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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 [alpha -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).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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, beta -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.

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.

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).

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).

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).

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).

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).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (RARalpha , -beta , and -gamma ) and retinoid X receptors (RXRalpha , -beta , and -gamma ) 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 RARalpha and RARbeta serve as signal relays in the RA-induced expression of IGFBP-3 in breast cancer cells (29). We have also found that RARbeta 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 RARbeta 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Endocrinol Metab 281(1):E138-E146
0193-1849/01 $5.00 Copyright © 2001 the American Physiological Society




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