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Original Studies |
Pituitary Research Unit (M.B., K.-C.L., K.K.Y.H.) and Neurobiology Research Program (T.P.I.), Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney New South Wales 2010, Australia; and Department of Medicine, Clinical Sciences Center (R.J.M.R.), Sheffield University, Sheffield S5 7AU, United Kingdom
Address all correspondence and requests for reprints to: Dr. Ken K. Y. Ho, Pituitary Research Unit, Garvan Institute of Medical Research, 384 Victoria Street, Sydney, New South Wales 2010, Australia. E-mail: k.ho{at}garvan.unsw.edu.au
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Abstract |
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The mRNA of full-length GHR and GHR-(1–279) were readily detectable in all tissues investigated, with liver, fat, muscle, and kidney showing high levels of expression. These two receptor isoforms were also detected in a range of human cell lines, with strongest expression in IM9, a lymphoblastoid cell line. In contrast, GHR-(1–277) message was expressed at low levels in liver, fat, muscle, kidney, and prostate and in trace amount in IM9 cells.
Full-length GHR was the most abundant isoform, accounting for over 90% of total receptor transcripts in liver, fat, and muscle for quantitative RT-PCR. However, liver had 2- to 4-fold more full-length receptor mRNA and 16- to 40-fold more GHR-(1–277) mRNA than fat and muscle, whereas the mRNA levels of GHR-(1–279) were similar in the three tissues. GHR-(1–279) constituted less than 4% in liver and 7–10% in fat and muscle. GHR-(1–277) accounted for 0.5% of total GHR transcripts in liver and less than 0.1% in the other two tissues. These data suggest that the absolute and relative abundance of mRNA of the three GHR isoforms may be tissue specific. The regulation of expression of exon 9 alternatively spliced GHR variants may provide a potential mechanism for modulation of GH sensitivity at the tissue level.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, two alternatively spliced transcripts at exon 9 of the GHR gene, GHR-(1–279) and GHR-(1–277), were identified in human liver (4, 5). GHR-(1–279) lacks the first 26 bp of exon 9 of the full-length receptor (GHRfl), whereas for GHR-(1–277), this exon is totally deleted (5). Both alternative splices result in a frame shift and a premature stop codon, so that the resulting messenger ribonucleic acids (mRNAs) encode GHR isoforms with intact extracellular and transmembrane domains but lacking more than 90% of the intracellular domain. These receptor variants have no signaling capacity and can inhibit GH action mediated by GHRfl in a dominant negative manner (5, 6). Furthermore, patients heterozygous for mutations that generated splicing out of exon 9 are GH insensitive (6, 7), providing evidence for a pathophysiological role for these truncated receptors.
Little is known about the distribution and abundance of the alternatively spliced (exon 9) GHR variants in human tissues. In the present study we developed quantitative RT-PCR assays specific for the full-length and truncated GHRs and examined their mRNA expression in a wide range of human tissues and cell lines.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human tissues used in this study were liver, fat, muscle, kidney, heart, prostate, fetal liver, lymphocytes, and skin fibroblasts. Cell lines included T47D (breast), HuH7 (liver), IM9 (lymphocyte), SaOS2 (bone), and PC3 (prostate). Tissue samples obtained at time of operative procedures after informed consent or at autopsy for heart and fetal liver were frozen in liquid nitrogen and stored at -70 C. Lymphocytes were purified from whole blood using Mono-Poly Resolving medium (ICN Biomedicals, Inc., Aurora, OH). Skin fibroblasts and cell lines were grown in culture using standard methods.
GHR isoform clones
Full-length GHR complementary DNA (cDNA; pGHR501.1) was a gift from Dr. William Wood (Genentech, Inc., San Francisco, CA). Clones of GHR-(1–279) and GHR-(1–277) were prepared as previously described (5). These clones were used for validating the specificity of PCR primers for the GHR isoforms and as DNA templates for synthesis of recombinant RNA standards for quantitative RT-PCR.
PCR primers
Primers for PCR (Fig. 1A
) were
obtained from Pacific Oligos (Lismore, Australia). A common forward
primer (F2) in exon 7 was used in the PCR for all three GHR isoforms,
whereas specific reverse primers were designed for each isoform. Primer
F1 was targeted to the first 26 bp of exon 9 of GHR, which is present
only in the full-length receptor (Fig. 1B). Primer V1 for GHR-(1–279)
comprised the last 10 bp in exon 8 and bp 27–38 in exon 9. Primer V2
for GHR-(1–277) was targeted at the splice junction between exons 8
and 10 and was devoid of exon 9. The PCR products for GHRfl,
GHR-(1–279), and GHR-(1–277) were 235, 223, and 226 bp in size,
respectively.
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Total RNA was purified from tissue samples (50–100 mg) and cell lines (3–4 x 107 cells) using TRIzol reagent (Life Technologies, Inc., Melbourne, Australia) as recommended by the manufacturer and stored in diethylpyrocarbonate-treated water at -70 C. The RNA concentration was estimated by absorbance at 260 nm. The integrity of RNA samples was checked by examining 28S and 18S bands using agarose gel electrophoresis.
RT-PCR
RT was performed using the Superscript Preamplification System (Life Technologies, Inc.) in which 5 µg total RNA and random hexamers were used. Template RNA was removed by digestion with ribonuclease H and T1 before PCR. Controls with water or no RT were included in all experiments.
PCR was performed with 2 µL RT reaction mixture in 50 µL PCR buffer
(10 mmol/L Tris-HCl, pH 8.3, and 50 mmol/L KCl) containing 1.5 U
AmpliTaq Gold (Perkin-Elmer Corp., Branchburg, NJ), 2
mmol/L MgCl2, 2 µmol/L deoxy-NTP, and 50 pmol
of the forward and reverse primers. After heating to 94 C for 12 min,
35 cycles were run, with denaturation at 94 C for 45 s, annealing
at 58 C for 30 s, and extension at 72 C for 30 s. The
reaction was completed with a final extension at 72 C for 5 min. To
control for sample loading, PCR for ß-actin was performed in parallel
using human ß-actin primers (Fig. 1a
) and the same temperature and
parameters, except with only 24 cycles. The PCR products were analyzed
by electrophoresis on 3% Super Fine Resolution agarose gel
(Amresco, Solon, OH) and UV visualization after ethidium bromide
staining. The band intensity was quantified by densitometry using
Photoshop (Adobe, San Jose, CA). Results represent the mean ±
SEM of at least 3 experiments.
Quantitative RT-PCR
Recombinant RNA standards were constructed for each GHR isoform
by PCR as described by Vanden Hueval et al. (8). Briefly, a
common forward mutant PCR primer (FM2) comprising the T7 promoter
sequence and the F2 primer sequence modified to include an
AatII restriction site (Fig. 1A) was used. PCR was carried
out with the FM2 primer and reverse primer specific for each GHR
isoform using the corresponding isoform clones as DNA templates. Then,
2 µL PCR product were used for synthesizing the RNA standards using
T7 RNA polymerase and MAXI-Script T7 Kit (Ambion, Inc.,
Austin, TX) transcription kit. RNA was purified by ethanol
precipitation, treated with RQ1 deoxyribonuclease (Promega Corp., Madison, WI), repurified, and suspended in 20 µL
diethylpyrocarbonate-treated water for storage at -70 C.
Concentrations of the RNA standards were determined by
A260 measurement.
For quantitative RT-PCR assays, recombinant RNA standards were added at predetermined amounts to 2 µg sample RNA. The RT-PCR reaction was then carried out as described above. At the end of the reaction, 18 µL PCR product were digested with 15 U AatII (Roche Molecular Biochemicals, Sydney, Australia) and separated by agarose gel electrophoresis. AatII cleaved the PCR products of the standards to generate bands 25 bp smaller than those of corresponding GHR isoforms in samples. Bands were visualized after ethidium bromide staining and quantified by densitometry. The amounts of GHR isoform mRNAs for liver, fat, and muscle (n = 3) were determined at equivalence points in the logarithmic plot of the ratio of standard to sample RNA vs. standard RNA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The specificity of PCR primers for the three GHR isoforms was
validated against cDNA clones containing the receptor isoforms. Using
primers F2 and Fl (Fig. 1A), a band was obtained only with the
full-length GHR (GHRfl), not with the truncated receptors (Fig. 2A). Similarly, a band was detected only
for GHR-(1–279) with primer V1 (Fig. 2B) and for GHR-(1–277) with
primer V2 (Fig. 2C). The identities of the PCR products were confirmed
by DNA sequencing (data not shown).
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mRNA expression of the three GHR isoforms in various human tissues
and cell lines was investigated using the isoform-specific RT-PCR
assays. GHRfl mRNA was detected in all tissues and cell lines, with
strongest expression in liver (Fig. 3).
The levels were also high in fat, muscle, and kidney (
75–80% of
that in liver; Fig. 4). Intermediate
levels were detected in heart and fetal liver (30–60%) and low levels
in lymphocyte, fibroblast, and prostate (
15%). GHRfl message was
also readily detectable in cell lines, with relatively similar levels
across cell lines at 55–80% that in liver. Uniform sample loadings
were verified by parallel RT-PCR assays of ß-actin (Fig. 3).
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). Liver still had the highest level of expression, whereas the
levels in fat, muscle, and fetal liver were 80–85% that in liver and
60% of that in kidney (Fig. 4). Lymphocyte, fibroblast, heart,
and prostate had the lowest levels, corresponding to 10–25% of that
in liver. The expression of GHR-(1–279) was highly variable in cell
lines, ranging from 20% in HuH7, a hepatoma cell line, to 75% in IM9,
a lymphoblastoid cell line.
Unlike GHRfl and GHR-(1–279), kidney had a higher level of
GHR-(1–277) mRNA expression (130%) than liver (Figs. 3 and 4). The
levels were around 75% in fat and muscle and 45–50% in prostate and
fetal liver, and were undetectable in lymphocyte, fibroblast, and
heart. GHR-(1–277) mRNA was expressed at very low levels in IM9,
SaOS2, and PC3 (<15%). No band was detected for T47D and HuH7.
Quantitative RT-PCR
To determine the abundance of the three GHR isoforms in human
tissues, we developed quantitative RT-PCR assays for each isoform using
isoform-specific recombinant RNA standards. These standards had
sequences homologous to the corresponding isoform mRNAs, except for the
introduced AatII restriction site (Fig. 1A). Quantitative
RT-PCR assays were performed with sample RNA in the presence of
increasing amounts of RNA standards, followed by AatII
digestion. Products of the standards, but not the samples, were
digested by AatII and appeared as bands 25 bp smaller than
those of the samples (Fig. 5A). The plot
of the log ratio of standard to sample vs. standard RNA was
linear (Fig. 5B).
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). When the three
tissues were compared, liver had approximately 2- and 4-fold more total
receptor transcripts than fat and muscle, respectively. The level of
GHRfl mRNA was highest in liver, intermediate in fat (56% that of
liver), and lowest in muscle (25%). GHR-(1–279) mRNA levels were
similar in the three tissues, whereas those of GHR-(1–277) in fat and
muscle were 2.5% and 6.2% of that in liver, respectively.
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). Despite differences in absolute
amounts, GHRfl was the predominant isoform, representing 90–96% of
the total GHR transcripts in the three tissues (Fig. 6A). In contrast,
the relative abundance of the two truncated isoforms in the three
tissues was quite different. The proportion of GHR-(1–279) in the
total transcripts was lowest in liver and highest in muscle, in which
its relative abundance was 3 times that in liver (Fig. 6B). For
GHR-(1–277), the relative abundance was highest in liver and least in
fat, where it was 25-fold lower than in liver, and was intermediate in
muscle, where it was 5-fold lower than in liver (Fig. 6C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transcripts for GHR-(1–279) and GHR-(1–277) were first identified in human liver (4, 5); however, their distribution in extrahepatic tissues has not received detailed studies. Using isoform-specific quantitative RT-PCR assays, we confirm previous reports that GHRfl and GHR-(1–279) transcripts are expressed in a wide range of human tissues (4), but demonstrate for the first time the existence of GHR-(1–277) mRNA in extrahepatic tissues. However, in contrast to the previous findings of very low levels of GHR-(1–279) in liver, muscle, and kidney, we found that this isoform was readily detectable in these tissues, an observation in agreement with Ross et al. (5), who also found GHR-(1–279) mRNA to be easily detectable in liver, although the absolute abundance was not reported.
Expression of mRNA for the three-receptor isoforms was highest in the major target tissues of GH (liver, fat, muscle and kidney), and the pattern of isoform expression varied widely among tissues, implicating a tissue-specific role for the isoforms. Interestingly, the GHR-(1–279) level in fetal liver was comparable to that in adult liver, whereas those of GHRfl and GHR-(1–277) were only half the levels in adult liver, suggesting that the exon 9 alternative splicing may be regulated developmentally. There are also major differences in the pattern of GHR isoform expression between normal tissues and their corresponding tumor cell lines. Whether alteration in alternative splicing of the GHR gene may be associated with tumorigenic transformation of normal tissues warrants further investigation.
As the amplification efficiencies of the three RT-PCR assays may vary due to different primer sets, we developed and used isoform-specific, quantitative RT-PCR assays to determine the absolute amounts of the receptor isoform mRNAs between and within tissues. Liver expressed 2- and 4-fold more total GHR transcripts than fat and muscle, respectively, an order consistent with that reported previously in these tissues (9, 10). When individual receptor isoforms were compared, the variable tissue expression patterns of the three isoforms were again evident. The data for GHR-(1–277) are the first quantitative comparison with the other isoforms in human tissues. Our quantitative RT-PCR data are consistent with those obtained from the standard RT-PCR assays, although the differences in relative abundance are much larger in the quantitative assays. The absolute values obtained with the quantitative RT-PCR give more reliable estimates than the standard RT-PCR, which rely on single relative densitometric comparisons.
Apart from absolute abundance, the proportions of the full-length and truncated GHRs mRNAs were also variable. The ratios of GHRfl to GHR-(1–279) and GHR-(1–277) were 27:1:0.1 in liver, 14:1:0.002 in fat, and 9:1:0.01 in muscle. The truncated receptors, GHR-(1–279) and GHR-(1–277), lack internalization signals in the cytoplasmic domain. They are able to heterodimerize with the full-length receptor and act as dominant negative inhibitors of GH signaling (5, 6, 11, 12), with the degree of inhibition increasing as the ratio of GHRfl to truncated receptor decreases (5, 6). The difference in relative abundance of GHR mRNA isoforms may have physiological significance if similar differences in GHR proteins are expressed in these tissues. For a 10:1 ratio of GHRfl to GHR-(1–279) like that observed in our studies, GH action was inhibited by up to 30% (5). Therefore, the present finding of different relative mRNA abundance of the full-length and truncated receptors between tissues suggests that regulation of alternative splicing at exon 9 of GHR may have a potential impact on the responsiveness to GH at the level of the individual tissue. Although there is ample evidence that GHR expression is regulated by various factors, including GH, insulin, estrogen, and glucocorticoids (1, 13, 14, 15), their impact on exon 9 alternative splicing has not been reported.
Tissue-specific expression of GHR protein isoforms may also determine the production of the GH-binding protein (GHBP), a soluble protein present in serum identical to the extracellular domain of GHR (16). In humans, GHBP is thought to be generated by proteolytic cleavage of the membrane-bound receptor (5, 17). Cells transfected with truncated GHR isoforms produced more GHBP than those expressing GHRfl (4, 5, 11, 18). It is likely that the absence of the intracellular domain that contains the motifs for internalization (19) renders the receptor isoforms more susceptible to proteolytic cleavage. Although the liver is considered a major source of GHBP in blood (15, 16), our finding that the relative abundance of truncated GHR isoform mRNAs is proportionally higher in fat and muscle suggests that extrahepatic tissues may also be a source of GHBP. Indeed, the observation that GHBP levels are highly related to body mass and fat mass and are reversed by weight loss support this view (15, 20, 21). Further studies of tissue distribution of the protein expression of GHR isoforms as well as receptor turnover are therefore desirable.
In summary, we have developed specific RT-PCR assays for quantitative assessment of expression of the full-length and exon 9 alternatively spliced GHR mRNAs in human tissues. These receptor transcripts have distinct tissue distribution patterns and relative abundance, suggesting a mechanism for tissue-specific regulation of GH action.
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Acknowledgments |
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Footnotes |
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Received February 14, 2000.
Revised April 13, 2000.
Accepted April 19, 2000.
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References |
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