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Characterization of Growth Hormone Receptor Messenger Ribonucleic Acid Variants in Human Adipocytes

Department of Pediatrics (Z.R., C.G.G.) and Division of Experimental Medicine (Y.W., C.G.G.), Faculty of Medicine, McGill University, Montréal, Québec, Canada H3Z 2Z3

Address all correspondence and requests for reprints to: Dr. Cynthia Gates Goodyer, Endocrine Research Laboratory, Room 415/1, McGill University-Montreal Children’s Hospital Research Institute, 4060 Ste. Catherine Street West, Montréal, Québec, Canada H3Z 2Z3. E-mail: cindy.goodyer{at}muhc.mcgill.ca.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Context: Human GH exerts profound effects on adiposity through its specific receptor, hGHR. Eight hGHR mRNAs are produced by the hGHR gene due to splicing from alternate 5'-untranslated region first exons into a common acceptor site upstream of the start codon in exon 2. Four transcripts (V2, V3, V5, V9) are ubiquitously expressed, whereas the other four (V1, V4, V7, V8) are expressed only in normal postnatal liver, suggesting that different promoter usage is a mechanism for developmental- and tissue-specific regulation of the hGHR gene.

Objective: Because it is unknown whether this occurs in adipocytes, we screened human adipocyte cDNA for hGHR mRNAs using 5'-rapid amplification of cDNA ends.

Results: Eighty-nine percent of the clones were V2-like, 3% were V3-like, and 8% were five new mRNA variants (VA-VE). All new 5'-untranslated region sequences mapped within the hGHR 5'flanking region. RT-PCR assays showed expression in multiple fetal and adult tissues, and, thus, they are not adipocyte specific. We compared expression of hGHR mRNAs in adult liver, adult fat, and the human preadipocyte SGBS cell line, using duplex RT-PCR. In liver, V1 and V2 are the major hGHR mRNAs, whereas in adipose, V2 predominates; VA and VC are expressed at similar lower levels in both. In SGBS preadipocytes, approximately 70% of hGHR mRNA is V2. During differentiation, total hGHR and V2 transcripts are markedly up-regulated [hGHR: 2.3 ± 0.2-fold (mean ± SE), P < 0.01; V2: 3.0 ± 0.8, P < 0.03], whereas other variants also increased but remained relatively minor transcripts.

Conclusions: We have identified five new hGHR mRNA variants. Because the V2 transcript is predominant in adipocytes at all developmental stages, the mechanisms regulating its expression should be examined.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
GH IS A key regulator of postnatal growth and metabolism (1, 2, 3). GH exerts these effects by binding to its specific cell surface receptor and triggering various intracellular signaling cascades, resulting in modulation of target cell activity (4). Thus, the receptor expression level is critical for tissue sensitivity to GH.

The GH receptor (GHR), a single transmembrane protein of the class I cytokine receptor family (4), is expressed in most cell types, and it is well known that its levels can be affected by development (5, 6, 7, 8, 9), nutritional status (10, 11), and hormones (10, 12, 13, 14, 15, 16, 17, 18). However, our understanding of the molecular mechanisms controlling GHR expression is incomplete. Studies in different species have shown a common feature: heterogeneity in the 5'-untranslated region (5'UTR) of GHR transcripts (10, 12, 13, 19, 20, 21, 22, 23, 24). This is generated by transcribing and splicing from different 5'-noncoding leader exons to a common splice acceptor site 9–11 bp upstream of the start codon in exon 2 of the GHR gene; thus, they all code for the same protein. In the human, nine 5'UTR mRNA variants (V1-V9) have been reported from screenings of adult liver and cardiac muscle cDNA libraries (19, 20). Although V6 is now considered to be an artifact (21), the other eight variants have been validated by genomic localization and expression profiling.

Chromosomal mapping of these 5'-noncoding exons determined that seven form two separate clusters, each approximately 2 kb in size: V2, V9, and V3 form module A, 140.8–142.4 kb upstream of exon 2, whereas V7, V1, V4, and V8 form module B, 15.8–17.9 kb upstream of exon 2, and V5 is located adjacent to exon 2 (Fig. 1A, revision of Ref.19) (21). Module A and V5 mRNAs are widely expressed; in contrast, module B transcripts are detected only in normal postnatal liver (19). Similar tissue-specific and developmentally specific expression patterns have been observed in other species, suggesting that the derivatives of these 5'UTR exons are likely to be the result of common regulatory mechanisms controlling GHR expression (12, 13, 19, 21, 25). Furthermore, recent studies demonstrated that the 5'UTR variant sequences have differential effects on translation efficiency (12, 26). Therefore, to understand what controls human GHR (hGHR) expression and thus hGH responsiveness, it will be essential to isolate all hGHR 5'UTR mRNA variants and to study how they are regulated in different tissues.

View larger version (34K): [in this window] [in a new window]   FIG. 1. A, Schematic of the hGHR gene structure. The 5'-noncoding exons (V1-V5, V7-V9, V3a/b, VA-VE) and the nine coding exons (open boxes) are shown. The ATG translation start site is located in exon 2. Seven of the 5'-noncoding exons are clustered in approximately 2-kb regions to form two separate modules: module A (V2-V9-V3) is 140.8–142.4 kb upstream of exon 2, whereas module B (V7-V1-V4-V8) is 15.8–17.9 kb upstream of exon 2. All of the exons were mapped within the 5'-upstream region of hGHR gene on chromosome 5 based on contig Hs5_6733 (GenBank no. NT_006576.15). Previous mapping of modules A and B exons (19 ) was undertaken with a BAC clone (hcit. 102E14) that must have had deletions and/or recombinations, resulting in the loss of approximately 104 kb to account for the differences observed with the 5'-flanking region in the present GenBank human genome sequence. B, Schematic representation of the chromosomal location of the VC homologous sequence on the mouse GHR gene. The 5'-flanking region of the mouse GHR gene contains a sequence showing homology to the newly characterized VC exon (mLC), located approximately midway between the L2 (V2-like) and L1 (V1-like) exons. C, 5'UTR sequences of the five new hGHR mRNA variants. Sequences of the newly identified 5'UTR noncoding exons are shown in uppercase letters. The numbers indicate the position and the length of each new 5'UTR based on the GenBank human genome database (no. NT_006576.15). Extra 5' nucleotides found in respective clones are illustrated in the shaded box of lowercase letters (spliced from VC), the boxed lowercase letters (spliced from V3), or the underlined italics (3' extension of V3a/b) (see details in Results). These sequences have been submitted to the GenBank nucleotide database under the following accession numbers: VA: DQ157455, VB: DQ157456, VC: DQ157457, and VE: DQ157458 [VD is ineligible for submission (<50 nt)].

	Materials and Methods

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5'-rapid amplification of cDNA ends (5'RACE)

5'RACE PCR amplification was performed on a Marathon-Ready cDNA pool from adipocytes of four lean and obese individuals (Clontech, Palo Alto, CA) using a 5'-anchor primer (Table 1) and an hGHR-specific reverse primer (Exon2_R4; Table 1). PCR products were cloned into the pCRII-TOPO vector (Invitrogen, Burlington, Ontario, Canada); inserts were checked by EcoRI digestion and sequenced using the ABI Prism BigDye terminator cycle sequencing kit (Applied Biosystem Inc., Foster City, CA).

Fetal tissues were obtained after therapeutic abortion [10.5–19.5 wk fetal age (29)]. Postnatal specimens (15–75 yr) were collected at surgery or within 4–10 h after death. McGill University Health Centre Ethics Committees approved the studies, and all patients provided informed consent. Tissues were flash frozen and stored at –70 C until analysis.

Human Simpson-Golabi-Behmel syndrome (SGBS) preadipocytes were cultured and differentiated into mature adipocytes (30). In brief, SGBS preadipocytes were maintained in DMEM/Ham’s F12 (Invitrogen) containing 10% fetal bovine serum (Cansera, Rexdale, Ontario, Canada) and antibiotics at 37 C with 5% CO₂. Differentiation was initiated when preadipocytes became confluent (d 0). Initially, confluent cells were incubated for 4 d in Quickdiff medium [serum-free, containing 20 nM insulin (Sigma, St. Louis, MO), 200 pM T₃ (Sigma), and 100 nM cortisol (Sigma), supplemented with 25 nM dexamethasone (Sigma), 500 µM 3-isobutyl-methyl-xanthine (Sigma),and 2 µM rosiglitazone (GlaxoSmithKline, West Sussex UK)]. Subsequently the cells were switched to adipogenic medium (containing insulin, T₃, and cortisol) and maintained for up to 16 d. Differentiation was visualized by accumulation of lipid, using Oil Red O staining. Samples were collected every 2–4 d during the time course study.

RNA preparation and RT-PCR assays

Total RNA was isolated from frozen tissues or cultured cells using TRIZOL reagent (Invitrogen); concentrations were determined by spectrophotometry and integrity verified by gel electrophoresis. To examine hGHR expression, 5 µg total RNA were reverse transcribed in 20 µl containing 1x reverse transcription (RT) buffer, 0.5 µM hGHR-specific antisense primer (Exon2_R3; Table 1) and 200 U Superscript II RT. Reactions were incubated at 42 C for 50 min and terminated at 70 C for 15 min. PCR assays were carried out in 25 µl containing 1x PCR buffer, 2 mM MgCl₂, 0.3 mM deoxynucleotide triphosphates, 0.2 µM 5'UTR hGHR variant-specific sense primers (Table 1), 0.2 µM hGHR antisense primer (Exon2_R3 or Exon2_R4; Table 1), 2.5 µl RT product, and 5 U Taq DNA polymerase (Invitrogen). After an initial incubation for 3 min at 94 C, the sample was amplified for 35 cycles comprised of 94 C for 30 sec, 66 C for 30 sec, and 72 C for 1 min. H₂O and no RT controls were always included for assessment of contamination. PCR products were cloned into the pCR2.1-TOPO vector and sequenced to confirm correct amplification.

Semiquantitative RT-PCR

Relative expression levels were determined by duplex RT-PCR, using the Quantum RNA 18S internal standards kit (Ambion Inc., Austin, TX): this includes 18S primers and a pair of 18S competimers to adjust for variations in PCR amplification and loading. In brief, 1 µg of total RNA was treated with RNase-free DNase I (Invitrogen) and used in a 20-µl RT reaction with random hexamers (Invitrogen) and Superscript II RT. Duplex PCRs were carried out with 2.3 µl of RT product (limiting the analysis to eight variants) mixed with sense primers specific to the hGHR 5'UTR noncoding regions and an antisense primer to coding exon 2, and Quantum RNA 18S internal standards with a predetermined ratio (primer to competimer of 1:4). The PCR profile was 94 C for 30 sec, 63–64 C for 30 sec, and 72 C for 1 min. After 34 cycles (preliminary tests showed to be always an exponential amplification), PCR products were resolved on a 2% agarose gel and densitometric analyses performed using GelDoc software (Sigma). Data are presented as the relative ratio of total hGHR mRNA or of each 5'UTR hGHR variant normalized to 18S RNA.

Statistical analyses

Statistical significance (P < 0.05) was determined using the Student’s t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Characterization of adipocyte hGHR mRNA variants

5'RACE of human adipocyte cDNA resulted in 97 hGHR-derived inserts; 86 were V2-like (i.e. matched the exon V2 sequence), three were V3-like, and eight were new variants (Table 2). Five novel 5'UTR sequences, designated VA-VE, were identified (Fig. 1, A and C). Both VB clones contained a 55 nt sequence at their 5'-end derived from V3, indicating that VB is an alternatively spliced product from exon V3. The VE sequence is an 111-nt 3'extension of exon V3a/b, which, given the hGHR genomic sequence, must be generated through the use of different GT splice sites. In contrast, the VA, VC, and VD clones showed no sequence homology with previously published hGHR 5'UTR variants. All three VA clones had an extra 15 nt at their 5'-ends that were the same as the 15 nt at the 3'-end of the VC sequence, suggesting that VA is an alternatively spliced product of exon VC.

By comparison with the GenBank human genome database, using the Blast algorithm (National Center for Biotechnology Information, Bethesda, MD), all five new 5'UTR sequences were mapped to discrete regions within the 5'-flanking region of the hGHR gene, between modules A and B (GenBank no. NT_006576.15) (Fig. 1A). It should be noted that previous mapping of the exons (19) was undertaken with a BAC clone (hcit. 102E14) that must have had deletions and/or recombinations resulting in the loss of approximately 104 kb to account for the differences observed with the 5'-flanking region in the present GenBank human genome.

To determine whether there are homologs in other species, we compared the new 5'UTR sequences to the mouse genome and known rat, bovine, and ovine GHR mRNA variants in the GenBank databases. Only VC exhibited any similarity (87%) with the mouse genome. Interestingly, the genomic location of this VC-like sequence (mLC, Fig. 1B) is also approximately midway between mouse L2 (V2-like) and L1 (V1-like).

Expression of the new 5'UTR variants in human tissues

Multiple human adult (liver, fat, kidney) and fetal (liver, fat, kidney, lung, skeletal muscle, placenta) tissues were examined to determine whether expression of the new 5'UTR transcripts was adipocyte specific. With VA, VB, and VE primers, amplicons of expected sizes were detected in all tissues (Fig. 2; data not shown); these products were sequenced, confirming the correct amplification. Occasionally a second minor slower migrating band was obtained with VA and VB primers. With the VC primer, two major amplicons were obtained in all tissues, suggesting alternative splice products: sequencing revealed that the lower band (223 bp, Fig. 2) is VC itself, whereas the upper band (348 bp) is VC+VA. This finding was consistent with the 5'RACE results, in which all three VA clones contained 15 nucleotides derived from VC at their 5' ends. No specific amplification from VD was detected in any tissue, even fat, suggesting that the PCR conditions were not optimal or the level of expression is extremely low. Thus, the RT-PCR screening confirmed the existence of four (VA, VB, VC, VE) new hGHR transcripts. They were expressed in every tissue tested and were not unique for adipocytes.

View larger version (62K): [in this window] [in a new window]   FIG. 2. Expression of new 5'UTR mRNA variants in human tissues. The expression patterns of the new mRNA variants (VA-VE) were examined by RT-PCR using total RNAs extracted from several human fetal and adult tissues. The cDNAs were amplified with sense primers specific to the individual noncoding exons and a common antisense primer to hGHR exon 2 (Table 1). The widely expressed V2 and V3 variants were used as positive controls; the two lower V3 bands represent V3 and V3+V3a/b, whereas the third band was minor and invariably expressed. PCR products were run on 2% agarose gels to visualize the specific amplicons, using the 100-bp DNA ladder as a migration marker (M). Representative gels are shown from studies of samples (n = 3) for each tissue. There was occasionally a second VA or VB band that could not be sequenced due to low expression levels. VC primers detected two bands due to alternative splicing (see details in Results). A specific VD product was never visualized, although nonspecific bands did occur occasionally (e.g. fetal and adult kidney gels), suggesting either technical problems with the PCR assay conditions or very low levels of expression in these tissues. The VE primers amplified a single amplicon of the expected size in all tissues. (V3* lane shows the results of using a different antisense primer.)

To determine the relative abundance of hGHR transcripts, we developed duplex RT-PCR assays. Because different primer pairs were used for each variant, the resultant data do not add up to total GHR but give a relative comparative value. Studies of adult liver and fat (Fig. 3, A and B) showed that whereas V2, V3, V9, VA, VC, and VC+A are expressed in both tissues, V1 was highly expressed only in postnatal liver. These data support previous reports that V1 is a postnatal liver-specific transcript (19, 31, 32). In adult liver, V1 and V2 transcripts form the majority of the hGHR mRNA pool, whereas in adult fat, V2 predominates. In both tissues, VA, VC, and VC+A are expressed at lower levels, similar to V3 and V9. Assays (n = 2) of fetal liver showed similar V2, V9, and V3 expression as in adult liver and fat, with a relatively low level of VA, VC, and VC+A, and no V1 (data not shown). Unfortunately, the fetal fat samples were insufficient for duplex assays.

View larger version (35K): [in this window] [in a new window]   FIG. 3. Relative expression of total hGHR and seven of its mRNA variants in adult liver and fat. The relative expression levels of total hGHR and seven of its mRNA variants (V2, V3, V9, V1, VA, VC, and VC+A) in human adult liver (hAL) or fat (hAF) tissue were estimated by semiquantitative duplex RT-PCR. A, The relative ratios of total hGHR or individual mRNA variants were normalized to 18S rRNA (quantum RNA 18S internal standard; Ambion). Each tissue type was analyzed using multiple samples (n = 3), and the data are presented as mean ± SE. *, P < 0.05; ***, P < 0.01. B, Representative gels of the semiquantitative RT-PCRs. Twelve microliters of PCR products were separated on 2% agarose gels using the 100-bp DNA ladder as a marker (M). Arrows indicate the expected 18S amplicon (489 bp), which was used as the internal control.

Zou et al. (33) reported previously that total GHR mRNA levels increased significantly during differentiation of mouse 3T3-L1 preadipocytes. However, similar studies during human adipocyte differentiation have not been published. We, therefore, obtained the SGBS human preadipocyte cell line (30) and assessed the quantitative changes in total hGHR mRNA as well as six of the most highly expressed variant transcripts during maturation, using duplex RT-PCR assays. At the predipocyte stage, the predominant variant (70%) was V2; other forms were either expressed at low levels (e.g. VA and VC+A: 10%) or were barely detectable (e.g. V3, V9, VC) (Fig. 4). Total hGHR and V2 mRNAs were markedly up-regulated in parallel from d 0–4 postinduction of differentiation, reaching a maximum by d 12 [hGHR: 2.3 ± 0.2-fold (mean ± SE), P < 0.01; V2: 3.0 ± 0.8, P < 0.03]. VA and VC+A mRNAs also increased but later, between d 4 and 12, whereas the other 5'UTR transcripts remained minor populations. These data clearly demonstrate that total hGHR gene expression increases significantly as human preadipocytes differentiate and that this is mainly due to the V2 transcript, especially during the earliest developmental stages.

View larger version (16K): [in this window] [in a new window]   FIG. 4. Relative expression of total hGHR and hGHR mRNA variants during SGBS adipocyte differentiation. Human SGBS preadipocytes were cultured to confluence and then differentiated into mature adipocytes (30 ). Day 0 represents the confluent preadipocyte stage before the induction of differentiation. Samples were collected at 2- to 4-d intervals during the differentiation process. The relative expression levels of total hGHR and six of its mRNA variants (V2, V3, V9, VA, VC, and VC+A) were assessed by semiquantitative RT-PCR using 18S rRNA as internal control (see Results and legend of Fig. 3 for details). Data are presented as the relative ratios of either total hGHR or its individual mRNA variants normalized to 18S rRNA. Each time point represents two (mean) or three (mean ± SE) differentiation experiments (n = 2 for d 4 and 6; n = 3 for d 0, 8, and 12).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

First isolated from adult liver and cardiac muscle cDNA libraries, eight hGHR mRNA variants have been well characterized to date, with four showing developmental (postnatal)- and tissue (liver)-specific expression (19, 20, 21). We were interested in examining the hGHR transcripts expressed in adipose tissue, another important hGH target, to know whether there might be adipocyte-specific variants. Using 5'RACE, we demonstrated that V2 is the predominant (89%) hGHR transcript in fat cells. This is strikingly different from what was reported for an adult liver cDNA library, in which V1 (47%), V2 (27%), and V3 (11%) were all highly expressed (20), or from the study of a cardiac muscle library, in which V2 (37.5%), V9 (25%), and V3 (16.7%) were all well represented (19). This difference was further confirmed by our semiquantitative RT-PCR comparison of the expression patterns of total hGHR and several of its mRNA variants in adult liver vs. fat. Although no other data from human adipose tissue have been reported, similar findings in the mouse were presented by Moffat et al. (25): the L2 GHR variant (V2-like) was present in 88% of the 5'RACE clones from adipose tissue of nonpregnant mice. Therefore, although liver, muscle, and adipose tissues are all major target organs for hGH and all express high levels of hGHR mRNA (5, 27, 34, 35), the mechanisms regulating hGHR expression in each tissue appear to be very different.

In our study, five new 5'UTR mRNA variants were isolated. Except for VA, which Wickelgren et al. also observed in adipose tissue (unpublished data, GenBank no. AY216680), the other four variants have never been reported. The new 5'UTR sequences all map within the 5'-flanking region of the hGHR gene. In a comparison with the mouse genome, the VC sequence showed high similarity (87%) to a homologous region of the mouse GHR 5'-flanking region. None of the other novel variants had significant similarities, and there were no homologies, with any of the known rat, ovine, or bovine GHR 5'UTR exons.

To determine whether these new variants were adipose-specific transcripts, we screened multiple fetal and adult tissues using RT-PCR. Unfortunately, the answer was no: four of the new variants [VA, VB, VC (and VC+A), VE] could be detected in all of the tissues tested. Although we were unable to amplify a VD transcript, the VD exon was localized to 1.2 kb downstream of module A, suggesting that it may be a low-expressing hGHR variant exon. Duplex RT-PCR assays confirmed these data and demonstrated that variants VA, VC, and VC+A are expressed in both adult liver and fat at levels similar to V3 and V9, whereas a more limited study of fetal liver suggests they may be expressed at lower levels early in gestation.

The possible functions of these widely expressed minor transcripts have long been a puzzle that is not limited to the human: there are multiple GHR mRNA variants expressed at low levels in the mouse and bovine as well [e.g. mL3–5 (25), b1C-1I (12)]. They do not appear to contribute significantly to GHR expression under normal physiological conditions. However, it cannot be excluded that, under certain pathophysiological conditions, the expression of one minor transcript will increase markedly in a specific tissue or several tissues, causing a major change in GHR expression and affecting sensitivity to GH. One other possibility is suggested by previous studies showing that different GHR 5'UTR sequences have profound effects on translational activities of test RNAs. Jiang and Lucy (12) found that, in cattle, the predominant nonhepatic GHR 5'UTR variant b1B (V2-like) had the poorest translational ability, whereas other ubiquitously expressed minor transcripts (e.g. b1H, b1I) were more efficiently translated. Moffat et al. (26) reported analogous observations for mouse GHR mRNA variants.

Seven of the previously identified hGHR 5'UTR exons form two small clusters in the 5'-flanking region of the hGHR gene. Three mechanisms have been suggested for regulation of their transcriptional activity: 1) an upstream common promoter that controls transcription from all exons within the cluster; 2) independent promoters that regulate transcription from individual exons; or 3) a combination of the two possibilities (12, 19, 25). To determine how the five new mRNA variants may be generated, we located their positions on the human genome sequence relative to the known hGHR coding and 5'non coding exons. VC and VA exons are clustered in a 1.2-kb region, 81.3 kb upstream of hGHR coding exon 2, approximately midway between modules A and B, suggesting that these two are regulated by a new promoter region. Both Wickelgren et al. (GenBank submission) and we (present study) found that the VA sequence had extra nucleotides at the 5'end that are homologous to the 3'end of VC, indicating that VA is an alternatively spliced product of VC. These speculations were confirmed by our RT-PCR tissue screening, in which the VC primer was able to consistently amplify two products (VC, VC+A), and by duplex RT-PCR quantification, in which the level of VA approximates the level of VC+A. Based on these data, we believe that the VC and VA transcripts are generated from a common promoter, located 5' to VC. Both VB and VE appear to arise from the hGHR transcript initiated from exon V3 because 5'RACE clones containing VB or VE all include 40–53 nt of the V3 sequence at their 5' end. However, RT-PCR with the V3 primer could not consistently amplify V3+VB or V3+VE products, suggesting that these transcripts have low expression levels in the tissues tested.

The new variants were not adipose tissue specific. However, fat tissue is a complex mix of mature adipocytes as well as preadipocytes, vascular cells, and pericytes (36). Therefore, we hypothesized that there might be differential expression of hGHR transcripts at specific developmental stages. Using the human preadipocyte SGBS cells, we found that total hGHR mRNA levels increased significantly during early stages (d 0–4) of SGBS cell differentiation and reached a maximum when the adipocytes matured (d 12). Although differentiation procedures for human and mouse cell lines are quite different, these data are consistent with what Zou et al. (33) observed during 3T3-L1 preadipocyte differentiation. They found, using RNase protection assays, that GHR mRNA levels began to increase after 3 d of differentiation and reached a maximum in mature adipocytes by d 7; the relative abundance of total GHR mRNA was 28 times greater in the mature adipocyte, compared with the preadipocyte. The difference in fold change levels achieved in each case (28 times in 3T3-L1, 2.4 times in SGBS) may be due to different sensitivity of the assay methods as well as the cells themselves.

We show here for the first time that the significant increase in total hGHR mRNA is primarily due to V2, the predominant hGHR transcript in adipose tissue. More interestingly, we observed specific variant expression patterns in preadipocytes vs. mature adipocytes. In the preadipocyte, the major transcript is V2; we also observed low levels of the VA and VC+A forms but could not consistently detect V3, V9, or VC. In the mature adipocyte, VA and VC+A transcripts represented a significant proportion of the total hGHR pool (35%) whereas V3, V9, and VC remained at low levels. These data suggest that certain hGHR 5'UTR transcripts may be more prominently expressed at certain developmental stages, perhaps leading to differential expression of hGHR and, ultimately, altered responses to hGH.

The widely expressed hGHR V9 transcript was first isolated from a cardiac muscle cDNA library (19). Two other V9-related mRNA variants, V9a and V9b, were observed by Wickelgren et al. in skeletal muscle (unpublished data, GenBank no. AF230800 and AF230801). By comparison with the human genome, we found that V9b is the 3' end of V9 and that V9a is an alternative splicing product of V9 (with the V9a exon located about midway between VA and module B). However, in our adipose 5'RACE screen, we did not obtain any V9-containing clones. In addition, semiquantitative RT-PCR assays of adult fat samples showed that V9 mRNAs were expressed at a relatively low level. Furthermore, few V9 transcripts could be detected in SGBS preadipocytes, and even after differentiating into mature adipocytes, V9 expression remained low. Thus, in contrast to muscle, it appears that the V9 hGHR transcript is minimally expressed in adipocytes.

Obesity has become a major epidemic health problem, especially in developed nations. To combat this, it will be important to have a thorough understanding of the mechanisms controlling the ontogeny as well as the mass of adipose tissue. hGH has long been known to be one of the most important negative regulatory hormones: hGH treatments significantly reduce body fat in GH-deficient children and adults (27, 37, 38, 39, 40, 41), although similar treatments of morbidly obese individuals who are not hGH deficient have been less successful for unknown reasons (28, 42, 43).

Whereas the effects of hGH on adipose tissue are known to be through its specific receptor, our understanding of how hGHR expression is regulated in adipocytes is surprisingly limited. It has been well documented that obese people have low hGH levels, due to the inhibitory effects of excess FFA on hGH secretion as well as increased hGH clearance (28, 44, 45). In contrast, there are no reports on their adipose tissue hGHR. Their relatively low responsiveness to chronic hGH therapy suggests decreased hGHR expression and/or function. However, Gleeson et al. (45) observed an increase in circulating IGF-I, positively correlated with GH binding protein levels after a single bolus of hGH in obese individuals. Whether this increase in hepatic responsiveness reflects conditions within the adipocyte remains to be determined.

Our present study begins to address this issue. However, more in-depth studies of the V2 promoter and primary human adipose tissues are needed to better understand what regulates hGHR expression in adipocytes and how this may alter adipocyte sensitivity to hGH.

	Acknowledgments

 
We gratefully acknowledge the gift of SGBS cells from Dr. M. Wabitsch (University of Ulm, Ulm, Germany).

	Footnotes

 
This work was supported by the Canadian Institutes of Health Research and a studentship from the Fonds de Recherches en Santé du Québec (to Y.W.).

The authors have no conflict of interest to declare.

First Published Online February 21, 2006

Abbreviations: GHR, GH receptor; hGHR, human GHR; 5'RACE, 5'-rapid amplification of cDNA ends; RT, reverse transcription; SGBS, Simpson-Golabi-Behmel syndrome; 5'UTR, 5'-untranslated region.

Received August 10, 2005.

Accepted February 9, 2006.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Flint DJ, Binart N, Kopchick J, Kelly P 2003 Effects of growth hormone and prolactin on adipose tissue development and function. Pituitary 6:97–102[CrossRef][Medline]
2. Nam SY, Lobie PE 2000 The mechanism of effect of growth hormone on preadipocyte and adipocyte function. Obes Rev 1:73–86[CrossRef][Medline]
3. Veldhuis JD, Roemmich JN, Richmond EJ, Rogol AD, Lovejoy JC, Sheffield-Moore M, Mauras N, Bowers CY 2005 Endocrine control of body composition in infancy, childhood, and puberty. Endocr Rev 26:114–146[Abstract/Free Full Text]
4. Piwien-Pilipuk G, Huo JS, Schwartz J 2002 Growth hormone signal transduction. J Pediatr Endocrinol Metab 15:771–786[Medline]
5. Goodyer CG, Figueiredo R, Krackovitch S, DeSouza Li L, Manalo J, Zogopoulos G 2001 Characterization of the growth hormone receptor in human dermal fibroblasts and liver during development. Am J Physiol Endocrinol Metab 281:E2313–E2320
6. Adams TE, Baker L, Fiddes RJ, Brandon MR 1990 The sheep growth hormone receptor: molecular cloning and ontogeny of mRNA expression in the liver. Mol Cell Endocrinol 73:135–145[CrossRef][Medline]
7. Li J, Owens JA, Owens PC, Saunders JC, Fowden AL, Gilmour RS 1996 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[Abstract]
8. Walker JL, Moats-Staats BM, Stiles AD, Underwood LE 1992 Tissue-specific developmental regulation of the messenger ribonucleic acids encoding the growth hormone receptor and the growth hormone binding protein in rat fetal and postnatal tissues. Pediatr Res 31:335–339[Medline]
9. Ymer SI, Herington AC 1992 Developmental expression of the growth hormone receptor gene in rabbit tissues. Mol Cell Endocrinol 83:39–49[CrossRef][Medline]
10. Talamantes F, Ortiz R 2002 Structure and regulation of expression of the mouse GH receptor. J Endocrinol 175:55–59[Abstract]
11. Wang Y, Eleswarapu S, Beal WE, Swecker Jr WS, Akers RM, Jiang H 2003 Reduced serum insulin-like growth factor (IGF) I is associated with reduced liver IGF-I mRNA and liver growth hormone receptor mRNA in food-deprived cattle. J Nutr 133:2555–2560[Abstract/Free Full Text]
12. Jiang H, Lucy MC 2001 Variants of the 5'-untranslated region of the bovine growth hormone receptor mRNA: isolation, expression and effects on translational efficiency. Gene 265:45–53[CrossRef][Medline]
13. Schwartzbauer G, Menon RK 1998 Regulation of growth hormone receptor gene expression. Mol Genet Metab 63:243–253[CrossRef][Medline]
14. Rhoads RP, Kim JW, Leury BJ, Baumgard LH, Segoale N, Frank SJ, Bauman DE, Boisclair YR 2004 Insulin increases the abundance of the growth hormone receptor in liver and adipose tissue of periparturient dairy cows. J Nutr 134:1020–1027[Abstract/Free Full Text]
15. Leung KC, Doyle N, Ballesteros M, Waters MJ, Ho KK 2000 Insulin regulation of human hepatic growth hormone receptors: divergent effects on biosynthesis and surface translocation. J Clin Endocrinol Metab 85:4712–4720[Abstract/Free Full Text]
16. Meinhardt U, Eble A, Besson A, Strasburger CJ, Sraer JD, Mullis PE 2003 Regulation of growth-hormone-receptor gene expression by growth hormone and pegvisomant in human mesangial cells. Kidney Int 64:421–430[CrossRef][Medline]
17. Mullis PE, Eble A, Marti U, Burgi U, Postel-Vinay MC 1999 Regulation of human growth hormone receptor gene transcription by triiodothyronine (T3). Mol Cell Endocrinol 147:17–25[CrossRef][Medline]
18. Vottero A, Kimchi-Sarfaty C, Kratzsch J, Chrousos GP, Hochberg Z 2003 Transcriptional and translational regulation of the splicing isoforms of the growth hormone receptor by glucocorticoids. Horm Metab Res 35:7–12[CrossRef][Medline]
19. Goodyer CG, Zogopoulos G, Schwartzbauer G, Zheng H, Hendy GN, Menon RK 2001 Organisation and evolution of the human growth hormone receptor gene 5' flanking region. Endocrinology 142:1923–1934[Abstract/Free Full Text]
20. Pekhletsky RI, Chernov BK, Rubtsov PM 1992 Variants of the 5'-untranslated sequence of human growth hormone receptor mRNA. Mol Cell Endocrinol 90:103–109[CrossRef][Medline]
21. Orlovskii IV, Sverdlova PS, Rubtsov PM 2004 [Fine structure, expression and polymorphism of the human growth hormone receptor gene]. Mol Biol (Mosk) 38:29–39
22. Adams TE 1995 Differential expression of growth hormone receptor messenger RNA from a second promoter. Mol Cell Endocrinol 108:23–33[CrossRef][Medline]
23. Domene HM, Cassorla F, Werner H, Roberts Jr CT, Leroith D 1995 Rat growth hormone receptor/growth hormone-binding protein mRNAs with divergent 5'-untranslated regions are expressed in a tissue-specific manner. DNA Cell Biol 14:195–204[Medline]
24. Zogopoulos G, Nathanielsz P, Hendy GN, Goodyer CG 1999 The baboon: a model for the study of primate growth hormone receptor gene expression during development. J Mol Endocrinol 23:67–75[Abstract]
25. Moffat JG, Dao H, Talamantes F 2000 Alternative 5'-untranslated regions of mouse GH receptor/binding protein messenger RNA are derived from sequences adjacent to the major L2 promoter. J Endocrinol 167:145–152[Abstract]
26. Moffat JG, Tran K, Talamantes F, Functional analysis of the mouse GH receptor/binding protein L2 promoter and 5' UTR L2 sequence. Program of the 81st Annual Meeting of The Endocrine Society, San Diego, CA, 1999, p 154 (Abstract P1-92)
27. Fisker S, Kristensen K, Rosenfalck AM, Pedersen SB, Ebdrup L, Richelsen B, Hilsted J, Christiansen JS, Jorgensen JO 2001 Gene expression of a truncated and the full-length growth hormone (GH) receptor in subcutaneous fat and skeletal muscle in GH-deficient adults: impact of GH treatment. J Clin Endocrinol Metab 86:792–796[Abstract/Free Full Text]
28. Franco C, Brandberg J, Lonn L, Andersson B, Bengtsson BA, Johannsson G 2005 Growth hormone treatment reduces abdominal visceral fat in postmenopausal women with abdominal obesity: a 12-month placebo-controlled trial. J Clin Endocrinol Metab 90:1466–1474[Abstract/Free Full Text]
29. Munsick RA 1984 Human fetal extremity lengths in the interval from 9 to 21 menstrual weeks of pregnancy. Am J Obstet Gynecol 149:883–887[Medline]
30. Wabitsch M, Brenner RE, Melzner I, Braun M, Moller P, Heinze E, Debatin KM, Hauner H 2001 Characterization of a human preadipocyte cell strain with high capacity for adipose differentiation. Int J Obes Relat Metab Disord 25:8–15[CrossRef][Medline]
31. Zou L, Burmeister LA, Sperling MA 1997 Isolation of a liver-specific promoter for human growth hormone receptor gene. Endocrinology 138:1771–1774[Abstract/Free Full Text]
32. Zogopoulos G, Albrecht S, Pietsch T, Alpert L, von Schweinitz D, Lefebvre Y, Goodyer CG 1996 Fetal- and tumor-specific regulation of growth hormone receptor mRNA expression in human liver. Cancer Res 56:2949–2953[Abstract/Free Full Text]
33. Zou L, Menon RK, Sperling MA 1997 Induction of mRNAs for the growth hormone receptor gene during mouse 3T3–L1 preadipocyte differentiation. Metabolism 46:114–118[CrossRef][Medline]
34. Hermansson M, Wickelgren RB, Hammarqvist F, Bjarnason R, Wennstrom I, Wernerman J, Carlsson B, Carlsson LM 1997 Measurement of human growth hormone receptor messenger ribonucleic acid by a quantitative polymerase chain reaction-based assay: demonstration of reduced expression after elective surgery. J Clin Endocrinol Metab 82:421–428[Abstract/Free Full Text]
35. Ballesteros M, Leung KC, Ross RJ, Iismaa TP, Ho KK 2000 Distribution and abundance of messenger ribonucleic acid for growth hormone receptor isoforms in human tissues. J Clin Endocrinol Metab 85:2865–2871[Abstract/Free Full Text]
36. Ailhaud G, Grimaldi P, Negrel R 1992 Cellular and molecular aspects of adipose tissue development. Annu Rev Nutr 12:207–233[CrossRef][Medline]
37. Brook CG 1973 Effect of human growth hormone treatment on adipose tissue in children. Arch Dis Child 48:725–728[Abstract/Free Full Text]
38. Carroll PV, Drake WM, Maher KT, Metcalfe K, Shaw NJ, Dunger DB, Cheetham TD, Camacho-Hubner C, Savage MO, Monson JP 2004 Comparison of continuation or cessation of growth hormone (GH) therapy on body composition and metabolic status in adolescents with severe GH deficiency at completion of linear growth. J Clin Endocrinol Metab 89:3890–3895[Abstract/Free Full Text]
39. Maison P, Griffin S, Nicoue-Beglah M, Haddad N, Balkau B, Chanson P 2004 Impact of growth hormone (GH) treatment on cardiovascular risk factors in GH-deficient adults: a metaanalysis of blinded, randomized, placebo-controlled trials. J Clin Endocrinol Metab 89:2192–2199[Abstract/Free Full Text]
40. Boguszewski CL, Meister LH, Zaninelli DC, Radominski RB 2005 One year of GH replacement therapy with a fixed low-dose regimen improves body composition, bone mineral density and lipid profile of GH-deficient adults. Eur J Endocrinol 152:67–75[Abstract/Free Full Text]
41. Leal CA 2004 Long-term challenges in growth hormone treatment. Horm Res 62(Suppl 4):23–30
42. Murray RD, Shalet SM 2002 Adult growth hormone replacement: lessons learned and future direction. J Clin Endocrinol Metab 87:4427–4428[Free Full Text]
43. Albert SG, Mooradian AD 2004 Low-dose recombinant human growth hormone as adjuvant therapy to lifestyle modifications in the management of obesity. J Clin Endocrinol Metab 89:695–701[Abstract/Free Full Text]
44. Miller KK, Biller BM, Lipman JG, Bradwin G, Rifai N, Klibanski A 2005 Truncal adiposity, relative growth hormone deficiency, and cardiovascular risk. J Clin Endocrinol Metab 90:768–774[Abstract/Free Full Text]
45. Gleeson HK, Lissett CA, Shalet SM 2005 Insulin-like growth factor-I response to a single bolus of growth hormone is increased in obesity. J Clin Endocrinol Metab 90:1061–1067[Abstract/Free Full Text]

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