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Endocrinology, doi:10.1210/en.2005-0742
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Endocrinology Vol. 147, No. 6 2944-2955
Copyright © 2006 by The Endocrine Society

Genetic Increase in Serum Insulin-Like Growth Factor-I (IGF-I) in C3H/HeJ Compared with C57BL/6J Mice Is Associated with Increased Transcription from the IGF-I Exon 2 Promoter

Martin L. Adamo, Xiuye Ma, Cheryl L. Ackert-Bicknell, Leah Rae Donahue, Wesley G. Beamer and Clifford J. Rosen

Department of Biochemistry (M.L.A., X.M.), The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3900; The Jackson Laboratory (C.L.A.-B., L.R.D., W.G.B., C.J.R.), Bar Harbor, Maine 04609-1500; and The Maine Center for Osteoporosis Research and Education (C.J.R.), Bangor, Maine 04401

Address all correspondence and requests for reprints to: Martin L. Adamo, Ph.D., Department of Biochemistry, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, MSC 7760, San Antonio, Texas 78229-3900. E-mail: adamo{at}biochem.uthscsa.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
C3H/HeJ (C3H) mice exhibit 30–40% higher serum IGF-I than do C57BL/6J (B6) mice, in association with increased bone mineral density and strength. These differences are inherited and thus provide a model for determining molecular mechanisms for genetic variation of serum IGF-I and downstream actions. We now report that increased serum IGF-I in C3H mice is associated with increased transcription from the minor exon 2 promoter in liver from female and male mice. The increase in hepatic IGF-I gene expression caused by increased abundance of IGF-I mRNA transcribed from the exon 2 promoter can quantitatively account for the increased serum IGF-I in C3H mice. Also, levels of both Ea and Eb IGF-I mRNAs are increased in livers of male C3H mice. Fasting lowered serum IGF-I and liver IGF-I mRNA levels in female mice of both strains. However, serum IGF-I and liver IGF-I mRNA levels remained higher in fasted C3H mice compared with fasted B6 mice. Levels of IGF-I transcripts initiated from exon 2 are also significantly increased in skeletal muscle, fat, ovaries, and kidneys of C3H mice. IGF binding protein (IGFBP)-5 mRNA levels are significantly higher in muscle and fat of C3H mice than in B6 mice. Levels of exon 1-containing transcripts are increased in whole femurs of male and female C3H mice. We conclude that increased transcription of the IGF-I gene occurs in a promoter- and tissue-specific manner in C3H mice. The increased IGF binding protein-5 mRNA levels in fat and muscle suggest that IGF-I signaling is increased in these tissues in C3H mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IGF-I REGULATES MUSCULOSKELETAL growth through autocrine/paracrine and endocrine mechanisms. Production of endogenous IGF-I in bone is important for bone formation in response to parathyroid hormone and prostaglandin E2 produced after bone fracture (1, 2, 3, 4, 5, 6). Moreover, targeted expression of IGF-I transgenes under control of osteoblast and muscle-specific promoters increases bone and muscle growth, respectively, without increasing serum IGF-I levels (7, 8). However, serum IGF-I levels, which reflect mainly liver production, also contribute to bone formation. Mice in which both the liver IGF-1 (Igf1) and acid labile subunit (Als) genes have been disrupted exhibit 90% reductions in serum IGF-I and clear deficiencies in chondrogenesis and bone formation despite normal levels of bone IGF-I mRNA (9). Consistent with important roles for both circulating and locally produced IGF-I in bone formation, mice in which the IGF-I receptor is disrupted in osteoblasts exhibit reduced bone formation rates (10).

Although genetically manipulated mice with deficiencies in IGF-I expression have been used to show impaired linear growth and reduced peak bone acquisition, the issue of whether allelic differences in IGF-I within the physiologic range impacts the skeleton has been much more difficult to address. Interestingly, it has been shown previously that serum IGF-I and volumetric bone mineral density (vBMD) are both complex traits that cosegregate in humans and mice (11). Also, we identified several quantitative trait loci (QTLs) from two inbred strains that are identical for serum IGF-I and femoral vBMD. In earlier work (12, 13), we showed that the C57BL/6J (B6) strain has 20–30% lower serum IGF-I and comparably reduced vBMD than C3H/HeJ (C3H) mice. In addition, C3H mice show greater rates of bone formation, lower skeletal resorption, and greater breaking strength than B6 (14, 15, 16, 17, 18). Thus, we hypothesized that allelic variation between C3H and B6 resulted in differences in circulating and skeletal IGF-I; this, in turn, could lead to alterations in the acquisition of peak bone mass. In this report, we have investigated tissue Igf1 gene expression to more fully understand the molecular genetic differences between these two strains with respect to IGF-I and bone phenotypes. Our results suggest that liver Igf1 gene transcription is increased selectively from promoter 2, which is classically thought of as the minor promoter, such that in C3H mice, the promoter 2 is as strongly expressed as promoter 1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
All mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Tissues were harvested from 8-wk-old female and male mice. Livers were obtained from retired breeders at 8–12 months of age. All tissues were stored frozen at –70 C until total RNA extraction performed using the RNASTAT reagent (Biotexc, Houston, TX). Femurs were ground under liquid nitrogen before homogenization in RNASTAT. RNA was quantified using the absorbance at 260 nm. In one experiment, 8-wk-old male and female mice were either fasted for 48 h or fed the NIH Rat and Mouse/Auto 6F diet, consisting of 18% protein, 6% fat, and 5% fiber (Richmond, IN). All procedures involving mice were reviewed and approved by the Institutional Animal Care and Use Committees of The Jackson Laboratory and the University of Texas Health Science Center (San Antonio).

IGF-I RIA
Serum IGF-I was measured by an RIA (ALPCO, Windham, NH). IGF binding proteins (IGFBPs) were first removed from the IGF-I by an acid dissociation step. This was followed by the addition of a neutralization buffer containing excess recombinant human IGF-II, allowing the IGF-II to bind to the IGFBPs before immunoassay with a human anti-IGF-I polyclonal antibody. The sensitivity of the assay was 10 ng/ml IGF-I; the interassay coefficient of variation based on normal standards and pooled serum of C3H and B6 was approximately 6%. There was no cross-reactivity with IGF-II. Standards were run in each assay as well as normal pools from both progenitors.

Solution hybridization/ribonuclease (RNAse) protection assays (RPAs)
Mouse IGF-I probes were used for RPAs. A probe that allowed simultaneous measurement of mature exon 1 and mature exon 2 IGF-I mRNAs was generated from a cDNA clone provided by Dr. G. Bell (19). ‘A 386-bp fragment of this cDNA consisting of 145 bp of exon 2 and 241 bp of exon 3 and the first part of exon 4 was subcloned into pGEM 2. The probe was linearized with EcoRI for run-off transcription with T7 RNA polymerase. To simultaneously quantify IGF-I exon 1 pre-mRNAs, pre-mRNAs generated from exon 2 transcription initiation, and mature exon 2 mRNAs transcribed from the various start sites, we also generated a probe consisting of the most 3' 126 bp of intron 1, the 72 bp of exon 2, and the first 188 bp of intron 2 prepared by PCR amplification of mouse genomic DNA. The PCR product was subcloned into pGEM-4Z, and the resulting vector was linearized with EcoRI for run-off transcription with T7 RNA polymerase. To measure levels of IGF-I mRNAs transcribed from the exon 1 promoter start sites, a genomic fragment containing the entire mouse exon 1 sequence and 76 bp of intron 1 was subcloned into pGEM4Z. Finally, to quantify transcripts encoding either Ea or Eb E-peptide, a 247-bp fragment corresponding to the 127 bp of the 3' end of exon 4, the 52-bp exon 5, and the initial 68 bp at the 5' end of exon 6 was ligated into pGEM 4Z. To measure the abundance of mRNA encoding fatty acid synthase (FAS), 144 bp of exon 1 of mouse FAS was PCR amplified and cloned into pGEM 4Z. The abundance of IGFBP-5 mRNA was measured using a plasmid containing 286 bp of mouse IGFBP-5 exon 1. Linearized vectors containing portions of the mouse 28S ribosomal RNA and mouse ß-actin RNA sequences were obtained from Ambion (Austin, TX).

Antisense RNA probes were biosynthetically labeled with 32P using procedures previously described (20). One microgram of linearized DNA was incubated with buffer, 0.5 mM ATP, CTP, and GTP, and 50 µCi {alpha}-[32P]-UTP (approximately 800 Ci/mmol, DuPont/NEN Life Science Products, Boston, MA), and unlabeled UTP to give a final concentration of 200 µM and the appropriate RNA polymerase. After reaction, the template was removed by deoxyribonuclease (DNAse) I digestion, and the labeled probe was extracted and purified with two rounds of ethanol precipitation. In the case of the 28S rRNA probe labeling, 0.5 mM of all four NTPs was used. Reagents for probe labeling were obtained from Promega (Madison, WI) and Ambion. For RPA, reagents were obtained from Ambion. Equal microgram quantities of total RNA were hybridized to approximately 200,000 cpm of probe for approximately 16 h, followed by digestion of unhybridized RNA with RNAses A and T1. Protected hybrids were extracted, precipitated, denatured, and electrophoresed through denaturing urea/polyacrylamide gels. Gels were dried and exposed to x-ray film to visualize protected probe bands that reflect the abundance of hybridizing RNA. Gels were also exposed to PhosphorImager screens and the intensity of PhosphorImager bands was determined using the system provided by Molecular Dynamics (Sunnyvale, CA). In analyzing data from the exon 2-exon 3-exon 4 probe, band intensity was normalized by adjusting for the number of radioactive U residues in a given protected band.

Statistics
Numerical data are shown as mean ± SEM. Significance of differences (P < 0.05) between strain means was determined using one-way ANOVA in the SIMSTAT3 package (Normand Peledeau, Provalis Research, Montreal, Quebec, Canada).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Hepatic IGF-I gene expression
RPA using the exon 2-based antisense RNA probe and liver RNA from B6 and C3H female mice revealed a protected band at approximately 240 nucleotides (nt) and a series of closely spaced bands above and blow the 300-nt marker (Fig. 1AGo). These results are the same as those obtained using a similarly constructed rat probe with rat liver RNA (21) and indicate the presence of exon 1 and exon 2 transcripts. The exon 1 transcripts protect the 241-bp exon 3–4 probe sequence common to all IGF-I mRNAs. The exon 2 transcripts are initiated at sites ranging from about 72 to about 52 bp upstream of the 3' end of exon 2, resulting in protection of 290–310 nt of the probe. The intensity of the bands reflecting exon 2 transcripts in C3H females at 8 wk is approximately twice that of B6 females (Fig. 1BGo), with about a 15% increase in the abundance of exon 1 transcripts in C3H females (not significant in this series of mice). The 2-fold increase in exon 2 transcripts in C3H female liver (P < 0.05) resulted in their abundance equaling that of exon 1 transcripts and in an overall 41% increase in liver IGF-I mRNA abundance. In view of the small increase in abundance of total exon 1 transcripts, we determined whether the abundance of exon 1 transcripts initiated from the various exon 1 start sites might be selectively altered in C3H females compared with B6 females using a probe complementary to the exon 1 sequence. Bands of approximately 240 nt and of approximately 350 nt were observed, which would be consistent with the expected size of start site 2 and start site 3 transcripts, respectively (data not shown), as characterized in rat liver (21). The intensity of bands did not show major differences between B6 and C3H female liver, thus confirming results obtained for total exon 1 transcripts using the exon 2-based probe (data not shown).


Figure 1
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  		FIG. 1. RPA of liver IGF-I exons 1 and 2, plus actin mRNAs from 8-wk-old B6 and C3H female mice. A, Autoradiograms of separate RPAs showing the position of protected probe bands corresponding to IGF-I mRNAs transcribed from the exon 1 and exon 2 promoters and actin mRNA, respectively. B, phosphor image quantification of RPA data from A. Protected bands were corrected for specific radioactivity and expressed as a fraction of the levels of exon 1 mRNAs in B6 mice (arbitrarily set to 1.00). Exon 2 mRNA abundance was significantly higher in C3H than in B6 liver (P < 0.05). Livers were from five separate B6 mice and four separate C3H mice.

 
To determine whether increased exon 2 transcription persists in older female animals, RPAs were performed on livers of retired breeder females (8–12 months old). Exon 2 mRNA transcripts were increased 2.3-fold in C3H retired breeders (P < 0.05 compared with B6), with a 30% increase in exon 1 transcripts (not significant) (Fig. 2Go). Importantly, ß-actin transcripts were not different between B6 and C3H livers in either the young or older females (Figs. 1Go and 2Go).


Figure 2
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  		FIG. 2. RPA of liver IGF-I mRNA in female retired breeder B6 and C3H mice. A, Autoradiograms of separate RPAs using the same antisense probes used for Fig. 1Go, which detect exon 2 and exon 1 transcripts, and actin mRNAs, respectively. B, Phosphor image quantification of RPA data from A. Protected bands were corrected for specific radioactivity and expressed as a fraction of the levels of exon 1 mRNAs in B6 mice (arbitrarily set to 1.00). Exon 2 mRNA abundance was significantly higher in C3H than in B6 liver (P < 0.05). Livers were from eight separate B6 mice and six separate C3H mice.

 
The increase in exon 2 transcripts in female C3H mice could be due to increased transcription, increased mRNA stability, or both. To address these possibilities, an antisense probe was used that simultaneously measures total exon 1 pre-mRNA transcripts, mature exon 2 transcripts, and exon 2 pre-mRNA. As seen in Fig. 3AGo, a 386-nt band was observed in RPAs of liver mRNA from both B6 and C3H females, reflecting protection of the full length of the probe insert by an mRNA containing contiguous intron 1, exon 2, and intron 2 sequence and thus reflecting total exon 1 pre-mRNA transcripts. In addition, bands of approximately 260 nt were obtained that were of greater abundance in female C3H liver than in female B6 liver (P < 0.05). These are the expected sized bands resulting from protection of the contiguous exon 2-intron 2 portion of the probe and thus are presumed to be exon 2 pre-mRNA. Moreover, a prominent ladder of bands was obtained of approximately 50–70 nt, which is the expected size of probe protected by correctly initiated and spliced exon 2 transcripts. As with the exon 2 pre-mRNA transcripts, the intensity of these bands was 2-fold greater in female C3H than in female B6 liver (Fig. 3BGo; P < 0.05). The intensity of the exon 1 pre-mRNA band did not vary significantly between strains. Thus, it would appear that the abundance of both pre-mRNA and mature mRNA resulting from exon 2 transcription initiation are increased in female C3H liver, leading to the conclusion that transcription from the exon 2 promoter is increased in C3H liver. Two other distinct bands were observed, whose abundance did not vary between strains. These bands could reflect mature exon 2 transcripts initiated from minor transcription start sites upstream of the major exon 2 start site located at approximately –72. Such minor upstream exon 2 start sites have been observed in rat liver (21, 22).


Figure 3
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  		FIG. 3. RPA of IGF-I exon 1 and exon 2 pre-mRNAs and mature exon 2 mRNAs in female B6 and C3H mice. A, Antisense probe complementary to contiguous intron 1, exon 2, and intron 2 sequence of the mouse IGF-I gene was used in RPAs. The positions of the protected probe fragments corresponding to total pre-exon 1 mRNAs, preexon 2 mRNAs, and mature exon 2 mRNAs initiated at the various exon 2 transcription start sites are indicated. The lanes labeled probe were carried through the assay in the absence of liver RNA or not carried through the assay but loaded on to the gel, respectively. The lane to the left (M) is an RNA marker of the indicated size bands. B, Phosphor image quantification of the autoradiogram shown in A. The levels of mature exon 2 mRNAs were divided by 100 to fit the same scale. The levels of mature exon 2-initiated transcripts and pre-IGF-I mRNAs initiated from the exon 2 promoter were significantly higher in C3H than in B6 mice. Livers were from five separate B6 mice and five separate C3H mice.

 
Fasting is known to decrease liver IGF-I mRNA levels in rats (22, 23). Fasting of female B6 and female C3H mice reduced serum IGF-I (Fig. 4AGo) and liver IGF-I mRNA levels (Fig. 4Go, B and C). However, exon 2 transcript levels and serum IGF-I levels were higher in fasted female C3H than in fasted female B6 mice (Fig. 4Go, B and C). Moreover, exon 2 mRNA levels in fasted C3H mice were the same as exon 2 transcripts in fed B6 mice. Thus, the strain differences in exon 2 transcripts were maintained in fasted mice. Although fasting reduced serum IGF-I levels in the male mice of both strains, serum IGF-I levels were higher in fasted male C3H than in fasted male B6 mice (Fig. 5AGo). However, fasting did not produce the expected decline in liver IGF-I in male mice of either strain (Fig. 5Go, B and C). Levels of FAS mRNA were lower in liver from both fasted male and female B6 and C3H mice compared with fed mice (Figs. 4BGo and 5BGo).


Figure 4
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  		FIG. 4. Effect of fasting on liver IGF-I mRNA and serum IGF-I levels in female B6 and C3H mice. Eight-week-old female mice were fed ad libitum or fasted for 48 h. Serum and liver were obtained when mice were killed and assayed for immunoreactive IGF-I (A) and IGF-I mRNA, 28S rRNA, and FAS mRNA using separate RPA as described in the text. B, Autoradiograms of RPAs for IGF-I mRNA, 28S rRNA, and FAS mRNA. C, Results of phosphor image quantitation of IGF-I RPAs, where the level of IGF-I mRNA in fed B6 is expressed as 1.00, and levels of IGF-I mRNA in the other groups are expressed as a fraction of this value. Data are for three mice from each group.

 

Figure 5
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  		FIG. 5. Effect of fasting on liver IGF-I mRNA and serum IGF-I levels in male B6 and C3H mice. Eight-week-old male mice were fed ad libitum or fasted for 48 h. Serum and liver were obtained at when mice were killed and assayed for immunoreactive IGF-I (A) and IGF-I and FAS mRNAs and 28S rRNA using separate RPA as described in the text. B, Autoradiograms of RPAs for IGF-I mRNA, 28S rRNA, and FAS mRNA. C, Results of phosphor image quantitation of IGF-I RPAs, where the level of IGF-I mRNA in fed B6 is expressed as 1.00, and levels of IGF-I mRNA in the other groups are expressed as a fraction of this value. Data are for three mice from each group, except for male C3H RPA data, for which data were for two mice.

 
Male mice were also used to determine whether there were any strain differences in alternative splicing involving exons 4–6. The levels of IGF-I mRNAs containing exon 4 spliced directly to exon 6 (Ea form) (19) were predominate over IGF-I transcripts containing exon 4 spliced to exon 5 and exon 5 spliced to exon 6 (Eb form) (19) in males of both strains. However, the levels of both Ea and Eb transcripts were increased in C3H compared with B6 livers (Fig. 6Go, A and B), indicating that there was normal alternative splicing of a greater number of IGF-I precursor transcripts in male C3H liver. Moreover, these results support our hypothesis that C3H mice express increased levels of full-length IGF-I-encoding mRNAs in liver.


Figure 6
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  		FIG. 6. RPA of liver IGF-I Ea and Eb mRNAs in 8-wk-old male B6 and C3H mice. An antisense RNA probe complementary to exon 4-5-6 sequence of the mouse IGF-I Eb mRNA was used in RPAs. The full-length protected band reflects Eb mRNAs. Ea mRNAs protect the exon 4 and exon 6 portions of this probe, as indicted in the autoradiogram (A). Both Ea and Eb mRNAs were significantly elevated in C3H mice (quantified data in B; P < 0.05). Data are for three separate B6 mice and three separate C3H mice. The abundance of each band is arbitrarily set at 1.00 for B6, and C3H is fold stimulation.

 
Extrahepatic IGF-I expression
In rats, several extrahepatic tissues also express exon 2-initiated transcripts, although at much lower abundance than in liver (24, 25). RPAs using the exon 2-based probe revealed that the same was true for female mouse tissues, with exon 2-initiated IGF-I gene expression ranging from about 2% of total IGF-I mRNA in lung to about 14% of the total in kidney. Skeletal muscle (Fig. 7Go), fat (Fig. 8Go), and kidney (Fig. 9Go) expressed significantly higher levels of exon 2 transcripts in C3H compared with B6 females (P < 0.05 in all three tissues). Ovarian exon 2 expression was extremely low in both strains but was significantly higher in C3H than in B6 (data not shown). Heart and lung exon 2 expression was low in B6 and C3H females, and although exon 2 transcripts appeared to be slightly increased in C3H lung, the effect did not reach statistical significance in these groups of mice (data not shown). Duodenum, brain, and spleen did not express detectable levels of exon 2 transcripts in either B6 or C3H mice under RPA conditions used in this study (data not shown).


Figure 7
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  		FIG. 7. RPA of skeletal muscle exon 1 and exon 2 mRNAs from 8-wk-old female B6 and C3H mice. The antisense probe that simultaneously measures exon 1 and exon 2 mRNAs and the probe that measures actin mRNA were employed in separate RPAs using RNA from skeletal muscle. A, Representative autoradiogram of the RPA done on muscles from five separate C3H mice and five separate B6 mice. B, Quantified data (phosphor image analysis) for 10 C3H and 10 B6 mice. Exon 2 transcripts were significantly higher in C3H muscles than in B6 muscles (P < 0.05).

 

Figure 8
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  		FIG. 8. RPA of adipose exon 1 and exon 2 mRNAs from 8-wk-old female B6 and C3H mice. The antisense probe that simultaneously measures exon 1 and exon 2 mRNAs and the probe that measures actin mRNA were employed in separate RPAs using RNA from adipose tissue. A, Representative autoradiogram of the RPA done on adipose tissue from five separate C3H mice and five separate B6 mice. B, Quantified data (phosphor image analysis) for the same C3H and B6 mice. Exon 2 transcripts were significantly higher in C3H adipose than in B6 adipose (P < 0.05).

 

Figure 9
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  		FIG. 9. RPA of kidney exon 1 and exon 2 mRNAs from 8-wk-old female B6 and C3H mice. The antisense probe that simultaneously measures exon 1 and exon 2 mRNAs and the probe that measures actin mRNA were employed in separate RPAs using RNA from kidney. A, Autoradiogram of the RPA done on kidneys from eight separate B6 mice and nine separate C3H mice. B, Quantified data (phosphor image analysis) for these mice. Exon 2 transcripts were significantly higher in C3H kidney than in B6 kidney (P < 0.05).

 
To determine whether there was increased IGF-I signaling in soft tissues of C3H mice compared with B6 mice, we measured the abundance of IGFBP-5 mRNA. IGFBP-5 mRNA is known to be increased by IGF-I in several cell types (26, 27, 28, 29, 30, 31). We found that there was a significantly higher abundance of IGFBP-5 mRNA in skeletal muscle (Fig. 10Go) and fat (Fig. 11Go) of female C3H mice compared with B6 (P < 0.05 in both tissues). However, IGFBP-5 mRNA levels were not increased in kidney, heart, spleen, or duodenum of C3H mice (data not shown).


Figure 10
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  		FIG. 10. RPA of skeletal muscle IGFBP-5 mRNA from 8-wk-old B6 and C3H mice. The IGFBP-5 antisense probe described in the text was employed in RPAs using RNA from skeletal muscle. A, Autoradiograms from two separate RPAs. B, Quantified data (phosphor image analysis) for a total of eight separate B6 muscles and 10 separate C3H muscles. IGFBP-5 mRNA was significantly higher in C3H muscle than in B6 muscle (P < 0.05).

 

Figure 11
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  		FIG. 11. RPA of adipose IGFBP-5 mRNA from 8-wk-old B6 and C3H mice. The IGFBP-5 antisense probe described in the text was employed in RPAs using RNA from adipose tissue. A, Autoradiogram of the RPA using adipose RNA from four separate B6 mice and three separate C3H mice. B, Quantified data (phosphor image analysis) from this autoradiogram. IGFBP-5 mRNA was significantly higher in C3H adipose than in B6 adipose (P < 0.05).

 
Analyses of a limited number of whole femur RNA samples from 8-wk female mice revealed a different regulation of IGF-I gene expression than in soft tissues. Femurs expressed mostly exon 1 transcripts that were increased on average by 5-fold in C3H females (P < 0.05). However, ß-actin mRNAs were also increased by almost 3-fold in female femurs, whereas 28S rRNA showed no increase (Fig. 12AGo). In male femurs, there was about a 2-fold increase in exon 1 transcripts (P < 0.05), with no strain differences in either actin mRNA or 28S rRNA (Fig. 12BGo).


Figure 12
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  		FIG. 12. RPA of exon 1 IGF-I and actin mRNAs, and 28S rRNAs, respectively, from femurs of 8-wk-old B6 and C3H mice. RPAs were conducted on RNA isolated from whole femur separately using the IGF-I antisense probe that simultaneously detects exon 1 and exon 2 mRNAs, the antisense probes for actin mRNA, and the antisense probe for 28S rRNA. A, Autoradiograms of RPAs on femoral RNA from three separate female B6 mice and three separate female C3H mice. B, Respective autoradiograms for three separate male B6 mice and three separate male C3H mice. C, Phosphor image analysis of gels. IGF-I exon 1 transcripts were significantly elevated in male and female C3H femurs compared with male and female C3H femurs, respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of this study strongly suggest that the increase in serum IGF-I levels in the C3H strain compared with the B6 strain is due to increased transcription of the liver IGF-I gene from the minor exon 2 promoter. Transcription from the exon 2 promoter produces an mRNA predicted to encode prepro-IGF-I with a 32-amino acid signal peptide that includes the amino-terminal five amino acids uniquely encoded by exon 2 in rats, humans, and mice (19, 32, 33). Immunoreactive IGF-I is secreted by cultured OVCAR-3 cells that express predominately exon 2 transcripts (34), and exon 2 mRNAs are translated in vitro into prepro-IGF-I that can be processed into pro-IGF-I by canine pancreatic microsomes (33). These results, as well as the fact that the magnitude of the increase in liver IGF-I mRNA is quantitatively similar to the magnitude of the increase in serum IGF-I, strongly suggest that the higher levels of liver IGF-I transcripts account for the increased serum IGF-I in C3H mice. Exon 2 transcripts are also increased in several extrahepatic tissues, which could contribute to increased serum IGF-I. In addition, increased extrahepatic IGF-I could act in an autocrine/paracrine manner. There is increased growth in the cortical bone compartments of C3H mice that yields increased vBMD (12). The increased vBMD in C3H mice could be due, in part, to increased serum IGF-I and/or increased bone IGF-I expression. In femurs, we found that exon 1 transcripts were increased in C3H mice, suggesting an alternative mechanism for increased IGF-I gene expression in bone. It is not known, however, whether the increase in exon 1 transcripts that we observed in femurs is due to expression by bone cells per se (i.e. osteoblasts, osteoclasts, or osteocytes) or is contributed by cellular components of the bone marrow such as osteoprogenitor cells. Previous work showed that the concentration of immunoreactive IGF-I in femurs, calvariae, and tibiae from C3H mice was on average 32% higher than in the same bones of B6 mice and, furthermore, that cultured calvarial osteoblasts from C3H mice secreted greater amounts of immunoreactive IGF-I into conditioned medium than did cells from B6 mice (13). In the current study, we found that the expression of exon 2-initiated IGF-I transcripts and IGFBP-5, an IGF-I stimulated gene (26, 27, 28, 29, 30, 31), were both increased in muscle and fat of C3H mice compared with B6 mice. An in vivo study showed that hypophysectomy strongly reduced IGFBP-5 mRNA in muscle and fat but did not reduce IGFBP-5 mRNA in heart or kidney (35). Infusion of IGF-I into the hypophysectomized rats increased the abundance of IGFBP-5 mRNA in skeletal muscle, heart, and adipose tissue, but not in kidney (35). In that study, spleen IGFBP-5 mRNA was not detectable. Interestingly, infusion of growth hormone produced a greater increase in muscle and adipose IGFBP-5 mRNA than IGF-I did, and this correlated with increased IGF-I mRNA in muscle and fat (35). However, infusion of GH did not increase IGFBP-5 mRNA in heart or kidney. The authors concluded that increased IGFBP-5 mRNA in muscle and adipose reflected either increased local IGF-I and/or a direct effect of GH. The lack of effect of GH on heart IGF-I was suggested to reflect lack of a statistically significant effect of GH on heart IGF-I mRNA. Thus, it was felt that despite the increased serum IGF-I caused by GH, stimulation of IGFBP-5 may be more dependent on increased local IGF-I expression (35). Small intestine was not examined in this study. In a different study, infusion of IGF-I into parenterally fed rats increased IGFBP-5 mRNA in jejunum, whereas GH infusion did not (36). The GH increased serum IGF-I levels but did not increase jejunal IGF-I. In addition, an intestinal IGF-I transgene increased intestinal IGFBP-5 mRNA (37). In our model, there is increased local expression of IGF-I mRNA from the exon 2 promoter in muscle and fat but not in spleen, heart, or duodenum, also suggesting that increased local IGF-I may be required for increased IGFBP-5 mRNA in C3H mice. Because IGF-I infusion can increase heart and intestinal IGFBP-5, it is possible that in our C3H mice, the increase in serum IGF-I was insufficient to stimulate heart and duodenum IGFBP-5 mRNA. The absence of a significant increase in IGFBP-5 mRNA in kidney, in which exon 2 transcripts were increased, is more difficult to explain. Although the effects were not reported as statistically significant, hypophysectomy reduced kidney IGF-I mRNA, and IGF-I and GH infusions restored kidney IGF-I without significantly altering kidney IGFBP-5 (35). Thus, either kidney IGFBP-5 may not be regulated by IGF-I, or the increase in serum and kidney IGF-I was not sufficient to regulate IGFBP-5. The increase in immunoreactive IGF-I (13) and increased vBMD in C3H bone (12) suggests that increased expression of IGF-I from the exon 1 promoter described herein may provide a mechanism for increased autocrine/paracrine IGF-I signaling in skeletal tissue of C3H mice.

Our conclusion that there is increased transcription from the exon 2 promoter in C3H mice is based on measurements of simultaneous increases in mature exon 2 transcripts and unprocessed exon 2 transcripts. Previous studies show an excellent correlation between measures of IGF-I pre mRNA transcript abundance and gene transcription rates assessed by nuclear run-on assay (38, 39, 40). Moreover, if unprocessed IGF-I transcripts were increased because of delayed splicing or decreased nucleocytoplasmic transport of the processed transcripts, this would be expected to reduce the abundance of mature processed transcripts. Our finding of increased levels of unprocessed and processed exon 2 transcripts strongly suggests that increased transcription initiation from the exon 2 promoter at least in part is responsible for increased liver IGF-I gene expression in C3H mice. In addition, we found that although a 48-h fast lowered the levels of liver exon 2 IGF-I mRNA transcripts in female mice of both strains, the levels of the exon 2 transcripts were still about 2-fold higher in fasted C3H mice than in fasted B6 mice. Because fasting reduces liver IGF-I mRNA in a posttranscriptional mechanism (23), this result is consistent with the presence of a transcriptional mechanism for increased liver IGF-I mRNA in C3H mice that is independent of food intake. The observation that liver IGF-I mRNA was not decreased in fasted male mice of either strain was unexpected. Because lipogenic enzyme mRNA expression showed the expected decline with fasting (41) in both male and female mice of both strains, our results cannot be explained by a generalized failure of male mouse liver gene expression to show the expected metabolic adjustments to fasting. Thus, additional study is required to provide a rational explanation for the failure of male liver IGF-I mRNA levels to decrease with fasting. If confirmed, it could indicate translational or posttranslational mechanisms in the decreased serum IGF-I in fasted male mice.

Our current results have not delineated the mechanism for the increase in exon 2 transcription in C3H mice. Preliminary data suggest that in vitro binding of nuclear proteins to proximal exon 2 promoter fragments is not different between C3H and B6 mice and that the activity of the proximal exon 2 promoter from the C3H Igf1 gene is not higher than that from B6 mice in transient transfection assays. However, further study using more extensive regions of the Igf1 gene from C3H and B6 mice in its chromatinized state will be required to elucidate potential cis-acting elements and/or trans-acting factors that may be responsible for tissue-specific increased IGF-I gene expression in C3H mice. The necessity of examining more extensive regions of the IGF-I gene is also indicated by the fact that exon 1 transcripts are significantly increased in bone and increased, albeit insignificantly in these studies, in soft tissues. This suggests that regions outside of the proximal exon 2 promoter are interacting with tissue-specific factors to regulate IGF-I gene expression. Another approach to determining the basis for increased exon 2 transcription in C3H mice is to ask which signaling pathways could be responsible. GH is the major stimulator of liver IGF-I transcription, and although there were early suggestions that GH may stimulate exon 2 transcripts more than it stimulates exon 1 transcripts (42), other studies have shown that GH stimulates both exon 1 and exon 2 transcripts equally well (43). Moreover, we have obtained results suggesting that even in the presence of the little (lit) mutation that causes isolated GH deficiency, liver exon 2 transcripts are higher in C3H-lit/lit than in B6-lit/lit mice (Ackert-Bicknell, C. L., C. J. Rosen, W. G. Beamer, and L. R. Donahue, unpublished observations). Thus, it appears unlikely that GH is causing these strain differences. Our genetic mapping studies indicate that four QTLs determine strain differences in serum IGF-I between C3H and B6 mice. The QTLs are on chromosomes 1, 6, 10, and 15. Moreover, an epistatic locus on chromosome 11 interacts with chromosome 6 alleles (44, 45). Recently, we found that the chromosome 6 alleles from C3H actually decrease serum and skeletal IGF-I when on the B6 background (46, 47, 48). Thus, the increased serum IGF-I and increased exon 2 transcription in C3H mice compared with B6 mice either reflects one of the other QTLs or results from the mixture of all four QTLs. Of interest, chromosome 10 contains the Igf1 gene, suggesting that cis-acting elements in the Igf1 gene and/or trans-acting factors binding to the gene may be responsible for increased IGF-I expression in C3H mice. It is possible, however that several genes within these various QTLs are interacting with tissue-specific factors to produce increases in transcription from the exon 2 promoter in some, but not all, soft tissues. In addition, tissue-specific factors are likely modifying genetic regulation of transcription from the exon 1 promoter, resulting in significant increases in transcription from the exon 1 promoter in bone, but insignificant increases in exon 1 transcription in soft tissues. Studies are currently underway, using gene sequencing and mouse genetic approaches, to determine the mechanisms responsible for strain differences in IGF-I expression. These studies could have major significance for human disease, in which variations in serum IGF-I in the normal range are strong predictors of risk for development of cancer and osteoporosis (11).


   Acknowledgments
 
We thank Wayne Matheny for preparation of the construct used in the RPA of IGF-I Ea and Eb mRNAs.


   Footnotes
 
This work was supported by National Institutes of Health Grant AR45433.

M.L.A., X.M., C.L.A.-B., L.R.D., W.G.B., and C.J.R. have nothing to declare.

First Published Online March 9, 2006

Abbreviations: B6, C57BL/6J; C3H, C3H/HeJ; DNase, deoxyribonuclease; FAS, fatty acid synthase; IGFBP, IGF binding protein; nt, nucleotide(s); QTL, quantitative trait locus; RNase, ribonuclease; RPA, RNAse protection assay; vBMD, volumetric bone mineral density.

Received June 20, 2005.

Accepted for publication February 27, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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