Regulation of IGF-I mRNA by GH: putative functions for class 1 and 2 message

D. C. O'Sullivan, T. A. M. Szestak, and J. M. Pell

The Babraham Institute, Cambridge CB2 4AT, United Kingdom


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study investigated mechanisms regulating hepatic insulin-like growth factor (IGF)-I class 1 and 2 mRNA levels. Lambs were treated with growth hormone (GH) either as an acute, single dose or over a longer term. Total hepatic unspliced, pre-mRNA levels increased after the single dose of GH but were attenuated after 8 days of GH, with exon 1- and 2-derived pre-mRNA levels displaying coordinate responses. Surprisingly, changes in total spliced, mature mRNA levels did not reflect those for pre-mRNA, instead being augmented after 8 days of GH. GH also induced a differential increase in the ratio of mature class 2-to-class 1 IGF-I mRNA; therefore, this must be predominantly via posttranscriptional mechanisms. Increases in the ratio of class 2-to-class 1 mRNA were observed in polysomal vs. total RNA preparations derived from GH-treated but not control lambs, indicating an increased proportion of class 2 transcripts engaged in translation. Our findings indicate that GH may stabilize mature class 2 transcripts or destabilize mature class 1 transcripts and that class 2 mRNA may have a greater translational potential. The following two main functions of hepatic class 2 IGF-I mRNA are suggested: an efficient "monitor" of GH status via providing a rapid negative feedback mechanism and a coordinator of endocrine-regulated tissue growth.

liver; transcription; alternative splicing; sheep; growth


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

INSULIN-LIKE GROWTH FACTOR I (IGF-I) regulates cell function, including proliferation, survival, and differentiation, and is therefore essential for normal development and growth (28, 38). Postnatally, IGF-I is found in large amounts in the circulation, most being maintained as a ternary complex that consists of IGF-I, IGF-binding protein-3 (IGFBP-3), and the acid-labile subunit (ALS), thus providing a reservoir of IGF-I peptide (39). Even though the importance of circulating IGF-I for growth regulation has been challenged (26, 27, 46), recent studies in which both hepatic IGF-I and ALS synthesis have been abolished using gene targeting have confirmed that circulating IGF-I is crucial for normal growth, that the ablated hepatic IGF-I synthesis is apparently not compensated by increased IGF-I synthesis by other tissues, and that the molecular form of circulating IGF-I is important (47). The liver therefore has a central role in the maintenance of circulating IGF-I and in directing the complex molecular interactions of IGF-I with its direct and indirect binding partners. Furthermore, the establishment of a growth hormone (GH)-responsive endocrine IGF network is crucial for successful adaptation from fetal to postnatal life (24) and coordinating tissue growth and development, with hepatic IGF-I being in large part responsible for this endocrine role. However, the mechanisms by which the liver achieves these unique functions are uncharacterized.

The IGF-I gene is complex: mature peptide is encoded by only exons 3 and 4, with alternative splicing occurring at both the 5' (exons 1 and 2)- and 3' (exons 5 and 6)-ends of the gene, generating multiple mRNA species (30). Exons 1 and 2 are each associated with unique promoters and have disperse vs. discrete start sites, generating class 1 and class 2 mRNAs, respectively; interestingly, the liver is the major site of promoter 2 activity, with promoter 1 only being active in most tissues (41). Our previous studies and those of others have demonstrated a preferential increase in hepatic IGF-I derived from exon 2 in animals treated chronically with GH (29, 36), with changed nutritional status (36), and during development (1, 25). However, the mechanism accounting for differential increases in class 2 mRNA are not yet identified; differential transcriptional regulation has not been demonstrated to date (e.g., see Ref. 3). Thus, despite sequence conservation across mammalian species, the functional significance of IGF-I promoter 2 and its mRNA is not established.

It is intriguing that the liver is responsible for circulating IGF-I and is also the major tissue for IGF-I promoter 2 activity; correlation between class 2 mRNA and circulating IGF-I concentrations provides compelling evidence for a functional relationship (36). GH is a key regulator of postnatal circulating IGF-I concentrations, and therefore the aim of this study was to investigate mechanisms by which GH regulates hepatic IGF-I class 1 and class 2 mRNA levels. Lambs, rather than rodents, were selected as the most appropriate model, because the GH-IGF-I axis is far more closely related between humans and sheep than between humans and rodents, and therefore our findings could have significance for clinical application. Because the mature, secreted IGF-I peptides derived from exons 1 and 2 are identical, our studies focused on 1) comparing transcriptional vs. nontranscriptional regulation of IGF-I exons 1 and 2 and 2) the potential translatability of class 1 and class 2 mRNAs.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Temporal effects of GH on pre- and mature IGF-I nascent and mature class 1 and class 2 transcript abundance were investigated by treatment of lambs with a single dose or 8 days of GH. Twenty 20-wk-old lambs were randomly allocated to the following four treatment groups: groups 1 and 2 were injected subcutaneously with vehicle control and groups 3 and 4 with bovine GH (0.2 mg · kg-1 · day-1 in carbonate-buffered saline: 150 mM NaCl and 25 mM Na2CO3, pH 9.4). Groups 1 and 3 were killed [via a lethal dose of pentobarbital sodium (0.7 mg/kg), followed by exsanguination] exactly 3 h after a single injection of GH or vehicle, and groups 2 and 4 were killed after 8 days of treatment with GH or vehicle, again exactly 3 h after the final dose. Samples of liver were rapidly dissected and frozen in liquid nitrogen. On the final day of treatment, jugular blood samples (10 ml) were taken every 30 min from 2 h before vehicle or GH administration until the animals were killed.

Extraction of total RNA. Total RNA was extracted from 1-g portions of frozen liver using the guanidinium isothyocyanate method (6). In addition, glycogen was removed from the final RNA pellet preparation by dissolving the precipitated RNA pellet in RNase-free water and incubation overnight at 4°C with the addition of sodium acetate to 3 M (pH 7.0). The RNA pellet was washed with 75% ethanol. Total RNA was quantified by absorbance at 260 nm (1.0 A = 40 µg/ml). RNA integrity was checked by size separation on a denaturing 1.4% agarose gel and visualization with ethidium bromide. To ensure that mRNA concentrations were also consistent, total RNA was hybridized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using Northern analysis (described below).

Preparation of polysomal RNA. Polysomal RNA was extracted from fresh liver as described by Foyt et al. (13) with the adaptations incorporated (12) using a discontinuous sucrose gradient for polysomal isolation.

Template-specific PCR to measure nascent and mature mRNA levels. Nascent (i.e., pre-mRNA) and mature class 1 and class 2 transcripts were quantified using a modified version of competitive RT-PCR termed competitive RS-PCR (RNA template-specific PCR; see Ref. 42). This method has the sensitivity of conventional RT-PCR with the advantage of a reduction in false positives caused by contamination with minute quantities of genomic DNA. Briefly, RNA was isolated from sheep liver and used as a template for reverse transcription to cDNA using 47-mer primers, of which only the first 17 nucleotides, at the 3' end, hybridize to the target. The remaining 30-nucleotide tag, being of unique sequence (termed T30), was then used as the downstream primer in all subsequent PCR reactions (35 cycles). The pairs and sequences of all primers are given in Fig. 1.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 1.   Location and design of primers for the development of RNA template-specific (RS)-PCR. The first three exons of the sheep insulin-like growth factor (IGF)-I gene are shown (not to scale); the shaded boxes depict exons (untranslated regions, light cross-hatch; translated regions, dark cross-hatch); the linking continuous lines depict introns. Dotted lines show alternate splicing of either exon 1 or 2 on to exon 3. Thick solid lines depict PCR primers (not to scale) used for the RS-PCR as described in MATERIALS AND METHODS. EX1, exon 1, EX2; exon 2; U, upstream primer; D, downstream primer; T, "tag"; numbers, e.g., 17 and 30, refer to number of nucleotides. Primer sequences were as follows: EX1U30, 5'-GCA CTT CAG AAG CAA TGG GAA AAA TCA GCA-3'; EX2U30, 5'-ACC CAC CCT GAC CTG CTG TAA AAG ATC TGG-3'; EX3D17T30, 5'-GAA CAC CGA TGA CAA GCT TAG GCA GTG ACC GCA TCC ACC AAC TCA GC-3'; IN1U30, 5'-GGG CTA TAG GGC ATG GAT ATG AAC TTT TGG-3'; IN1D17T30, 5'-GAA CAC CGA TGA CAA GCT TAG GCA GTG ACC CCG CTT TCC ATA TAC AG-3'; IN2U30, 5'-TTT GCA GGT TTT GAG CCC AGT TCC CTG TTC-3'; IN2D17T30, 5'-GAA CAC CGA TGA CAA GCT TAG GCA GTG ACC GAG AGA AAG AGA GAC TG-3'; and T30, 5'-GAA CAC CGA TGA CAA GCT TAG GCA GTG ACC-3'.

Quantification of the four RS-PCR products (i.e., specific for introns 1 and 2 and exons 1 and 2) was achieved using artificial mRNA internal standards (termed cRNAs). The four cRNAs (generated as described in Refs. 4 and 18) contained the same PCR primer sequences as those of the targets described above, were spiked into extracted RNA samples in a dilution series, and were distinguished from the target by size. Competitive RS-PCR (35 cycles) was then performed with the selected primers.

Reverse transcription. cRNA at decreasing concentrations [doubling dilutions of cRNA starting with exon 1 (2.49 × 10-17 mol), exon 2 (2.77 × 10-17 mol), intron 1 (9.65 × 10-20 mol), and intron 2 (1.60 × 10-19 mol)] was added to eight reactions, each containing 0.1 µg total hepatic RNA, 0.5 mM dNTPs, 1.0 unit RNase inhibitor, 1.0 µM 47-mer downstream primer, and 10.0 units Moloney murine leukemia virus reverse transcriptase (Promega, Southampton, Hants, UK).

PCR. Each reverse transcription reaction (10% portion) was used in the subsequent PCR reactions, each consisting of 1.5 mM MgCl2, 200 µM dNTPs, 0.3 µM of each primer, and 2.0 units Taq Start polymerase (Sigma Chemical, Poole, Dorset, UK). PCR reactions were electrophoresed on a 1.4% agarose gel.

Quantification of products. The relative amounts of the two PCR products were quantified by ethidium bromide staining using a Gel Doc 1000 (Bio-Rad, Hercules, CA). The ratio of log target product to the log cRNA product was plotted against the log number of moles of cRNA added to the PCR mixture; the number of moles of target product was equivalent to the moles of cRNA (on the abscissa) when the log ratio was 0.0. Amounts of mRNA associated with exon 1, intron 1, and exon 2 could be obtained directly from these calculations; however, nascent transcripts from intron 2 will contain nascent message derived from the promoters associated with both exons 1 and 2, and therefore the true value for class 2 nascent transcripts was obtained after subtraction of class 1 nascent levels. Relative amounts of pre-mRNA and mature mRNA levels derived from both exon 1 and exon 2 were confirmed in selected samples by RNase protection assay using single-exon riboprobes (which contain intronic sequence); similar changes in message abundance were observed from both techniques (data not shown).

Northern analyses. Total RNA was electrophoresed on formaldehyde 1.4% agarose gels, blotted on an MSI nylon transfer membrane (Micron Separations, Westbrook, MA), and ultraviolet cross-linked using a Stratalinker (Stratagene, La Jolla, CA). Asymmetric PCR was used for IGF-I probe synthesis. Initially, double-stranded DNA templates were generated by standard PCR for exons 1, 2, and 4 (primers: exon 1, upstream 5'-TGT GAC ATT GCT CTC AA-3', downstream 5'-CTT CAA GAA ATC ACA AAA G-3'; exon 2, upstream 5'-TAA TAC CCA CCC TGA CC-3', downstream 5'-TGT AGG TGT AAC CAT TT-3'; exon 4, upstream 5'-GGC TCG AGC AGT CGG AG-3', downstream 5'-AGC CTT GGG CAT GTC GG-3') and GAPDH (upstream primer: 5'-AAT ACT GGC AAA GTG GAC-3'; downstream primer 5'-AGA TGG TGA TGG CCT TTC-3'), which was followed by the asymmetric PCR using the downstream primer for each exon and 32P-labeled dCTP as part of the nucleotide mix. Hybridization and washing of blots was performed using Church and Gilbert's (7) reagents.

RNase protection assays. RNase protection assays were used to quantify IGF-I transcripts derived from the leader exons 1 and 2 and the common coding exons 3 and 4 of the IGF-I gene. Antisense riboprobes were generated from cDNA templates specific for exons 1-3-4 or exons 2-3-4, yielding two protected bands after an RNase protection assay, with the full-length band (404 or 356 bases) corresponding to the class 1 or 2 transcripts, respectively, and the smaller band (341 bases) being equivalent to mRNA protected by the common coding exons 3 and 4 (assumed to represent mRNA derived from the reciprocal leader exon). Intensities of protected bands were visualized on X-ray film and quantified using an image analyzer (Ceescan, Cambridge, UK); the intensity of each protected band was corrected for the number of U nucleotides. Total IGF-I mRNA levels could be calculated from the sum of each of the two protected bands. All procedures were carried out exactly as described previously (36).

Measurement of plasma IGF-I concentrations. Plasma IGF-I concentrations were determined in acid-ethanol-extracted samples with an additional overnight precipitation of IGFBPs at -20°C after initial incubation and centrifugation of protein in acid-ethanol as previously described (36). The anti-IGF-I antiserum was raised against human IGF-I in rabbits at the Babraham Institute and had a titer of 1:50,000.

Statistical analysis. Where appropriate, data were analyzed using two-way ANOVA, with treatment time (single dose vs. 8 days) and GH as main effects. ANOVA was followed by standard post hoc t-tests using the pooled SE. However, for Figs. 2-4, individual group errors (as SE) are also presented to show the variation associated with each treatment group.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 2.   Total hepatic IGF-I unspliced, pre-mRNA (A) and spliced, mature RNA (B) in lambs treated with either a single dose or 8 days of growth hormone (GH). Bars with different letters are significantly different (at least, P < 0.02). S.E.D., standard error of the difference.



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 3.   Exon 1- and 2-associated hepatic IGF-I unspliced, pre-mRNA (A) and spliced, mature mRNA (B) in lambs treated with either a single dose (s) or 8 days (8d) of GH. Data are expressed as the degree of increase above mean values for lambs treated with saline (Sal; absolute values in RESULTS). Bars with different letters are significantly different (at least, P < 0.02).



View larger version (62K):
[in this window]
[in a new window]
 
Fig. 4.   Association of hepatic IGF-I mRNA with polysomes in lambs treated with a single dose or 8 days of GH. A: Northern blots showing mRNA transcripts in total (25 µg) and polysomal (5 µg) RNA, determined using a common coding exon 4 probe. B: representative RNase protection assays (derived from n = 4/group) to determine exon 1- and exon 2-specific association with total (50 µg) and polysomal (10 µg) RNA using exon 1-3-4 or 2-3-4 riboprobes as indicated in MATERIALS AND METHODS. Sal, saline control; C: quantification of RNase protection bands displayed in B, expressed as a ratio of class 2 to class 1 mRNA. Bars with different letters are significantly different (at least P < 0.05).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Differential effects of acute and short-term GH administration on total hepatic pre- and mature IGF-I mRNA levels. The first aim of the current study was to determine whether pre- and mature mRNA levels exhibited similar changes in response to GH, inferring transcriptional vs. posttranscriptional regulation of IGF-I mRNA abundance, respectively (3, 16, 21). Total hepatic pre-IGF-I mRNA levels are presented in Fig. 2A and were about two orders of magnitude lower than mature levels (Fig. 2B). As expected, a significant increase in pre-mRNA levels (6-fold; P < 0.001) was observed in response to the single dose of GH. Surprisingly, after GH treatment for 8 days, total pre-mRNA levels were attenuated, decreasing significantly (P < 0.02) to a value only fourfold above mean control levels. Total hepatic mature IGF-I mRNA levels were ~32 × 10-19 mol mRNA/µg total RNA in the saline control groups and increased by ~2.5-fold 3 h after the single dose of GH (P < 0.001). In contrast to pre-mRNA, total IGF-I message levels did not decrease after 8 days of GH treatment but were augmented further to 3.5-fold above mean control values (P < 0.002 compared with the single dose of GH), therefore demonstrating temporal regulation of IGF-I mRNA levels by GH.

Circulating IGF-I concentrations. Mean circulating IGF-I concentrations were on average 207.4 ng/ml in saline-treated lambs and exhibited no change after only 3 h of GH treatment (i.e., the single dose). However, concentrations increased significantly (P < 0.001) by 2.4-fold after 8 days of GH [single-dose saline, 201.4; 8 days of saline, 213.4; single-dose GH, 214.6; 8 days of GH, 506.9; pooled SED (n = 5), 23.3 ng/ml]. Thus, even though pre- and mature IGF-I mRNA levels respond after 3 h of GH treatment, circulating levels of IGF-I do not increase after such a short time period.

Promoter 1- and 2-derived IGF-I mRNA: coordinate regulation of pre-mRNA but differential regulation of mature mRNA. We determined the contributions of mRNA derived from the individual promoters 1 and 2 to the changes in total pre- and mature hepatic IGF-I mRNA (represented in Fig. 3 as a degree of increase above mean saline-treatment levels). Promoter 1- and 2-derived pre-mRNA levels were equivalent in saline-treated control animals (intron 1, 0.072; intron 2, 0.080 × 10-19 mol mRNA/µg total RNA; means ± SE, n = 10). After the single dose of GH, both increased, with the increase for intron 2 exceeding that for intron 1 (5.3- vs. 7.1-fold), although not significantly. After 8 days of GH treatment, the increases in both promoter 1- and 2-associated pre-mRNA levels were attenuated; the decrease in promoter 2 pre-mRNA but not in promoter 1 pre-mRNA was significant (P < 0.02). Thus, in overall terms, the responses of promoter 1- and 2-derived IGF-I pre-mRNA appeared qualitatively coordinate, but the magnitude of the changes observed for promoter 2-derived mRNA exceeded those for promoter 1-derived message.

Changes in mature mRNA derived from exons 1 and 2 are presented in Fig. 3B, with mean absolute control values being as follows: exon 1, 24.07 ± 1.54; exon 2, 7.65 ± 1.73 × 10-19 mol mRNA/µg total RNA, means ± SE, n = 10. Exon 1-derived mRNA levels demonstrated only a modest increase of ~1.9-fold (P < 0.001) in response to the single dose of GH, and this was not increased by GH treatment for 8 days. In marked contrast, exon 2-derived mature IGF-I mRNA levels increased fivefold (P < 0.001) after the single dose of GH, and this was augmented further (P < 0.01) after 8 days of GH to an eightfold increase. Thus, in absolute terms, GH treatment changed the balance of class 1 and class 2 mRNA, inducing a dominance of class 2 mature mRNA in a temporally regulated manner. Because these changes could not be correlated with changes in pre-mRNA, they support the hypothesis that distinct mechanisms regulate unspliced, pre-mRNA vs. spliced, mature mRNA levels for IGF-I; of potential importance, exon 2-derived mRNA appears more sensitive to the posttranscriptional form of control.

Differential polysomal association of class 1 and 2 IGF-I mRNA. We next examined whether polysomal association, as an indicator of mRNA translatability (12, 13), was equivalent between class 1 and 2 mRNAs. Initially, Northern analysis was performed on total and polysomal RNA by using a specific exon 4 cDNA probe, which would detect all classes of IGF-I mRNA (Fig. 4A). For animals treated with vehicle or GH, the most abundant transcript in total RNA preparations (Fig. 4A, left) was detected at 7.5 kb, in agreement with previous studies (30), with weaker hybridization to 0.8- to 1.2-kb transcripts; after 8 days of GH treatment, the intensities of all these transcripts were increased. In marked contrast, no hybridization to the 7.5-kb transcript could be detected in polysomal preparations (Fig. 4A, right), even though more intense bands were detected at 0.8 to 1.2 kb in animals treated with GH for 8 days. Similar patterns of hybridization were also observed using exon 1-only or exon 2-only cDNA probes, i.e., no 7.5-kb transcript in polysomal preparations (data not shown); also, it appeared that the exon 2 probe hybridized more strongly to the 0.8- to 1.2-kb transcripts in the 8-day GH-treated animals. Consistency for RNA preparations was confirmed using GAPDH Northern blots (data not shown); however, Northern analysis yielded blots that were difficult to quantify reliably for IGF-I mRNA, and therefore RNase protection assays were used to further confirm relative abundances of class 1 and 2 mRNA in total and polysomal preparations.

Representative RNase protection assays to show class 1 and class 2 mRNA levels in the total mRNA population and associated with polysomes are shown in Fig. 4B. Quantified data derived from all samples are shown in Fig. 4C as the ratio of the abundance of class 2 to class 1 in total and polysomal preparations. In vehicle-treated animals, the ratio of class 2 to class 1 mRNA in total and polysomal RNA preparations was ~0.6 in both total and polysomal preparations. In animals given a single dose of GH, this ratio increased significantly to 0.69 in total RNA (P < 0.05) and further (P < 0.01) to 0.84 in polysomal RNA. These changes were augmented further in animals treated with GH for 8 days, with polysomal class 2 to class 1 mRNA significantly exceeding that for total RNA (P < 0.05). Overall, therefore, GH induced a mean 13% increase in the ratio of class 2 to class 1 mRNA derived from polysomes vs. total RNA in liver, indicating a modest preferential association of class 2 with protein synthetic machinery.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The two IGF-I gene promoters are independently regulated and encode unique mRNAs that are conserved across different species, implying differential functional importance; however, none has yet been identified unequivocally. We have made the following novel observations on GH regulation of IGF-I gene expression: 1) the preferential increase in class 2 IGF-I mature mRNA that occurs after several days of treatment is the result of posttranscriptional rather than transcriptional mechanisms, whereas after acute treatment regulation is largely transcriptional; and 2) class 2 mature mRNA has a greater overall translational potential than class 1 mRNA. These observations are of significance in terms of our understanding of IGF-I gene expression and also for clinical treatment with GH.

Determination of an optimal therapeutic dose of GH for deficient patients to maximize potential clinical benefit while minimizing risks associated with excessive GH exposure has proved difficult; responsiveness to GH varies over time, and endogenous GH and IGF-I secretion and sensitivity vary with age in normal patients (11). Reproduction of normal physiological patterns of pulsatile GH secretion is not possible, although a single daily subcutaneous injection of GH is optimal for improved growth rate in children (19, 37), as has been used in the current study. Serum IGF-I concentrations have been used as a marker for excess GH, particularly after the observations that the risk of developing some forms of carcinoma is correlated with circulating IGF-I levels (5, 15). Of potential concern, serum IGF-I concentrations have continued to increase gradually when patients are given a constant dose of GH (9) or have remained elevated in patients even after a reduction in GH dose (45). Our study may provide a mechanism to account for sustained IGF-I synthesis, since hepatic mature mRNA levels "accumulated" after several days of GH. In support of this hypothesis, we observed a continual increase in circulating IGF-I concentrations in lambs treated with a constant dose of GH for 10 wk from 200 to >1,000 ng/ml, and GH binding to hepatic receptors also increased (36).

A key finding from this study is the modest increase in class 1 IGF-I mature transcripts vs. the larger increase in class 2 mature transcripts in response to GH, despite coordinate changes in their respective pre-mRNA levels. Pre-mRNA is processed, migrating from the nucleus very soon after such maturation; thus, usually only fully spliced RNA is exported from the nucleus (10, 20, 23, 32). Therefore, the unspliced pre-mRNA levels measured in this study represent the overall IGF-I transcription rate; this is supported by the excellent correlation of IGF-I transcription, quantified independently using nuclear run-on assays, with measurements of IGF-I pre-mRNA during development (21), fasting (16), and GH treatment (3). We therefore conclude that GH regulates transcription from promoters 1 and 2 of the ovine IGF-I gene in a coordinate manner, as originally suggested by Bichell et al. (3). Thus the differential levels of mature class 1 and 2 mRNAs suggest posttranscriptional regulation.

GH therefore could change the relative rates of 1) splicing of promoter 1- and 2-derived mRNA, 2) transport of mature class 1 and 2 mRNAs from the nucleus, or 3) mature class 1 or 2 mRNA degradation. The current experiment has not addressed pre-mRNA splicing or mRNA export, although splicing has been suggested as a regulatory mechanism controlling IGF-I mRNA abundance in fasted rats (49). The data described here are also consistent with the hypothesis that IGF-I class 2 mRNA may be more stable than class 1 mRNA or that exon 1-derived mRNA is specifically destabilized. mRNA degradation is certainly established as important in the regulation of cytoplasmic mRNA levels (8, 44). Sequences at both the 5' and 3' ends of mRNA, the secondary structure, and association with RNA-binding proteins may all determine mRNA stability (33) by inhibiting or accelerating mRNA degradation. The size heterogeneity of IGF-I mRNA species observed on Northern blots is because of the use of different polyadenylation sites containing potentially destabilizing AU-rich regions within the 3'-untranslated region (3'-UTR) encoded by exon 6 (17). Specific stabilization of IGF-I mRNA by insulin, without changes in transcription rate, has been reported recently (14). However, at present, there is no evidence to support the hypothesis that the choice of 5' splicing determines the length of the 3'-UTR (30), and therefore it is not possible to associate GH regulation of class 1 and 2 mRNA with current mechanisms suggested for IGF-I mRNA degradation.

Polysomal association of IGF-I mRNA was determined to establish the number of transcripts potentially engaged in translation. Northern analysis revealed that only the smaller-size transcripts (0.8-1.2 kb) were associated with polysomal fractions, and these appeared to be enriched in GH-treated animals. These findings are in contrast with those of Thissen and Underwood (43), who detected the 7.5-kb IGF-I mRNA in polysomal fractions, but are in agreement with Foyt et al. (13). It is possible that the polyadenylation, which is in large part responsible for the size difference between the 0.8- to 1.2-kb and 7.5-kb mRNAs, in some manner inhibits the initiation of translation. Quantitative aspects of class 1 and class 2 IGF-I mRNAs associated with polysomes indicated that GH treatment enriched the proportion of class 2-to-class 1 transcripts by a modest but significant amount, suggesting that the proportion of the two mRNAs may have a role in determining overall IGF-I translational efficiency. In agreement with our findings for control animals, Foyt et al. (13) observed no enrichment of polysomes with class 2 mRNA. However, despite observing a small increase, Foyt et al. (12) later concluded that the relative proportions of exon 2 transcripts between total and polysomal populations were not statistically significantly different in response to changed GH status. Nielsen et al. (34) have reported the existence of specific mRNA-binding proteins for IGF-II that interact with 5' regions in a leader exon-specific manner; even though this would provide a mechanism for differential translatability of IGF-I class 1 and 2 mRNA, no such RNA-binding proteins have been reported for IGF-I to date.

Yang et al. (48) measured IGF-I translation directly in vitro using synthetic IGF-I RNAs; class 2 and class 1 start site 4 mRNAs were translated severalfold more efficiently than mRNAs derived from upstream, longer class 1 start sites. Because start site 4 usage is extremely low (13, 36, 41), it would be expected that class 2 transcripts are the most efficiently translated in vivo. RNA secondary structure is a major determinant of translation initiation (22), and molecular modeling (M-fold software; Babraham Bioinformatics Department) of class 1 and 2 IGF-I 5'-UTR implied extensive opportunity for a secondary structure in class 1 but not class 2 5'-UTR.

Ovine class 1 and 2 transcripts encode pre-IGF-I peptides containing 49 and 33 residues of the NH2-terminal signal sequence, respectively, of which the COOH-terminal 28 residues are encoded by the common exon 3. Usually, signal peptides are highly variable and share little in common except a hydrophobic core of ~7-15 amino acids, the cleavage sequence of two alanines at -1 and -3, and a positively charged NH2-terminal (2, 40). It is therefore surprising that the IGF-I class 1 and 2 signal peptide sequences display a high degree of conservation across species. The signal peptide hydrophobic core appears to be of key importance (31), and in the case of IGF-I this is derived from the common coding exon 3. The splicing of exon 2 to exon 3 only adds another five residues, which are not likely to be structurally significant, whereas exon 1 splicing adds another 21 residues. Molecular modeling (Insight-II Software; MSI, San Diego, CA) suggested that the additional residues derived from exon 1 generate another hydrophobic patch that is surrounded by charged residues and that the presence of a proline residue in the class 1 signal peptide introduces additional structural complexity. This initially translated sequence may impede secretory efficiency and therefore act as another level of controlling hepatic IGF-I synthesis.

The physiological function of hepatic IGF-I has been subject to controversy. In the absence of hepatic IGF-I synthesis (26, 27, 46), circulating IGF-I concentrations are markedly reduced, confirming the liver as principally responsible for endocrine IGF-I. Furthermore, increased GH concentrations were observed, supporting a role for either circulating or hepatic IGF-I in negative feedback mechanisms for GH synthesis. Surprisingly, however, mice lacking hepatic IGF-I synthesis grew at normal rates, suggesting that liver-derived endocrine IGF-I was not relevant for growth regulation and casting doubt on the original "somatomedin hypothesis." However, in important recent studies, the ALS has also been ablated, generating growth-retarded mice (47). Because the ALS is responsible for stabilizing the circulating IGF-I pool, these studies support a role for circulating endocrine IGF-I in growth regulation but crucially also suggest that the molecular form of IGF, i.e., association with IGFBPs and formation of ternary complex, is important. Hepatic class 2 IGF-I mRNA is important for the GH stimulation of circulating IGF-I concentrations, but the mechanism by which this occurs has not been identified to date. Our findings suggest that GH stimulation of hepatic IGF-I class 2 mRNA is temporally regulated via posttranscriptional rather than transcriptional mechanisms and that class 2 IGF-I mRNA may be translated and secreted with greater efficiency than exon 1-derived IGF-I. We therefore propose the hypothesis that exon 2-derived IGF-I represents a form of IGF-I that can be synthesized and secreted rapidly in response to GH with the function of coordinating postnatal growth and providing an efficient GH feedback mechanism.


    ACKNOWLEDGEMENTS

We thank Anthony Jones and staff for expert care of the lambs and John Coadwell (Babraham Bioinformatics) for the molecular modeling.


    FOOTNOTES

This work was funded as part of a Competitive Strategic Grant to The Babraham Institute from the Biotechnology and Biological Sciences Research Council.

Address for reprint requests and other correspondence: J. M. Pell, The Babraham Institute, Cambridge, CB2 4AT UK (E-mail: jenny.pell{at}bbsrc.ac.uk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

March 27, 2002;10.1152/ajpendo.00016.2002

Received 15 January 2002; accepted in final form 8 March 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Adamo, ML, Ben-Hur H, Roberts CT, Jr, and LeRoith D. Regulation of start site usage in the leader exons of the rat insulin-like growth factor-I gene by development, fasting, and diabetes. Mol Endocrinol 5: 1677-1686, 1991[Abstract].

2.   Bernstein, HD. Protein targeting: getting into the groove. Curr Biol 8: R715-R718, 1998[ISI][Medline].

3.   Bichell, DP, Kikuchi K, and Rotwein P. Growth hormone rapidly activates insulin-like growth factor I gene transcription in vivo. Mol Endocrinol 6: 1899-1908, 1992[Abstract].

4.   Celi, FS, Zenilman ME, and Shuldiner AR. A rapid and versatile method to synthesize internal standards for competitive PCR. Nucleic Acids Res 21: 1047-1052, 1993[ISI][Medline].

5.   Chan, JM, Stampfer MJ, Giovanucci E, Gann PH, Ma J, Wilkinson P, Hennekens CH, and Pollak M. Plasma insulin-like growth factor-I and prostate cancer risk: a prospective study. Science 279: 563-566, 1998[Abstract/Free Full Text].

6.   Chomczynski, P, and Sacchi N. 1989 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159, 1989.

7.   Church, GM, and Gilbert W. Genomic sequencing. Proc Natl Acad Sci USA 81: 1991-1995, 1984[Abstract].

8.   Day, DA, and Tuite MF. Post-transcriptional gene regulatory mechanisms in eukaryotes: an overview. J Endocrinol 157: 361-371, 1998[Abstract/Free Full Text],.

9.   De Boer, H, Blok GJ, Popp-Snijders C, Stuurman L, Baxter RC, and van der Veen E. Monitoring of growth hormone replacement therapy in adults, based on measurements of serum markers. J Clin Endocrinol Metab 81: 1371-1377, 1996[Abstract].

10.   Derman, E, Goldberg S, and Darnell E, Jr. HnRNA in HeLa cells: distribution of transcript sizes estimated from nascent molecule profiles. Cell 9: 465-472, 1976[ISI][Medline].

11.   Drake Howell, SJ, Monson JP, and Shalet SM. Optimizing GH therapy in adults and children. Endocr Rev 22: 425-450, 2001[Abstract/Free Full Text].

12.   Foyt, HL, Lanau F, Woloshak M, LeRoith D, and Roberts CT, Jr. Effect of growth hormone on levels of differentially processed insulin-like growth factor I mRNAs in total and polysomal mRNA populations. Mol Endocrinol 6: 1881-1888, 1992[Abstract].

13.   Foyt, HL, LeRoith D, and Roberts CT, Jr. Differential association of insulin-like growth factor 1 mRNA variants with polysomes in vivo. J Biol Chem 266: 7300-7306, 1991[Abstract/Free Full Text].

14.   Goya, L, de la Puente A, Ramos S, Martín MA, Escriváí F, Alvarez C, and Pascual-Leone AM. Regulation of IGF-I and -II by insulin in primary cultures of fetal rat hepatocytes. Endocrinology 142: 5089-5096, 2001[Abstract/Free Full Text].

15.   Hankinson, SE, Willet WC, Colditz GA, Hunter DJ, Michaud DS, Deroo B, Speizer FE, and Pollak M. Circulating concentrations of insulin-like growth factor-I and risk of breast cancer. Lancet 351: 1393-1396, 1998[ISI][Medline].

16.   Hayden, JM, Marten NW, Burke EJ, and Straus DS. The effect of fasting on insulin-like growth factor-I nuclear transcript abundance in rat liver. Endocrinology 134: 760-768, 1994[Abstract].

17.   Hepler, JE, Van Wyk JJ, and Lund PK. Different half-lives of insulin-like growth factor I mRNAs that differ in length of 3' untranslated sequence. Endocrinology 127: 1550-1552, 1990[Abstract].

18.   Heuvel, JPV, Tyson FL, and Bell DA. Construction of recombinant RNA templates for use as internal standards in quantitative RT-PCR. Biotechniques 14: 395-398, 1993[ISI][Medline].

19.   Hindmarsh, PC, Stanhope R, Preece MA, and Brook CG. Frequency of administration of growth hormone---an important factor in determining growth response to exogenous growth hormone. Horm Res 33: 83-89, 1990[ISI][Medline].

20.   Jackson, DA, Pombo A, and Iborra F. The balance sheet for transcription: an analysis of nuclear RNA metabolism in mammalian cells. FASEB J 14: 242-254, 2000[Abstract/Free Full Text].

21.   Kikuchi, K, Bichell DP, and Rotwein P. Chromatin changes accompany the developmental activation of insulin-like growth factor I gene transcription. J Biol Chem 267: 21505-21511, 1992[Abstract/Free Full Text].

22.   Kozak, M. Regulation of translation in eukaryotic systems. Annu Rev Cell Biol 8: 197-225, 1992[ISI].

23.   Kramer, A. The structure and function of proteins involved in mammalian pre-mRNA splicing. Annu Rev Biochem 65: 367-409, 1996[ISI][Medline].

24.   Li, J, Owens PC, Saunders JC, Fowden AL, and Gilmour RS. The ontogeny of hepatic growth hormone receptor and insulin-like growth factor I gene expression in the sheep fetus during late gestation: developmental regulation by cortisol. Endocrinology 137: 1650-1657, 1996[Abstract].

25.   Lin, W, and Oberbauer AM. Alternative splicing of insulin-like growth factor 1 mRNA is developmentally regulated in the rat and mouse with preferential exon 2 usage in the mouse. Growth Horm IGF Res 8: 225-233, 1998[ISI][Medline].

26.   Liu, JL, and LeRoith D. Insulin-like growth factor I is essential for postnatal growth in response to growth hormone. Endocrinology 140: 5178-5184, 1999[Abstract/Free Full Text].

27.   Liu, JL, Yakar S, and LeRoith D. Mice deficient in liver production of insulin-like growth factor I display sexual dimorphism in growth hormone-stimulated postnatal growth. Endocrinology 141: 4436-4441, 2000[Abstract/Free Full Text].

28.   Liu, JP, Baker J, Perkins AS, Robertson EJ, and Efstratiadis A. Mice carrying null mutations of the genes encoding insulin-like growth factor I (Igf-1) and type 1 IGF receptor (Igf1r). Cell 75: 59-72, 1993[ISI][Medline].

29.   Lowe, WL, Jr, Roberts CT, Lasky SR, and LeRoith D. Differential expression of alternate 5' untranslated regions in mRNAs enoding rat insulin-like growth factor I. Proc Natl Acad Sci USA 84: 8946-8950, 1987[Abstract].

30.   Lund, PK. Insulinlike growth factor I: molecular biology and relevance to tissue-specific expression and action. Recent Prog Horm Res 49: 125-148, 1994[ISI][Medline].

31.   Matoba, S, and Ogrydziak DM. Another factor besides hydrophobicity can affect signal peptide interaction with signal recognition particle. J Biol Chem 273: 18841-18847, 1998[Abstract/Free Full Text].

32.   Mattaj, IW, and Engelmeier L. Nucleocytoplasmic transport. Annu Rev Biochem 67: 265-306, 1999[ISI][Medline].

33.   Mitchell, P, and Tollervey D. mRNA stability in eukaryotes. Curr Opin Genet Dev 10: 193-198, 2000[ISI][Medline].

34.   Nielsen, J, Christiansen J, Lykke-Andersen J, Johnsen AH, Wewer UM, and Nielsen FC. A family of insulin-like growth factor-II mRNA-binding proteins represses translation in late development. Mol Cell Biol 19: 1262-1270, 1999[Abstract/Free Full Text].

35.   Pell, JM, Elcock C, Harding RL, Morrell DJ, Simmonds AD, and Wallis M. Growth, body copmposition, hormonal and metabolic status in lambs treated long-term with growth hormone. Br J Nutr 63: 341-445, 1990.

36.   Pell, JM, Saunders JC, and Gilmour RS. Differential regulation of transcription initiation from insulin-like growth factor-I (IGF-I) leader exons and of tissue IGF-I expression in response to changed growth hormone and nutritional status in sheep. Endocrinology 132: 1797-1807, 1993[Abstract].

37.   Phillip, M, Hershkovitz E, Belotserkovsky O, Lieberman E, Limoni Y, and Zadik Z. Once vs twice daily injections of growth hormone in children with idiopathic short stature. Acta Paediatr 87: 518-520, 1998[ISI][Medline].

38.   Powell-Braxton, L, Hollingshead P, Warburton C, Dowd M, Pitts-Meek S, Dalton D, Gillett N, and Stewart TA. IGF-I is required for normal embryonic growth in mice. Genes Dev 7: 2609-2717, 1993[Abstract].

39.   Rajaram, S, Baylink DJ, and Mohan S. Insulin-like growth factor binding proteins in serum and other biological fluids: regulation and functions. Endocr Rev 18: 801-831, 1997[Abstract/Free Full Text].

40.   Rusch, SL, and Kendall DA. Protein transport via amino-terminal targeting sequences: common themes in diverse systems. Mol Membr Biol 12: 295-307, 1995[ISI][Medline].

41.   Shemer, J, Adamo ML, Roberts CT, Jr, and LeRoith D. Tissue-specific transcription start site usage in the leader exons of the rat insulin-like growth factor-I gene: evidence for differential regulation in the developing kidney. Endocrinology 131: 2793-2799, 1992[Abstract].

42.   Shuldiner, AR, Tanner K, Moore CA, and Roth J. RNA template-specific PCR: an improved method that dramatically reduced false positives in RT-PCR. Biotechniques 11: 760-763, 1991[ISI][Medline].

43.   Thissen, JP, and Underwood LE. Translational status of the insulin-like growth factor-I messenger RNAs in liver of protein-restricted rats. J Endocrinol 132: 141-147, 1992[Abstract].

44.   Wickens, M, Anderson P, and Jackson RJ. Life and death in the cytoplasm: messages from the 3'end. Curr Opin Dev Gen 7: 220-232, 1997.

45.  Wiren L, Wilhelmson L, McKenna S, and Hember-Stahl EA. A comparison of the quality of life of GHD adults and a random sample population in Sweden. 22nd Int Symp Growth Hormone Growth Factors, Vienna, 1996.

46.   Yakar, S, Liu JL, Stannard B, Butler A, Accili D, Sauer B, and LeRoith D. Normal growth and development in the absence of hepatic insulin-like growth factor I. Proc Natl Acad Sci USA 96: 7324-7329, 1999[Abstract/Free Full Text].

47.  Yakar S, Ooi G, Boisclair Y, and LeRoith D. Inactivation of both liver IGF-I and the acid labile subunit genes cause a marked reduction in serum IGF-I and postnatal growth retardation. 83rd Ann Meet Endocr Soc OR5.5, 2001.

48.   Yang, H, Adamo ML, Kovai AP, McGuinness MC, Ben-Hur H, Yang Y, LeRoith D, and Roberts CT, Jr. Alternate leader sequences in insulin-like growth factor I mRNAs modulate translational efficiency and encode multiple signal peptides. Mol Endocrinol 9: 1380-1395, 1995[Abstract].

49.   Zhang, J, Chrysis D, and Underwood LE. Reduction of hepatic insulin-like growth factor I (IGF-I) messenger ribonucleic acid (mRNA) during fasting is associated with diminished splicing of IGF-I pre-mRNA and decreased stability of cytoplasmic IGF-I mRNA. Endocrinology 139: 4523-4530, 1998[Abstract/Free Full Text].


Am J Physiol Endocrinol Metab 283(2):E251-E258
0193-1849/02 $5.00 Copyright © 2002 the American Physiological Society