help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ye, P.
Right arrow Articles by D’Ercole, A. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ye, P.
Right arrow Articles by D’Ercole, A. J.
Endocrinology Vol. 138, No. 12 5466-5475
Copyright © 1997 by The Endocrine Society

ARTICLES

Regulation of Insulin-Like Growth Factor I (IGF-I) Gene Expression in Brain of Transgenic Mice Expressing an IGF-I-Luciferase Fusion Gene1

P. Ye2, Y. Umayahara3, D. Ritter, T. Bunting, H. Auman, P. Rotwein and A. J. D’Ercole

Department of Pediatrics, University of North Carolina at Chapel Hill (P.Y., T.B., H.A., A.J.D’E.), Chapel Hill, North Carolina 27599; and Departments of Biochemistry and Medicine, Washington University School of Medicine (Y.U., D.R., P.R.), St. Louis, Missouri 63110

Address all correspondence and requests for reprints to: Dr. A. J. D’Ercole, Department of Pediatrics, CB 7220, 509 Burnett-Womack, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7220.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin-like growth factor I (IGF-I) plays an important role in the development and function of the central nervous system (CNS). Little is known, however, about the factors and mechanisms involved in regulation of CNS IGF-I gene expression. To facilitate our goal to define mechanisms of IGF-I gene regulation in the CNS, we generated several lines of transgenic (Tg) mice that express firefly luciferase (LUC) under control of a 11.3-kb fragment from the 5' region of the rat IGF-I gene. Consistent with expression of the native IGF-I gene in murine brain, expression of the transgene predominated in neurons and astrocytes and used promoter 1, the major IGF-I promoter in the CNS and in most tissues. Transgene messenger RNA and protein expression rapidly increased after birth and peaked at postnatal (P) day 4 in all brain regions studied. LUC activities in all regions then gradually decreased to 0.5–4% of their peak values at P31, except for the olfactory bulb, which maintained about one third of its maximal activity. Compared with littermate controls, administration of dexamethasone decreased LUC activity and transgenic IGF-I messenger RNA abundance, whereas GH significantly increased the expression of the transgene. Addition of GH to cultured fetal brain cells from Tg mice for 12 h also increased LUC activity in a dose-dependent manner (77–388%). These results show that this IGF-I promoter transgene is expressed in a fashion similar to the endogenous IGF-I gene, and thus indicates that the transgene contains cis-elements essential for developmental, GH, and glucocorticoid regulation of IGF-I gene expression in the CNS. These Tg mice should serve as an useful model to study mechanisms of IGF-I gene regulation in the brain.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DURING central nervous system (CNS) development, insulin-like growth factor (IGF) I, a member of the insulin family, is widely expressed in a distinct temporal and spatial pattern. In rodent brain, IGF-I expression begins on or before embryonic (E) day 14 and gradually increases to a peak during the first postnatal week. IGF-I expression then declines significantly in most brain regions but continues throughout life (1, 2). Although IGF-I messenger RNA (mRNA) is predominately located in neurons and astrocytes, it also is expressed by cultured oligodendrocyte progenitors and mature oligodendrocytes (3) and may be produced by glial progenitors, because its mRNA is detected in the subventricular zone (the site of origin for these cells) in early postnatal development (4).

Our studies and the data of others have demonstrated that IGF-I plays an important role in CNS development. IGF-I promotes the proliferation and/or survival (5, 6, 7) and differentiation (5, 8) of oligodendrocytes and their progenitors, as well as stimulating myelination (9, 10, 11, 12). IGF-I also enhances the proliferation and/or survival of a variety of neurons and their precursors (13, 14, 15, 16), and stimulates neuronal differentiation and function (14, 17, 18).

Although the temporal and spatial coordinates of IGF-I gene expression in the CNS have been characterized, the mechanisms of the gene regulation are largely undefined. The paucity of studies on this topic may reflect the relatively low abundance of IGF-I mRNA in the adult brain, and the lack of neural cell lines expressing the gene. To facilitate our goal of defining the regulatory mechanisms controlling IGF-I gene expression in brain, we generated several lines of transgenic (Tg) mice that carry a firefly luciferase (LUC) reporter gene driven by rat IGF-I 5' regulatory sequences (IGF-I/LUC Tg mice). We observe high expression of the transgene within the CNS and in several other organs during development. We additionally find that the spatial and developmental pattern of IGF-I/LUC transgene mRNA and LUC enzymatic activity replicates that of endogenous IGF-I expression in the brain, and demonstrate that the transgene is regulated by two trophic factors, GH and glucocorticoids, in a fashion similar to the native IGF-I gene. Our results thus indicate that this transgene contains the cis-elements responsible for appropriate developmental and hormonal regulation in the CNS. These IGF-I/LUC Tg mice should serve as an useful model for further analysis of the regulation of IGF-I gene expression in the brain.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of Tg mice
The transgene (Fig. 1Go) used to generate Tg mice, designated IGF-I/LUC Tg mice, is comprised of 1) 11.3 kb 5' flanking regulatory region of the rat IGF-I gene; 2) an internal ribosomal entry sequence (IRES) derived from murine encephalomyocarditis virus (19, 20); 3) a firefly LUC reporter gene from plasmid pGL2 basic (Promega Biotech, Madison, WI); and 4) a SV40 sequence, also from plasmid pGL2 basic. The latter provides an additional splice site and a polyadenylation signal and site. The contiguous IGF-I gene sequence includes approximately 5.3 kb upstream of exon 1 (promoter 1), exon 1, the intron between exons 1 and 2 [which contains promoter 2 (21)], exon 2, intron 2, and the first 48 bp of exon 3. This 11.3-kb rat IGF-I gene fragment was linked to the IRES to ensure appropriate initiation of translation for LUC (19, 20). The approximately 14.5- kb transgene fragment was excised from the plasmid backbone using the restriction enzyme SalI, and gel-membrane purified. Classical microinjection technology was performed in the Transgenic Mice Facility of the Program in Molecular Biology and Biotechnology at the University of North Carolina at Chapel Hill.



View larger version (16K):
[in this window]
[in a new window]
  		Figure 1. Schematic diagram of 5' native rat IGF-I gene, 11.3-kb IGF-I/LUC transgene, and possible transgenic mRNAs. Top, Diagram of 5' portion of rat IGF-I locus. Promoters 1 and 2 (P1 and P2, respectively), and exons 1–3 are indicated, as are several restriction enzyme sites. Arrows over exon 1 represent major transcription start sites, whereas arrows over exon 2 indicate 5' minor and 3' major start sites, respectively. Middle and lower, Transgene and transgenic mRNA, respectively. Transgene comprises 11.3 kb from 5' portion of rat IGF-I gene, including 5.3 kb of P1, P2, exon 1, exon 2, and first 48 bp of exon 3. Transgene also includes an IRES, firefly LUC reporter gene, and a SV40 sequence, which provides an additional intron and a polyadenylation signal and site. Promoter 1-derived mRNAs would consist of exon 1, exon 3, IRES, and LUC; whereas promoter 2-derived mRNAs would consist of exon 2, exon 3, IRES, and LUC.

 
IGF-I/LUC Tg founder mice were identified by Southern hybridization analysis. The Tg mice then were bred as heterozygotes and routinely identified by PCR of tail genomic DNA (see below). Subsequently, homozygotes were generated by mating male and female heterozygous Tg mice. Heterozygous and homozygous Tg mice differed only in that homozygous Tg mice exhibit about 2-fold higher LUC activity, and in this report, only heterozygous Tg mice were studied. To obtain larger numbers of heterozygous Tg mice for study, homozygous Tg mice were often bred with normal mice. There were no histological abnormalities observed in the brain or other organs of IGF-I/LUC Tg mice, and the development of Tg mice appeared normal. Mice were maintained with 12 h light/12 h dark cycles at 22 C. To study the hormone regulation of Tg and native IGF gene expression in vivo, Tg mice were given ip injections of human GH (hGH, Genentech, South San Francisco, CA) or dexamethasone (Sigma Chemical Co., St. Louis, MO) for designated times. All procedures used were approved by the institutional review committee of the University of North Carolina at Chapel Hill.

Southern hybridization analysis and PCR
For Southern hybridization analysis, genomic DNA was extracted from mouse tails as described by Strauss et al. (22). After digestion with the restriction enzyme BamHI, aliquots of 7 µg DNA were electrophoresed on 1% agarose gels, transferred onto a GeneScreen membrane (DuPont NEN, Boston, MA), UV cross-linked, and hybridized with radiolabeled DNA probes (see below) in Church’s buffer (0.5 M sodium phosphate, pH 7.1, 7% SDS, and 0.1 mM EDTA) and washed at high stringency (40 mM sodium phosphate with 0.2% SDS at 55–60 C for 60 min).

For PCR, tails were digested with proteinase K (0.4 mg/ml) in 50 mM Tris-HCl, pH 7.4, 1 mM EDTA at 55 C for 6 h. After inactivation of proteinase K at 95–100 C for 10 min, supernatants were collected by centrifugation and subjected to PCR using 20 mer oligonucleotide primers. The amplified 393-bp DNA fragment corresponded to bp 511–893 of the LUC gene (Promega Catalog, p. 226, 1997).

Probes
For Southern hybridization assay, a 0.6-kb BamHI DNA fragment from the IRES was gel-purified and radiolabeled using a random primed DNA labeling Kit (Boehringer Mannheim, Indianapolis, IN) and 32P-labeled deoxycytidine triphosphate (Amersham, Arlington Heights, IL).

For RNase protection assay, DNA templates for E1 and E2 probes were generated by PCR and cloned into pBluescript (Stratagene, La Jolla, CA). The identity of the probe templates was confirmed by DNA sequencing. The 32P-labeled RNA probes were generated using T7 RNA polymerase and 32P-labeled cytidine triphosphate. The E1 probe, complementary to the 3' part of rat IGF-I exon 1 (85 bp), the 5' portion of exon 3 (48 bp), and the 5' end of the IRES (20 bp), detects promoter 1-derived transgenic mRNA. The E2 probe is complementary to the 3' part of rat IGF-I exon 2 (75 bp), the initial 48 bp of exon 3, and the first 20 bp of the IRES, and detects promoter 2-derived transgenic mRNA (Fig. 1Go).

RNase protection assay
Total RNA was extracted from tissues using the single-step method (23). Solution-hybridization RNase protection experiments were performed as described by us previously (24) using total RNA and 32P-labeled antisense RNA probes from the cloned DNA templates outlined above. Protected 32P-labeled probe fragments were separated on 6% polyacrylamide/8.3 M urea gels, and visualized by autoradiography. The intensity of autoradiography signal was quantitated using a densitometer (Molecular Dynamics, Sunnyvale, CA) and normalized to ethidium bromide staining of 18S ribosomal RNA from a duplicate RNA sample loaded on a 1% agarose-formaldehyde gel.

Measurement of LUC activity
Brains and other organs were rapidly removed from IGF-I/LUC Tg mice or control littermates, dissected, frozen in liquid nitrogen, and stored at -80 C until use. Proteins were extracted, and LUC activity was measured using the Luciferase Assay System (Promega) and a luminometer (Monolight 2010, Analytical Luminescence Laboratory, Ann Arbor, MI), according the manufacturer’s protocol. Tissues were homogenized with a prechilled mortar and pestle, lysed with 200–400 µl 1x lysis buffer (25 mM Tris-phosphate, pH 7.8, 2 mM dithiothreitol, 2 mM 1, 2-diaminocyclohexane-N, N,N,-tetraacetid acid, 10% glycerol, and 1% Triton X-100), and centrifuged for 5 min at 4 C. The supernatants were collected, and protein concentration was determined using BCA Protein Assay Kit (Pierce, Rockford, IL) with BSA as a standard. Protein (400 µg) was mixed with 100 µl LUC assay reagent [470 mM luciferin, 270 mM coenzyme A, 530 mM ATP, 20 mM tricine, 0.1 mM EDTA, 33.3 mM dithiothreitol, 2.67 mM MgSO4, 1.07 mM (MgCO3)4 Mg(OH)2-5H2O] at 25 C, and light intensity was measured with a luminometer over 10 sec.

Immunohistochemistry
Brains were obtained from IGF-I/LUC Tg and normal mice at postnatal (P) day 14, after transcardic perfusion with 4% paraformaldehyde in PBS, pH 7.4, and postfixation overnight at 4 C in the same fixative plus 20% sucrose. Serial, 10-µm thick, sagittal sections were cut with a cryostat and mounted onto gelatin-coated slides. After washing with PBS, the sections were incubated with a polyclonal antibody against LUC (1:800 dilution; Biogenesis, Franklin, MA) and a biotinylated secondary antibody. Antibody-antigen complexes were detected by the ABC kit conjugated with alkaline phosphatase (Vector Labs., Burlingame, CA) and visualized by incubation with nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl-phosphate (BCIP).

Aggregated brain cell culture
Cell cultures were prepared using the modified methods of Russo et al. (25) and Marienhagen et al. (26). The brains were removed and collected from E18 Tg mice, digested with trypsin at 37 C for 20 min, and dissociated into a single cell suspension by repeated trituration. Cell clumps were removed by passing the suspensions sequentially through 70-µm and 40-µm nylon screens. The cells were collected by centrifugation, resuspended in DMEM (Sigma), seeded onto 12-well plates coated with 1% soft agar, and cultured in DMEM supplemented with 4.5 g/L glucose and 10% FCS (Sigma). After about 9 h in culture, the cells aggregated. Cells then were exposed to hGH for 12 or 24 h. Proteins were extracted as described above and analyzed for LUC activity.

Statistic analyses
Statistical comparisons were made using Student’s t test or the Wilcoxon rank sum test for unpaired samples.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Six founder Tg mice were identified by Southern blot analysis, and each was bred to generate lines of Tg mice. Lines 1, 5, 12, and 20 expressed high levels of LUC enzymatic activity in brain and varying degrees of activity in testis, spleen, heart, lung, and kidney, whereas lines 2 and 17 expressed LUC only in testis (Table 1Go). The high- level expression of transgene in testes is consistent with previous observation on IGF-I gene expression in this organ in rodents (27, 28). No LUC activity was found in liver or fat in any Tg lines. The development of IGF-I/LUC Tg mice was normal, and there were no morphological abnormalities observed in the brain or other organs (data not shown).


View this table:
[in this window]
[in a new window]
  		Table 1. LUC activity in various organs of different lines of adult IGF-I/LUC Tg mice

 
To examine the spatial and temporal patterns of expression of the IGF-I/LUC transgene in the CNS, brains from P0 (day of birth) to P31 Tg mice were dissected into six regions: olfactory bulb, cerebral cortex, hippocampus, diencephalon, cerebellum, and brain stem, and LUC activity was determined. The patterns of transgene expression were similar for the three lines studied (lines 1, 5, and 12), and only data from line 1 are shown (Fig. 2Go). LUC activity in all six brain regions was low at birth and rapidly increased to a peak at P4 [cerebral cortex (886 ± 152 U/µg protein; mean ± SE), olfactory bulb (627 ± 195), and brain stem (645 ± 85) exhibited the highest activity at P4, followed by diencephalon (175 ± 27), cerebellum (118 ± 75), and hippocampus (114 ± 30)]. By P8–P10, LUC activity in all regions had decreased significantly, and by P31 LUC expression had declined to <4% of peak values in all tissues except for olfactory bulb, which maintained about a third of its highest activity. There were no differences in LUC expression in the brain between male and female IGF-I/LUC Tg mice, and therefore, LUC enzymatic activity from both sexes was combined. Localization of IGF-I/LUC transgene expression in brain was determined using immunohistochemical staining for LUC. The immunostaining was found predominately in the cytoplasm of neurons and astrocytes, as judged by their morphology (Fig. 3Go).



View larger version (28K):
[in this window]
[in a new window]
  		Figure 2. Regional brain LUC activity in line 1 IGF-I/LUC Tg mice during development. Brains of IGF-I/LUC Tg mice were collected and dissected at indicated ages. LUC activity was measured in olfactory bulb (OLF), cerebral cortex (CTX), hippocampus (HIP), diencephalon (DIE), brain stem (BS), and cerebellum (CB). Values are means ± SE from three to eight animals.

 


View larger version (119K):
[in this window]
[in a new window]
  		Figure 3. Immunostaining of LUC in cerebral cortex (A and B) and cerebellum (C and D). Brains from P14 IGF-I/LUC Tg (B and D) and normal mice (A and C) were perfused with 4% paraformaldehyde in PBS by intracardiac injection and sagittally sectioned. Sites of LUC expression were recognized by a polyclonal antibody against LUC protein and visualized by colorimetric reaction using NBT/BCIP. Arrows in B indicate labeled neurons, and arrowheads in D point to labeled Purkinje cells. Bar = 20 µm.

 
We next studied transgene expression at the mRNA level by RNase protection assay, using 32P-labeled single-stranded riboprobes complementary to correctly spliced transgene-derived transcripts. Using E1 riboprobes, which are complementary to 5' portions of transgenic and native IGF-I mRNA derived from promoter 1, appropriately sized transcripts were detected in whole brain and in several brain regions, including cerebral cortex, brain stem, and diencephalon, but were not seen in liver, heart, spleen, or lung (Fig. 4Go). By contrast, when E2 riboprobes were used that are complementary to 5' portions of transgenic and native IGF-I mRNAs derived from promoter 2, no transgene-derived mRNA was found in any organs studied (data not shown). These observations indicate that transcripts directed by rat IGF-I promoter 1 predominate in these Tg mice, consistent with the expression pattern of the endogenous IGF-I gene in rodents (29). Additional time course studies showed that transgene mRNA was expressed in these tissues from P4–P31, in a manner similar to the expression of native IGF-I mRNA (Fig. 4Go and data not shown).



View larger version (74K):
[in this window]
[in a new window]
  		Figure 4. Expression of IGF-I/LUC Tg mRNA in brains of developing and adult Tg mice. Total RNA (20 µg) isolated from P10 and P200 Tg mice was used in a RNase protection assay with 32P-labeled E1 riboprobes, as described in Materials and Methods. Yeast tRNA was included as a negative control. Protected 32P-labeled probe fragments were visualized by autoradiography after gel electrophoresis. Arrows at left indicate native IGF-I and Tg mRNA bands, respectively. NS, Nonspecific. Exon 1 probe and Tg and endogenous mRNAs that it protects are diagrammed below figure.

 
To examine GH regulation of transgene expression in the brain, P17 and P31 IGF-I/LUC Tg mice (line 1) were given hGH (1.5 µg/g body weight) ip every 12 h for 36 h. Animals were killed 12 h after the last injection. Compared with PBS-treated littermate controls, LUC activity in P17 mice treated with hGH was significantly increased in olfactory bulb (P < 0.001), cerebral cortex (P < 0.01), hippocampus (P < 0.001), and brain stem (P < 0.05) (Fig. 5Go). A similar trend was also noted in several brain regions in hGH-treated P31 Tg mice, but those results did not reach statistical significance (Fig. 5Go). After hGH injection steady state levels of the transgene mRNA were also increased in P17 brain stem and cerebral cortex, the only two tissues examined (Fig. 6Go). Compared with PBS-treated Tg littermate controls, 2-fold and 2.5-fold increases in the transgene mRNA were observed in brain stem (P < 0.02) and cerebral cortex (P < 0.05), respectively. Similarly, the abundance of native IGF-I mRNA was increased by approximately 50% in hGH-treated brain stem, as compared with control littermates.



View larger version (26K):
[in this window]
[in a new window]
  		Figure 5. Effect of GH on LUC activity in brains of P17 (open bars) and P31 (filled bars) IGF-I/LUC Tg mice. Tg mice were given three ip injections of hGH (1.5 µg/g body weight) at 12-h intervals. Twelve hours after last injection, brains were dissected, and tissues were lysed for measurement of LUC activity. Results are presented as percentage of values measured in PBS-treated Tg littermates (means ± SEM of four mice). A dotted line is drawn at 100% to facilitate comparison. Statistical comparisons were made using Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with PBS-treated littermate controls.

 


View larger version (82K):
[in this window]
[in a new window]
  		Figure 6. Effects of GH on expression of IGF-I/LUC transgene mRNA in cerebral cortex (CTX) and brain stem (BS). A, A representative autoradiograph of RNase protection assay for of IGF-I/LUC transgene mRNA in CTX and BS. Total RNA (20 µg), isolated from P17 Tg mice treated with hGH or PBS, was used in a RNase protection assay with a 32P-labeled E1 riboprobe, as described in Materials and Methods. Mouse and rat liver RNA and yeast tRNA were used as positive and negative controls, respectively. Protected 32P-labeled probe fragments were visualized by autoradiography following gel electrophoresis. Arrows at left indicate native IGF-I and transgene mRNA bands, respectively. Each lane was loaded with a sample from a different animal. Bottom, Ethidium bromide-stained 18S ribosomal RNA bands corresponding to brain RNA samples shown above. B, Quantitative analysis of IGF-I/LUC transgene expression. Autoradiograph shown in Fig. 6Go and results of two other similar experiments were scanned and intensity of bands was quantitated using a densitometer. Mean ± SEM of three samples is shown. Statistical comparisons with PBS-treated littermate controls were made using Student’s t test. Veh, Vehicle.

 
To further examine the effects of GH on transgene expression, we treated aggregated brain cultures derived from E18 Tg mice with hGH and measured LUC activity. Compared with nontreated controls, LUC enzymatic activity was markedly increased after exposing these cells to hGH for 12 h at concentrations of 10 ng/ml (~415% of control, P < 0.05), 100 ng/ml (~488%, P < 0.01), and 1000 ng/ml (~177%; P = NS) (Fig. 7Go). Smaller increases were observed following 24 h of GH treatment. The decline in response to GH at 1000 ng/ml may be a result of GH receptor down-regulation, or alternatively an inhibition of GH receptor dimerization, a known receptor response in the presence of GH excess (30).



View larger version (29K):
[in this window]
[in a new window]
  		Figure 7. Effect of GH on LUC activity in aggregated E18 brain cultures. Brains from E18 Tg mice were dissociated with trypsin. Single cell suspensions were collected, resuspended, and seeded into 12-well plates coated with 1% agar. After 9 h in culture, cultures were exposed to hGH for 12 or 24 h. Proteins were extracted, and LUC enzymatic activity activity was measured. Mean ± SEM of three samples is shown. Statistical comparisons with nontreated controls were made using Student’s t test. *, P < 0.05; **, P < 0.01.

 
Next we examined the effect of glucocorticoids on transgene expression during development. P7 Tg mice and control mice were killed 6 h after a single ip injection of dexamethasone (1 µg/g body weight). LUC activities in all regions studied except olfactory bulb and diencephalon were significantly decreased (Fig. 8Go). Compared with PBS-treated Tg littermates, LUC activity declined by approximately 46% in cerebral cortex (P < 0.05), approximately 36% in brain stem (P <= 0.001), approximately 33% in hippocampus (P < 0.01), and approximately 25% in cerebellum (P < 0.05). Dexamethasone treatment consistently increased LUC activity by approximately 42% in diencephalon, but these increases were not statistically significant.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 8. Effect of dexamethasone on LUC activity in brain. P7 Tg mice were given a single ip injection of dexamethasone (1 µg/g body weight) and killed 6 h later. Brains were dissected, and LUC activity measured. Results are presented as a percentage of values obtained in PBS-treated Tg littermates and represent means ± SEM of four mice. A dotted line is drawn at 100% to facilitate comparison. Statistical comparisons were made using Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with PBS-treated littermate controls.

 
Consistent with the changes in LUC activity, dexamethasone treatment significantly decreased the abundance of transgene mRNA in cerebral cortex, as revealed by RNase protection analysis (Fig. 9Go). Compared with PBS-treated littermate controls, dexamethasone decreased the abundance of transgene mRNA to 42% of initial values (P < 0.01) in cerebral cortex, but had only a modest affect on endogenous IGF-I mRNA (80% of initial values; P = NS).



View larger version (45K):
[in this window]
[in a new window]
  		Figure 9. Dexamethasone decreases levels of transgene IGF/LUC mRNA in cerebral cortex. Total RNA (20 µg) isolated from P7 Tg mice 6 h after a single ip injection of dexamethasone (1 µg/g body weight) was hybridized with 32P-labeled E1 probe, as described in Materials and Methods. A, Liver RNA and yeast tRNA (20 µg) were used as positive and negative controls, respectively. Arrows at right indicate native IGF-I and transgene mRNA bands, respectively. Bottom, Ethidium bromide-stained 18S ribosomal RNA bands from same samples. B, Autoradiograph and results of two other similar experiments were scanned and intensity of bands was quantitated using a densitometer. Mean ± SEM of six control and four treated samples is shown. Statistical comparisons were made using Wilcoxon rank sum test. Dex, Dexamethasone; Veh, vehicle. Note that SEM for Dex treatment is too small to appear on figure.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have generated several lines of Tg mice with a LUC reporter driven by the rat IGF-I promoter, and have found that these Tg mice express LUC activity in the brain. The spatiotemporal pattern of transgene expression and the predominance of transcripts derived from promoter 1 parallel native IGF-I gene expression during development, as revealed by in situ hybridization (2) and RNase protection assay (Ref. 29, 31 and this study). We also have shown that GH and glucocorticoids regulate expression of the transgene in a manner similar to that of the endogenous IGF-I gene during brain development. Our results, thus, demonstrate that this 11.3-kb rat IGF-I gene fragment contains the necessary cis-elements for appropriate developmental and hormonal regulation in the CNS.

GH is a major regulator of IGF-I gene expression in liver, as well as in several other organs (21). In adult hypophysectomized rats, brain IGF-I mRNA levels are reduced by approximately 60% and are restored to near normal values by intracerebral GH injection (32) or by systemic GH administration (33). Recently, the GH receptor and its mRNA have been detected in the developing rodent brain (34, 35, 36). The cellular distribution of the GH receptor and its mRNA has been mapped to neurons and astrocytes, and its expression peaks in the first week of postnatal life when IGF-I expression is maximal (4, 16), indicating a correlation of GH receptor and IGF-I expression. We find that short-term GH treatment also significantly increases expression of the IGF-I/LUC transgene both in vivo and in cultured brain cells. Our data are thus consistent with the distribution and ontogeny of GH receptor, and strongly support the hypothesis that GH plays a role in the regulation of brain IGF-I gene expression during development.

In previous studies we have shown that GH rapidly induces IGF-I transcription in the liver of hypophysectomized rats, and have found a correlation between activation of gene transcription and the appearance of a chromatin alteration, manifested as a DNase I hypersensitive site (HS) located in the second intron of the rat IGF-I gene (24). The transgene constructed for the current study contains this HS, termed HS7 (24), and also contains HS2 through HS6 and HS8 (37). One of these latter sites, HS3, maps to the proximal part of IGF-I promoter I (24), within a region that is required for high-level promoter activity after transient transfection into cultured cells (38, 39, 40, 41, 42, 43), and thus, probably contributes to transgene function. Although our results also are consistent with the hypothesis that HS7 plays a role in GH-induced IGF-I transcription, more direct studies will be required to confirm this idea.

Although the origin of the GH that regulates endogenous IGF-I gene expression in brain during development is not clear, it may be produced in brain, rather than the pituitary. This hypothesis is supported by the findings that GH-like immunoreactive material can be extracted from several regions of rat brain (44, 45, 46), and that GH mRNA is detected in basal cortex, hippocampus, and caudate nucleus-putamen by in situ hybridization histochemistry (47). During development, brain GH immunoreactivity increases transiently to a peak during the late fetal and early neonatal periods, the same time when expression of the GH receptor and IGF-I are maximal (4, 16, 34, 35). However, circulating GH also may contribute to regulation of IGF-I gene expression in brain, because in rats hypophysectomy reduces both brain GH levels (48) and IGF-I transcript abundance (32), and systemic administration of GH increases brain IGF-I mRNA (33). These latter observations suggest that GH crosses the blood-brain barrier (BBB) to influence IGF-I gene expression. Our results show that systemic treatment with GH significantly increases IGF-I gene and transgene expression in brains of P17 mice, whereas it causes only a minor rise in LUC activity in brains of P31 mice. The latter finding suggests that during early brain development, circulating GH can traverse an immature BBB and influence brain IGF-I expression and brain development. With aging the mature BBB may limit access of circulating GH to the CNS and thus prevent effects on brain IGF-I gene expression. This latter speculation is further supported by our finding that incubation with GH rapidly stimulates LUC activity in cultured brain cells.

We also found that dexamethasone treatment significantly reduces transgene expression in the developing cerebral cortex, hippocampus, brain stem, and cerebellum, indicating that glucocorticoids may be involved in brain IGF-I gene expression during development. This idea is supported by in vitro evidence that dexamethasone reduces IGF-I mRNA abundance by approximately 60% and approximately 40% in primary cultures of rat neurons and astrocytes, respectively (49).

IGF-I gene activity in liver gradually increases in the first month after birth and remains high throughout adult life (50), and hepatic IGF-I production is the major source of serum IGF-I (51). In contrast, fat exhibits high IGF-I gene expression during the first 2 weeks of postnatal life, and then this expression gradually decreases (52). Surprisingly, we did not detect LUC activity or transgene transcripts in either tissue in any lines of our Tg mice. The reasons for the absence of transgene expression in liver and fat are not clear. One possibility is a position effect of transgene insertion into the mouse genome that limits its expression in certain tissues, but such a result seems unlikely to have occurred in all six lines of Tg mice. Other possibilities are that the transgene contains inhibitory elements that are active in liver and fat, or alternately that it lacks regulatory elements necessary for expression in these tissues. At present we have no evidence for either of these latter alternatives, but both can be addressed through construction of new lines of Tg mice.

Consistent with the findings of others showing abundant expression of IGF-I mRNA in rodent testis (27, 28), we also demonstrated that a high expression of LUC transgene in testis. It has been shown that IGF-I is expressed in mouse testis and ovary in a distinct pattern during development (28). In testis, IGF-I mRNA is located in the interstitial compartment of developing mice, whereas it predominates in the seminiferous epithelium of P35 mice. In ovary IGF-I mRNA is detected in granulosa cells but not in oocytes. Whether the LUC transgene expression in testis has same distribution pattern, and whether ovary expresses the transgene needs further investigation. Nonetheless, the fact that all lines of transgenic mice express a high level of LUC activity in testis suggests that the 11.3-kb IGF-I 5' regulatory region contains appropriate regulatory elements for the testis.


   Footnotes
 
1 This work was supported by NIH Grants HD-08299 (to A.J.D) and DK-37449 (to P.R.). Back

2 Supported by NIH training Grant T32DK-07129. Back

3 Current address: Division of Molecular Medicine, Department of Medicine, Oregon Health Sciences University, Portland, Oregon 97201-3098. Back

Received June 25, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Bach MA, Shen-Orr Z, Lowe, Jr, WL, Roberts, Jr, CT, LeRoith D 1991 Insulin-like growth factor I mRNA levels are developmentally regulated in specific regions of the rat brain. Mol Brain Res 10:43–48[Medline]
  2. Bartlett WP, Li X-S, Williams M, Benkovic S 1991 Localization of insulin-like growth factor-1 mRNA in murine central nervous system during postnatal development. Dev Biol 147:239–250[CrossRef][Medline]
  3. Shinar Y, McMorris FA 1995 Developing oligodendroglia express mRNA for insulin-like growth factor-I, a regulator of oligodendrocyte development. J Neurosci Res 42:516–527[CrossRef][Medline]
  4. Bartlett WP, Li X-S, Williams M 1992 Expression of IGF-I mRNA in the murine subventricular zone during postnatal development. Mol Brain Res 12:285–291[Medline]
  5. Masters BA, Werner H, Roberts, Jr, CT, LeRoith D, Raizada MK 1991 Insulin-like growth factor I (IGF-I) receptors and IGF-I action in oligodendrocytes from rat brains. Regul Pept 33:117–131[CrossRef][Medline]
  6. McMorris FA, Smith TM, DeSalvo S, Furlanetto RW 1986 Insulin-like growth factor I/somatomedin C: a potent inducer of oligodendrocyte development. Proc Natl Acad Sci USA 83:822–826[Abstract/Free Full Text]
  7. Barres BA, Hart IK, Coles HS, Burne JF, Voyvodic JT, Richardson WD, Raff MC 1992 Cell death and control of cell survival in the oligodendrocyte lineage. Cell 70:31–46[CrossRef][Medline]
  8. McMorris FA, Dubois-Dalcq M 1988 Insulin-like growth factor I promotes cell proliferation and oligodendroglial commitment in rat glial progenitor cells developing in vitro. J Neurosci Res 21:199–209[CrossRef][Medline]
  9. Beck KD, Powell-Braxton L, Widmer HR, Valverde J, Hefti F 1995 Igf1 gene disruption results in reduced brain size, CNS hypomyelination, and loss of hippocampal granule and striatal parvalbumin-containing neurons. Neuron 14:717–730[CrossRef][Medline]
  10. Carson MJ, Behringer RR, Brinster RL, McMorris FA 1993 Insulin-like growth factor I increases brain growth and central nervous system myelination in transgenic mice. Neuron 10:729–740[CrossRef][Medline]
  11. Ye P, Carson J, D’Ercole AJ 1995 Insulin-like growth factor-I influences the initiation of myelination: studies of the anterior commissure of transgenic mice. Neurosci Lett 201:235–238[CrossRef][Medline]
  12. Ye P, Carson J, D’Ercole AJ 1995 In vivo actions of insulin-like growth factor-I (IGF-I) on brain myelination: studies of IGF-I and IGF binding protein-1 (IGFBP-1) transgenic mice. J Neurosci 15:7344–7356[Abstract]
  13. DiCicco-Bloom E, Black IB 1989 Depolarization and insulin-like growth factor-I (IGF-I) differentially regulate the mitotic cycle in cultured rat sympathetic neuroblasts. Brain Res 491:403–406[CrossRef][Medline]
  14. Zackenfels K, Oppenheim RW, Rohrer H 1995 Evidence for an important role of IGF-I and IGF-II for the early development of chick sympathetic neurons. Neuron 14:731–741[CrossRef][Medline]
  15. Ye P, Xing Y, Dai Z, D’Ercole AJ 1996 In vivo actions of insulin-like growth factor-I (IGF-I) on cerebellum development in transgenic mice: evidence that IGF-I increases proliferation of granule cell progenitors. Dev Brain Res 95:44–54[Medline]
  16. Torres-Aleman I, Pons S, Areevalo MA 1994 The insulin-like growth factor I system in the rat cerebellum: developmental regulation and role in neuronal survival and differentiation. J Neurosci Res 39:117–126[CrossRef][Medline]
  17. Knusel B, Hefti F 1991 Trophic actions of IGF-I, IGF-II and insulin on cholinergic and dopaminergic brain neurons. Adv Exp Med Biol 293:351–360[Medline]
  18. Ang LC, Bhaumick B, Juurlink BHJ 1993 Neurite promoting activity of insulin, insulin-like growth factor I and nerve growth factor on spinal motoneurons is astrocyte dependent. Dev Brain Res 74:83–88[CrossRef][Medline]
  19. Ghattas IR, Sanes JR, Majors JE 1991 The encephalomyocarditis virus internal ribosome entry site allows efficient coexpression of two genes from a recombinant provirus in cultured cells and in embryos. Mol Cell Biol 11:5848–5859[Abstract/Free Full Text]
  20. Jang SK, Davies MV, Kaufman RJ, Wimmer E 1989 Initiation of protein synthesis by internal entry of ribosomes into the 5' nontranslated region of encephalomyocarditis virus RNA in vivo. J Virol 63:1651–1660[Abstract/Free Full Text]
  21. Rotwein P 1991 Structure, evolution, expression and regulation of insulin-like growth factors I and II. Growth Factors 5:3–18[Medline]
  22. Strauss M 1995 Preparation of genomic DNA. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds) Current Protocols in Molecular Biology. John Wiley, New York, pp 2.2.1
  23. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  24. Bichell DP, Kikuchi K, Rotwein P 1992 Growth hormone rapidly activates insulin-like growth factor I gene transcription in vivo. Mol Endocrinol 6:1899–1908[Abstract/Free Full Text]
  25. Russo VC, Edmondson SR, Mercuri FA, Buchanan CR, Werther GA 1994 Identification, localization, and regulation of insulin-like growth factor binding proteins and their messenger ribonucleic acids in the newborn rat olfactory bulb. Endocrinology 135:1437–1446[Abstract]
  26. Marienhagen K, Pedersen P-H, Terzis AJA, Laerum OD, Arnold H, Bjerkvig R 1994 Interactions between fetal rat brain cells and mature brain tissue in vivo and in vitro. Neuropathol Appl Neurobiol 20:130–143[Medline]
  27. Oliver JE, Aitman TJ, Powell JF, Wilson CA, Clayton RN 1989 Insulin-like growth factor I gene expression in the rat ovary is confined to the granulosa cells of developing follicles. Endocrinology 124:2671–2679[Abstract/Free Full Text]
  28. Baker J, Hardy MP, Zhou J, Bondy C, Lupu F, Bellve AR, Efstratiadis A 1996 Effect of an Igf1 gene null mutation on mouse reproduction. Mol Endocrinol 10:903–918[Abstract/Free Full Text]
  29. Adamo ML, Neuenschwander S, LeRoith D 1994 Structure, expression, and regulation of the IGF-I gene. Adv Exp Med Biol 343:1–11
  30. Postel-Vinay MC, Finidori J, Goujon L, Sotiropoulos A, Dinerstein H, Edery M, Kelly PA 1994 Molecular biology of the growth hormone receptor. Endocrinol Metab 1[Suppl A]:3–7
  31. Rotwein P, Burgess SK, Milbrandt JD, Krause JE 1988 Differential expression of insulin-like growth factor genes in rat central nervous system. Proc Natl Acad Sci USA 85:265–269[Abstract/Free Full Text]
  32. Hynes MA, Van Wyk JJ, Brooks PJ, D’Ercole AJ, Jansen M, Lund PK 1987 Growth hormone dependence of somatomedin-C/insulin-like growth factor-I and insulin-like growth factor-II messenger ribonucleic acids. Mol Endocrinol 1:233–242[Abstract/Free Full Text]
  33. Wood TL, Berelowitz M, Gelato MC, Roberts, Jr, CT, LeRoith D, Millard WJ, McKelvy JF 1991 Hormonal regulation of rat hypothalamic neuropeptide mRNAs: effect of hypophysectomy and hormone replacement on growth-hormone-releasing factor, somatostatin and the insulin-like growth factors. Neuroendocrinology 53:298–305[Medline]
  34. García-Aragón J, Lobie PE, Muscat GEO, Gobius KS, Norstedt G, Waters MJ 1992 Prenatal expression of the growth hormone (GH) receptor/binding protein in the rat: a role for GH in embryonic and fetal development. Development 114:869–876[Abstract]
  35. Lobie PE, García-Aragón J, Lincoln DT, Barnard R, Wilcox JN, Waters MJ 1993 Localization and ontogeny of growth hormone receptor gene expression in the central nervous system. Dev Brain Res 74:225–233[Medline]
  36. Zhai Q, Lai Z, Roos P, Nyberg F 1996 Characterization of growth hormone binding sites in rat brain. Acta Paediatr 406[Suppl]:92–95
  37. Kikuchi K, Bichell DP, Rotwein P 1992 Chromatin changes accompany the developmental activation of insulin-like growth factor I gene transcription. J Biol Chem 267:21505–21511[Abstract/Free Full Text]
  38. Adamo ML, Lanau F, Neuenschwander S, Werner H, LeRoith D, Roberts, Jr, CT 1993 Distinct promoters in the rat insulin-like growth factor-I (IGF-I) gene are active in CHO cells. Endocrinology 132:935–937[Abstract/Free Full Text]
  39. McCarthy TL, Thomas MJ, Centrella M, Rotwein P 1995 Regulation of insulin-like growth factor I transcription by cyclic AMP in fetal rat bone cells through an element with exon1:protein kinase A-dependent control without a consensus cyclic AMP response element. Endocrinology 136:3901–3908[Abstract]
  40. Thomas MJ, Umayahara Y, Shu H, Centrella M, Rotwein P, McCarthy TL 1996 Identification of the cAMP response element that controls transcriptional activation of the insulin-like growth-I gene by prostaglandin E2 in osteoblasts. J Biol Chem 271:21835–21841[Abstract/Free Full Text]
  41. Hall LJ, Kajimoto K, Bichell D, Kim SW, James PL, Counts D, Nixon LJ, Tobin G, Rotwein P 1992 Functional analysis of the rat insulin-like growth factor I gene and identification of an IGF-I gene promoter. DNA Cell Biol 11:301–313[Medline]
  42. Pao C-I, Zhu J-I, Robertson DG, Lin KM, Farmer PK, Begovic S, Wu G-J, Phillips LS 1995 Transcriptional regulation of the rat insulin-like growth factor-I gene involves metabolism-dependent binding of nuclear proteins to a downstream region. J Biol Chem 270:24917–24923[Abstract/Free Full Text]
  43. An MR, Lowe, Jr, WL 1995 The major promoter of the rat insulin-like growth factor-I gene binds a protein complex that is required for basal expression. Mol Cell Endocrinol 114:77–89[CrossRef][Medline]
  44. Hojvat S, Baker G, Kirsteins L, Lawrence AM 1982 Growth hormone immunoreactivity in the rodent and primate CNS: distribution, characterization and presence post hypophysectomy. Brain Res 239:543–557[CrossRef][Medline]
  45. Pacold ST, Kirsteins L, Hojvat S, Lawrence AM, Hagen TC 1978 Biologically active pituitary hormones in the rat brain amygdaloid nucleus. Science 199:804–806[Abstract/Free Full Text]
  46. Lechan RM, Nestler JL, Molitch ME 1981 Immunohistochemical identification of a novel substance with human growth hormone-like immunoreactivity in rat brain. Endocrinology 109:1950–1962[Abstract/Free Full Text]
  47. Gossard F, Dihl F, Pelletier G, Dubois PM, Morel G 1987 In situ hybridization to rat brain and pituitary gland of growth hormone cDNA. Neurosci Lett 79:251–256[CrossRef][Medline]
  48. Hojvat S, Emanuele NV, Kirsteins L, Lawrence AM 1986 Impact of endocrine manipulations on brain-based rat growth hormone. Neuroendocrinology 44:355–360[CrossRef][Medline]
  49. Adamo M, Werner H, Farnsworth W, Roberts, Jr, CT, Raizada M, LeRoith D 1988 Dexamethasone reduces steady state insulin-like growth factor I messenger ribonucleic acid levels in rat neuronal and glial cells in primary culture. Endocrinology 123:2565–2570[Abstract/Free Full Text]
  50. Lund PK, Moats-Staats BM, Hynes MA, Simmons JG, Jansen M, D’Ercole AJ, Van Wyk JJ 1986 Somatomedin-C/insulin-like growth factor-I and insulin-like growth factor-II mRNAs in rat fetal and adult tissues. J Biol Chem 261:14539–14544[Abstract/Free Full Text]
  51. Schwander JC, Hauri C, Zapf J, Froesch ER 1983 Synthesis and secretion of insulin-like growth factor and its binding protein by the perfused rat liver: dependence on growth hormone status. Endocrinology 113:297–305[Abstract/Free Full Text]
  52. Peter MA, Winterhalter KH, Böni-Schnetzler M, Froesch ER, Zapf J 1993 Regulation of insulin-like growth factor-I (IGF-I) and IGF-binding proteins by growth hormone in rat white adipose tissue. Endocrinology 133:2624–2631[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
S. Eleswarapu, Z. Gu, and H. Jiang
Growth Hormone Regulation of Insulin-Like Growth Factor-I Gene Expression May Be Mediated by Multiple Distal Signal Transducer and Activator of Transcription 5 Binding Sites
Endocrinology, May 1, 2008; 149(5): 2230 - 2240.
[Abstract] [Full Text] [PDF]

Home page
 Exp. Biol. Med.Home page
S. S. Daftary and A. C. Gore
IGF-1 in the Brain as a Regulator of Reproductive Neuroendocrine Function
Experimental Biology and Medicine, May 1, 2005; 230(5): 292 - 306.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y. Wang and H. Jiang
Identification of a Distal STAT5-binding DNA Region That May Mediate Growth Hormone Regulation of Insulin-like Growth Factor-I Gene Expression
J. Biol. Chem., March 25, 2005; 280(12): 10955 - 10963.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. Woelfle, J. Billiard, and P. Rotwein
Acute Control of Insulin-like Growth Factor-I Gene Transcription by Growth Hormone through Stat5b
J. Biol. Chem., June 13, 2003; 278(25): 22696 - 22702.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
N. D. Aberg, B. Carlsson, L. Rosengren, J. Oscarsson, O. G. P. Isaksson, L. Ronnback, and P. S. Eriksson
Growth Hormone Increases Connexin-43 Expression in the Cerebral Cortex and Hypothalamus
Endocrinology, October 1, 2000; 141(10): 3879 - 3886.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
C. Benbassat, L. N. N. Shoba, M. Newman, M. L. Adamo, S. J. Frank, and W. L. Lowe Jr.
Growth Hormone-Mediated Regulation of Insulin-Like Growth Factor I Promoter Activity in C6 Glioma Cells
Endocrinology, July 1, 1999; 140(7): 3073 - 3081.
[Abstract] [Full Text]

Home page
 Physiol. Rev.Home page
E. E. Muller, V. Locatelli, and D. Cocchi
Neuroendocrine Control of Growth Hormone Secretion
Physiol Rev, April 1, 1999; 79(2): 511 - 607.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ye, P.
Right arrow Articles by D’Ercole, A. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ye, P.
Right arrow Articles by D’Ercole, A. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals