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Department of Pediatrics, Division of Endocrinology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7220
Address all correspondence and requests for reprints to: A. Joseph D’Ercole, M.D., The University of North Carolina, Department of Pediatrics, Division of Endocrinology, CB 7220, 509 Burnett-Womack, Chapel Hill, North Carolina 27599-7220.
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Abstract |
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200%), whereas
cerebella of IGF-II/IGF-I Tg mice had the greatest increase in IGFBP-5
mRNA (350%). The increase in IGFBP-5 mRNA correlated with the
regional expression of the transgene during development. The abundance
of IGFBP-5 protein was also found to be increased in both IGF-I Tg
mouse lines. The influence of IGF-I on IGFBP-5 expression was specific
because we found no evidence of changes in IGFBP-2, IGFBP-4, or
cyclophilin expression. Furthermore, as judged by in situ
hybridization histochemistry, IGF-I appeared to increase both the
number of IGFBP-5-expressing cells and the magnitude of their
expression, an observation that was especially marked in the molecular
layer and white matter of the cerebellum. These data indicate that
IGF-I regulates IGFBP-5 expression in vivo and is consistent
with the in situ hybridization data of others showing that
IGFBP-5 expression is temporally and spatially related to that of
IGF-I. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IGF-I’s actions are modulated by a family IGF binding proteins (IGFBP; IGFBP-1 to IGFBP-6). IGFBP-5 is abundantly expressed in brain. Its expression is maximal in first two weeks of postnatal life (6, 7, 8) and occurs in a spatotemporal pattern that coincides with the expression of brain IGF-I (8, 9, 10). In addition, brain IGFBP-5 expression is often associated with the proliferation of neural precursors, as in external granular layer of cerebellum and in paraventricular zone. These data led us to postulate that IGF-I regulates the expression of IGFBP-5, which may in turn augment IGF-I’s actions in brain development in an autocrine or paracrine fashion.
To begin to test our hypothesis, we have examined the expression of IGFBP-5 in two distinct lines of IGF-I Tg mice: metallothionein-I (MT-I)/IGF-I and IGF-II/IGF-I Tg mice. The MT-I/IGF-I Tg mice, in whom the transgene is driven by a mouse MT-I promoter, exhibit the highest levels of IGF-I transgene expression in cerebral cortex, (followed by hippocampus > diencephalon > brainstem and cerebellum; Ref 4). The IGF-II/IGF-I Tg mice, whose transgene is directed by a mouse IGF-II promoter, have the highest transgene expression in cerebellum, followed by brainstem and diencephalon, hippocampus, and cerebral cortex (2). Our studies show that the steady-state levels of IGFBP-5 messenger RNA (mRNA) are increased in the brains of IGF-I Tg mice, compared with those of normal littermate controls. These increases in IGFBP-5 mRNA abundance correlate with the expression of transgenes and are due to increases in the number of expressing cells, as well as increased expression in individual cells. Furthermore, IGFBP-5 polypeptide was increased in IGF-I Tg mice. These data indicate that IGF-I up-regulates brain IGFBP-5 expression in vivo at both the mRNA and protein levels.
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Materials and Methods |
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Northern blot hybridization analysis
Total RNA was extracted from various brain regions using the
acidic guanidinium thiocyanate-phenol-chloroform method (11) and
quantified spectrophotometrically at 260 nm. Aliquots of 20 µg total
RNA were electrophoresed on 1% agarose gels, transferred onto a
GeneScreen membrane (DuPont-NEN, Boston, MA) and UV cross-linked. After
stained with 0.02% methylene blue and photography to quantify the
amount of RNA transferred, the membranes were hybridized with
radiolabeled single stranded DNA (ssDNA) probes (see below) in
Church’s buffer (0.5 M sodium phosphate, pH 7.1/7%,
SDS/0.1 mM EDTA), and specific message bands were detected
by autoradiography. After autoradiography, membranes were stripped in
20 mM sodium phosphate/0.5 mM EDTA at 80–85C for 30–60
min and hybridized with a second probe.
Quantification was performed using a computer-assisted image analysis system (Image-Pro, Media Cybermetics, Silver Spring, MD). To ensure the accuracy of the changes in mRNA abundance and equal loading of RNA, the message levels were normalized to the cyclophilin (CYC) mRNA abundance or to the amount of 18S rRNA on the membrane. The abundance of CYC mRNA closely paralleled the amount of 18S rRNA transferred, as estimated by methylene blue staining.
Probes
IGFBP-5, CYC, and IGF-I DNA fragments were PCR amplified and
used as templates for probes. The IGFBP-5 DNA fragment corresponded to
bp 558-1201 of rat IGFBP-5 cDNA (12), CYC DNA fragment corresponded to
bp 106-517 of the rat CYC cDNA (13), and IGF-I DNA fragment
corresponded to bp 179-537 of human IGF-I cDNA (14). ssDNA probes were
generated from these templates by linear PCR using their respective 3'
end primers and 32P-labeled deoxy-CTP (Amersham, Arlington
Heights, IL), as previously reported (15, 16).
125I-IGF-I ligand and Western blot analysis
Brains of Tg and normal littermate mice were dissected and
frozen in liquid nitrogen. To extract protein, about 50 mg frozen
tissue was pulverized, lysed, and sonicated in lysis buffer (20
mM Tris-HCl, pH 7.4, 2% Triton X-100 and 10 mM
EDTA). Supernatants were collected by centrifugation at 12,000 rpm in a
microcentrifuge for 5 min at 4C. Aliquots of 80 µg protein were
separated on 12.5% polyacrmide gel and transferred onto nitrocellulose
membrane (Schleicher & Schuell, Keene, NH). For ligand blot analysis,
membranes were incubated with a ligand hybridization buffer containing
125I-IGF-I (5 x 105 cpm/ml), as
previously described (17). Western blot analysis was performed using a
chemiluminescence Western blotting kit (Boehringer Mannheim,
Indianapolis, IN) and a polyclonal anti-IGFBP-5 antibody (1:500,
provided by Dr. David Clemmons, University of North Carolina),
according to the manufacturer’s protocol.
In situ hybridization histochemistry (ISHH)
ISHH was performed as previously described (4). Briefly, after
fixation with 4% paraformaldehyde in PBS, the sagittal frozen-sections
(10 µm) were treated with 0.2 N HCl, extensively washed
with PBS and hybridized with IGFBP-5 antisense digoxigenin-riboprobe
generated using Genius RNA Labeling kit (Boehringer Mannheim,
Indianapolis, IN). The hybridization buffer contained 75% formamide,
10% dextran sulfate, 3 x SSC, 1 x Denhart’s, 50
mM sodium phosphate, pH 7.4, and approximately 0.5 ng/µl
of the probe. After incubation with the probe for 16–18 h at 55 C, the
sections were washed with 2 x SSC and 1 x SSC at 55 C,
followed by a wash with 0.2 x SSC for 1 h at 55C. The sites
of IGFBP-5 mRNA were revealed by incubating with antidigoxigenin
antibody conjugated with alkaline phosphatase (1:250) and
5-bromo-4-chloro-3-indolyl phosphate with nitroblue tetrazolium at room
temperature. Liver, which expresses little IGFBP-5 (Ref. 18 and our
unpublished data), was used as a negative control.
Statistics
Statistic comparisons were made using the Student’s
t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). In normal adult mice,
cerebellum exhibited the highest levels of IGFBP-5 mRNA, followed by
diencephalon, brainstem, hippocampus, and cerebral cortex. Compared
with their normal littermates, the abundance of IGFBP-5 mRNA was
significantly increased in most brain regions of both lines of IGF-I Tg
mice. In MT-I/IGF-I Tg mice, cerebral cortex exhibited the greatest
increase in IGFBP-5 mRNA abundance (by 92% over control,
P < 0.05), followed by hippocampus (63%,
P < 0.05), and brainstem (50%, P
< 0.05) (Figs. 1A and 2, top
panel). Diencephalon and cerebellum also exhibited 67% and
14% increases, respectively, but these increases were not
significant. In IGF-II/IGF-I Tg mice cerebellum had the greatest
increase in IGFBP-5 mRNA abundance (by 254%, P <
0.01), followed by brainstem (137%, P < 0.05) and
cerebral cortex (58%, P < 0.01) (Figs. 1B and 2,
bottom panel). Diencephalon showed an approximately 149%
increase in IGFBP-5 mRNA, but this was not significant because of high
variation among animals. These regional increases in brain IGFBP-5
expression in the IGF-I Tg mice correspond to the magnitude of IGF-I
transgene expression that we have found in these brain regions (2, 4).
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). The expression of the transgenes again exhibited a
similar regional pattern and correlated with the increase in IGFBP-5
mRNA abundance (Figs. 1 and 3). In
MT-I/IGF-I Tg mice, the highest levels of IGF-I transgene expression
were found in cerebral cortex, followed by hippocampus (100% of
cerebral cortex), diencephalon (30%), brainstem (25%), and
cerebellum (6%). While IGF-II/IGF-I Tg mice had the highest
transgene expression in cerebellum, followed by brainstem (26% of
cerebellum), diencephalon (23%), cerebral cortex (21%), and
hippocampus (2%).
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, top panel). In contrast,
the abundance of IGFBP-5 mRNA in cerebellum only modestly decreased
during development and the changes were not significant (Fig. 4, bottom panel). Compared with their normal littermates,
however, the abundance of IGFBP-5 mRNA gradually increased both in the
cerebral cortex of MT-I/IGF-I Tg mice and in the cerebellum of
IGF-II/IGF-I Tg mice, reaching about 200–300% of normal levels at P42
(Fig. 4). In contrast, the abundance of IGFBP-2 and IGFBP-4 mRNAs in
MT-I/IGF-I Tg mice did differ from their normal littermate (data not
shown).
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. In normal
mice, large and modestly stained IGFBP-5 positive cells were evenly
distributed throughout the cerebral cortex (Fig. 5A). In hippocampus
dentate gyrus neurons were also modestly stained (Fig. 5C). Purkinje
cells and internal granular cells were the major cerebellar cells
expressing IGFBP-5, but molecular layer cells and white matter cells
also expressed low levels of IGFBP-5 (Fig. 5E). Consistent with our
unpublished Northern data and the data of others (18), liver exhibited
little IGFBP-5 signal (Fig. 5G). Compared with normal littermate
controls, the intensity of the signal and the number of labeled cells
were significantly increased in the IGF-I Tg mice (Fig. 5, B, D, and
F). This was particularly evident in the CB of IGF-II/IGF-I Tg mice
where the expression of IGFBP-5 in molecular layer and white matter
cells was much more prominent (Fig. 5E).
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), but a 24-kDa IGFBP was observed
after extended exposure (data not shown). Compared with their
littermate controls, cerebral cortex of MT-I/IGF-I Tg mice exhibited a
marked increase in the abundance of 32-kDa IGFBP (3.2-fold greater
than controls, P < 0.01) but not in the 24-kDa IGFBP
(Figs. 6A and 7). Similarly, the amount
of 32 kDa was increased in the cerebella of IGF-II/IGF-I Tg mice (by
2-fold, P < 0.05) (Figs. 6A and 7).
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32 kDa), we employed Western immunoblot to confirm that the changes
observed in Western ligand blot analysis were due to the changes in the
IGFBP-5 abundance. Normal serum, which contains little, if any,
IGFBP-5, but abundant IGFBP-2 and -4, exhibited no immunoreactive
signal (Fig. 6B). In both normal and IGF-I Tg mice, a single band of 32
kDa was observed in cerebral cortex (Fig. 6B). In cerebellum, however,
an additional band with a molecular weight slightly larger than 32 kDa
was observed. Consistent with the results of Western ligand blot
analysis, the abundance of IGFBP-5 was significantly increased in
cerebral cortex of MT-I/IGF-I Tg mice (by 2.5-fold,
P < 0.01), as compared with normal controls (Fig. 6B
and Fig. 8). In the cerebellum of
IGF-II/IGF-I Tg mice, both bands of approximately 32 kDa were increased
in abundance (Fig. 6B and 8), and when we combined the abundance of
both bands, cerebellar IGFBP-5 abundance was increased by approximately
2.1-fold (P < 0.01).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The up-regulation of IGFBP-5 gene expression by IGF-I appears to be specific because we did not observe changes in the abundance of mRNAs for IGFBP-2, IGFBP-4, or cyclophilin. Similarly, the abundance of IGFBP-4 protein (presumed to be the 24-kDa band) did not differ in the cerebral cortex of IGF-I Tg mice and normal controls. Similar results also were observed in cultured aortic smooth muscle cells (19) and in thyroid cell line (20). Duan et al. (19) showed that addition of IGF-I to culture medium greatly stimulates the synthesis of IGFBP-5, but not IGFBP-2, in a dose- and time-dependent manner. This stimulation of IGFBP-5 expression by IGF-I appears to be cell specific because IGF-I increases IGFBP-5 mRNA abundance in both cultured porcine and human aortic smooth muscle cells but not human intestinal smooth muscle and fetal skin fibroblasts. Our ISHH data suggest that most IGFBP-5 expressing cells are affected. Whether the expression of IGFBP-5 in a subgroup of neurons and other IGFBP-5 expressing cells is not regulated by IGF-I, however, remains to be determined.
The regional IGFBP-5 expression in mouse brain has not been quantitatively analyzed previously. We found that the highest level of IGFBP-5 mRNA was expressed in the cerebellum of normal adult mice, followed by brainstem, diencephalon, hippocampus, and cerebral cortex. Similar results were observed in rat (6). ISHH analysis of cerebellum shows that Purkinje cells and internal granular cells are major cells expressing IGFBP-5 mRNA, whereas molecular layer cells and white matter cells express low levels of IGFBP-5 mRNA. Identification of the cells expressing IGFBP-5 in molecular layer and white matter may provide clues to IGFBP-5 actions. Bondy and Lee (8), using ISHH, showed that IGFBP-5 mRNA is transiently expressed in external granular cells during early cerebellum development. This expression, however, disappears with the inward migration of cells, and gradually increases in internal granular cells layer (Golgi cells) with age. In addition, they did not observe Purkinje cell IGFBP-5 expression. Stenvers et al (6), also using ISHH, did not detect a IGFBP-5 signal in either cerebral cortex or hippocampus in adult rat, although they demonstrated high expression of IGFBP-5 in both regions by Northern blot hybridization. Therefore, our data show a distinctive distribution pattern of IGFBP-5 expression in mouse brain that differs somewhat from these reports in the rat brain. The discrepancies may reflect a species-specific expression of brain IGFBP-5, or alternatively, a different sensitivity of the method employed.
Previous reports indicate that the expression of IGFBP-5 is temporally and spatially correlated to IGF-I mRNA expression during brain development (8, 9, 10). Consistent with the known developmental decrease in IGF-I mRNA levels in cerebral cortex, our data show that the expression of IGFBP-5 mRNA also gradually decreases with age in the cerebral cortex of normal mice. We, however, observed no such decrease in cerebellar IGFBP-5 mRNA during development, despite decreases in the IGF-I mRNA levels during this same time period (10, 21). The high levels of IGFBP-5 mRNA in cerebella, however, are likely explained by relatively stable IGF-I protein content in developing cerebellum. Torees-Aleman et al. (21) observed little change in rat cerebellar IGF-I content during development despite the fall of cerebellar IGF-I mRNA. Transport of IGF-I to the cerebellum from the inferior olives appears to explain the stable levels of cerebellar IGF-I (22), and in turn the relatively high and stable abundance of IGFBP-5 mRNA during development that we observed.
IGFBP-5 is capable of potentiating IGF-I’s actions on cell proliferation and growth in culture. When IGFBP-5 is associated with extracellular matrix, it can augment IGF-I’s actions on fibroblast growth (23). Andress and Birnbaum (24) showed that osteoblast-derived IGFBP-5 potentiates the stimulatory effects of IGF-I or IGF-II on mitogensis of cultured osteoblasts. Consistent with these in vitro data, high levels of IGFBP-5 expression is often found coincident with IGF-I expression and with cellular proliferation in the developing brain (8). High IGFBP-5 expression also has been found in the cerebellum of the Weaver mutant mice (25) and in the areas surrounding lesions during recovery from hypoxia-ischmia injury (26). In this study, we have further demonstrated that IGF-I up-regulates the expression of brain IGFBP-5 in vivo, supporting our hypothesis that IGFBP-5 plays an important role in augmenting IGF-I’s stimulatory actions on CNS development.
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Acknowledgments |
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Footnotes |
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2 Supported by NIH Training Grant T32-DK-07129.
Received August 12, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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