatment (C), respectively. Data represent means ± SE;
n = 5 samples. Means not bearing the same letter are
significantly different (P < 0.05).
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With respect to the expression of the IGFBP-5 gene, a single injection
of RA to the VA-deficient quail significantly increased the mRNA levels
by ~50% in brain and liver (P < 0.05) and more than
fourfold in heart and lung (P < 0.01) (Fig. 6,
right). An administration of retinol also induced an
increase in the mRNA levels of IGFBP-5 of about twofold in lung
(P < 0.05), but not in brain, liver, or heart. In
kidney, treatment with either RA or retinol did not alter the
expression levels of IGFBP-5 mRNA.
To determine whether the inductive effect of RA or retinol on the
expression of the IGFBP-5 transcript depended on the retinoid status of
the animals, we performed an experiment with the VA-sufficient quail
that was similar to the experiment done with the VA-deficient quail.
Regardless of retinoid status, an injection of RA (0.1 mg/bird) into
the VA-sufficient quail also caused a significant increase in the
levels of IGFBP-5 mRNAs in both lung and heart after 4 h
(P < 0.01; Fig. 7,
right). The magnitude of induction was less than that seen
in the VA-deficient quail, partly reflecting the differences in the
basal mRNA levels of the two control groups (+VA vs.
VA). In
addition, retinol treatment did not affect the IGFBP-5 mRNA levels in
all of the tissues examined. Furthermore, just as in the case of the
VA-deficient quail, neither RA nor retinol treatment altered the
expression of the IGFBP-2 gene in any of the VA-sufficient tissues
examined in this study (Fig. 7, left).

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Fig. 7.
Injection of retinol or RA into the VA-sufficient Japanese quail
induces the overexpression of the IGFBP-5 transcript.
All-trans retinol palmitate (+VA) or all-trans RA
(+RA) (0.1 mg/bird) was intramuscularly injected into the VA-sufficient
quail (21 days of age). After 4 h, these quail were killed, and
their tissues were dissected out to measure IGFBP-2 and IGFBP-5 mRNAs
by use of RNase protection assay. Each IGFBP-2 or IGFBP-5 mRNA is shown
(top), and the quantification expressed as values relative
to the abundance in the vehicle treatment (C) are presented
(bottom), respectively. Data represent means ± SE;
n = 5 samples. Means not having a common letter are
significantly different (P < 0.05).
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DISCUSSION |
It has been established from mammalian studies that IGFBPs share a
relatively high similarity in amino acid sequences but differ in
posttranslational modifications, cellular locations, and affinity for
IGFs. Each IGFBP is expressed in a species- and tissue-specific manner
and is also subject to differential developmental and hormonal
regulation (15, 34). This suggests that each IGFBP may
play specific roles in the regulation of IGF-mediated cell growth and
metabolism in defined tissues. In the present study, we found that the
expression patterns of the genes for IGFBP-2 and IGFBP-5 also show a
tissue-specific manner in the case of Japanese quail. Therefore, IGFBPs
in the bird may also have different roles in the regulation of
IGF-mediated actions.
Feeding 1-day-old quail a VA-deficient diet caused rapid decreases in
the levels of both serum retinol and hepatic retinyl palmitate and
consequently reduced the animals' growth rate. To elucidate the
involvement of the biochemical and physiological factors involved in
this process, we examined the effects of VA deficiency on the gene
expression of IGF-I, IGF-I receptor, and insulin receptor in our
preceding study and found the possible involvement of the IGF system in
VA-supported growth (12). To gain further insight into the
actions of the IGF system in VA-supported growth, we examined VA
regulation of IGFBP-2 and IGFBP-5 gene expression and found for the
first time that the expression of these two genes is differentially
affected by changes in VA nutritional status in the in vivo system used
in the present study. For example, the gene expression of IGFBP-5 is
very sensitive to the change of VA status, in contrast to that of
IGFBP-2. VA deficiency caused a significant decrease in the levels of
IGFBP-5 mRNA in many tissues in a manner similar to that of the effects
of VA on the expression of IGF-I (12), with a single
injection of RA increasing the IGFBP-5 mRNA levels more than
fourfold that of the control value in some tissues after 4 h.
IGFBPs have been suggested to have both inhibitory and stimulatory
effects on cell proliferation, depending on the cell types studied, and
these effects can occur in either an IGF-dependent or -independent
manner (20, 23). IGFBP-2 is thought to exert inhibitory
effects mainly on IGF-stimulated cell proliferation in normal cell
types (27). From our results, the possibility that IGFBP-2
was involved in the retarded growth of Japanese quail caused by the VA
deficiency may be quite low. On the other hand, the positive
relationship between the changes in the expression of IGFBP-5 and IGF-I
genes in the VA-deficient quail suggested that IGFBP-5 may have
stimulatory effects on quail growth in either an IGF-dependent or
-independent way. It is worth noting that the decrease in body weight
was preceded by a decrease in the expression of IGFBP-5 mRNA. Thus,
taken together with the fact that both serum IGF-I concentrations and
tissue IGF-I mRNA levels decreased in the VA-deficient quail
(12), these results further indicate that the IGF system
may be associated with the signal transduction of VA in the growth
process, although the exact physiological consequence of the decrease
or increase in the expression levels of IGFBP-2 and IGFBP-5 is not known.
Numerous studies in mammals have been dedicated to elucidation of the
hormonal and metabolic regulation mediating the expression of IGFBPs.
Among them, insulin, IGFs, growth hormone, cAMP, dexamethasone, progesterone, and nutritional status have been shown to be major regulators of IGFBP gene expression (34). With respect to
the regulation of IGFBP-2 gene expression, fasting (24,
35), hypophysectomy (25), and diabetes
(24) have been reported to increase the levels of IGFBP-2
in the rat. Much less information is available on the expression of
IGFBP-5 in an in vivo system. The results presented in this study
indicate that dietary VA levels also modulate the expression of IGFBP-2
and IGFBP-5 genes in Japanese quail. However, this regulation was
highly specific to each tissue. VA deficiency caused a significant
decrease in the levels of IGFBP-2 mRNA in lung, but not in the other
tissues examined. In the levels of IGFBP-5 mRNA, a decrease occurred in
liver, heart, and lung but was not seen in brain, testis, or kidney.
Similar tissue-specific expression of IGFBP-2 mRNA also was observed in
rats after fasting (35), hypophysectomy (19),
and diabetes (3). For example, fasting increased the
levels of IGFBP-2 mRNA in liver, but not in kidney or brain. From these
results, it can be speculated that different regulatory factors, which
show cell and binding protein specificity, will affect the regulation
of IGFBP-2 and IGFBP-5 expression. With respect to tissue specificity,
lung is the only tissue in which both IGFBP-2 and IGFBP-5 mRNA levels
were reduced by the VA deficiency, strongly indicating that lung is the
tissue most sensitive to a change in VA status. These results also
suggest that IGFBP-2 and IGFBP-5 may be essential factors modulating
the normal function of the lung.
Another aspect of our findings concerns the effects of RA on the
expression of IGFBP-2 and IGFBP-5. RA, serving as the active form of
VA, is known to promote cell proliferation and differentiation. The
demonstration of nuclear RA receptors (RAR
, -
, and -
) and retinoid X receptors (RXR
, -
, and -
) that function as
transcriptional regulatory factors suggests that retinoids affect
growth by modulating gene expression (17, 18). Recent
studies have demonstrated the important effects of RA on the expression
of IGFBPs in many cell lines, including up- or downregulation,
depending on either the cell line or the type of IGFBPs (2, 6,
13, 14). For example, IGFBP-2 mRNA levels are decreased by RA in
the human neuroblastoma cell line SK-N-SH (22) and primary
cultured rat osteoblasts (26) but are increased in
primary-cultured rat hepatocytes (26). IGFBP-5 mRNA levels
are induced by RA in rat osteoblast cells (6) but are
reduced in human osteoblast cells (41). However, no
published information exists regarding the effects of RA on the gene
expression of the IGFBPs in any in vivo system. In the present study,
we show for the first time that RA can rapidly induce the expression of
IGFBP-5, whereas it does not exert any effect on the IGFBP-2 mRNA
levels in our in vivo system with Japanese quail. This inductive effect
of RA on IGFBP-5 gene expression also appeared in a tissue-specific
manner, as observed in the effect of VA deficiency on this gene's
expression. IGFBP-5 mRNA levels were significantly increased by RA in
brain, liver, lung, and heart but were unaffected in testis and kidney.
Furthermore, we have shown that administration of RA to the control (VA
sufficient) quail also rapidly induced the expression of IGFBP-5 mRNA
at a magnitude less than that seen in the VA-deficient group. This rapid increase in the IGFBP-5 mRNA levels in some tissues induced by RA
treatment, together with the decrease of IGFBP-5 transcript in the
corresponding tissues of the VA-deficient quail, suggests an important
role for IGFBP-5 in mediating the physiological actions of VA in
Japanese quail.
The mechanisms under which VA regulates the expression of IGFBP-2 and
IGFBP-5 are not known. It is known that the molecular actions of RA are
mediated primarily by its nuclear receptors. It has been reported
recently that RAR
and RAR
serve as signal relays in the
RA-induced expression of IGFBP-3 in breast cancer cells
(29). We have also found that RAR
mRNA levels were
reduced by VA deficiency and were rapidly induced by administration of RA in the same in vivo model used in the present study (11a), suggesting the possible involvement of RAR
or other RARs and RXRs in the regulation of IGFBP-2 and IGFBP-5 by VA. The fact that
IGFBP-5 mRNA was induced as quickly as 4 h after the injection of
RA suggests that RA regulates the IGFBP-5 gene directly, whereas the
IGFBP-2 gene may be indirectly regulated by VA nutrition in the same
way as the de novo synthesis of other factors. Moreover, previous
studies have shown that RA may modulate its effects through a variety
of mechanisms, including transactivation by specific nuclear receptors
to alter the rate of gene transcript (17, 18) and by
posttranscriptional mechanisms involving changes in RNA stability or
processing (40). In the case of the regulation of the
IGFBP-5 gene by RA, either transcriptional (6) or
posttranscriptional (30) mechanisms have been reported.
Whether the effects of VA on the mRNA levels of IGFBP-2 and IGFBP-5 in
the present study occur at the transcriptional or posttranscriptional
levels or some combination of the two awaits further study. To
elucidate these issues, more extensive study is currently under
investigation using a rat in vivo system.
In conclusion, the results presented here clearly indicate that dietary
VA levels modulate the IGFBP-2 and IGFBP-5 genes in many different
tissues of Japanese quail. The decrease in the mRNA levels
of IGFBP-2 and IGFBP-5 caused by VA deficiency and the induction
of IGFBP-5 transcript by the administration of VA strongly suggest that
the IGF system may play an important role in expressing the
physiological actions of VA.
 |
ACKNOWLEDGEMENTS |
We thank Teruo Ebihara (Ebihara Quail Farm, Tochigi, Japan) for
kindly providing day-old Japanese quails.
 |
FOOTNOTES |
This work was supported in part by a Grant-in-Aid for Japan Society for
the Promotion of Science (JSPS) Fellows (no. 98218) to Z. W. Fu
from the Ministry of Education, Science, Sports, and Culture of Japan.
JSPS is gratefully acknowledged for providing a postdoctoral fellowship
to Z. W. Fu.
Address for reprint requests and other correspondence: H. Kato,
Laboratory of Nutritional Biochemistry, Dept. of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, Univ. of
Tokyo, Bunkyo-ku, Tokyo, 113-8657, Japan (E-mail:
akatoq{at}mail.ecc.u-tokyo.ac.jp).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 27 October 2000; accepted in final form 26 February 2001.
 |
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