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Endocrinology, doi:10.1210/en.2006-0772
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Endocrinology Vol. 148, No. 1 45-53
Copyright © 2007 by The Endocrine Society

Isolated Autosomal Dominant Growth Hormone Deficiency: Stimulating Mutant GH-1 Gene Expression Drives GH-1 Splice-Site Selection, Cell Proliferation, and Apoptosis

Souzan Salemi, Shida Yousefi, Didier Lochmatter, Andrée Eblé, Johnny Deladoëy, Iain C. A. F. Robinson, Hans-Uwe Simon and Primus E. Mullis

Paediatric Endocrinology (S.S., D.L., A.E., J.D., P.E.M.), University Children’s Hospital, Inselspital, and Department of Pharmacology (S.Y., H.-U.S.), University of Bern, CH- 3010 Bern, Switzerland; and National Institute for Medical Research (I.C.A.F.), Mill Hill, London NW7 1AA, United Kingdom

Address all correspondence and requests for reprints to: Prof. Primus E. Mullis, M.D., Paediatric Endocrinology, Diabetology and Metabolism, University Children’s Hospital, Inselspital, CH-3010 Bern, Switzerland. E-mail: primus.mullis{at}insel.ch.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The majority of mutations that cause isolated GH deficiency type II (IGHD II) affect splicing of GH-1 transcripts and produce a dominant-negative GH isoform lacking exon 3 resulting in a 17.5-kDa isoform, which further leads to disruption of the GH secretory pathway. A clinical variability in the severity of the IGHD II phenotype depending on the GH-1 gene alteration has been reported, and in vitro and transgenic animal data suggest that the onset and severity of the phenotype relates to the proportion of 17.5-kDa produced. The removal of GH in IGHD creates a positive feedback loop driving more GH expression, which may itself increase 17.5-kDa isoform productions from alternate splice sites in the mutated GH-1 allele. In this study, we aimed to test this idea by comparing the impact of stimulated expression by glucocorticoids on the production of different GH isoforms from wild-type (wt) and mutant GH-1 genes, relying on the glucocorticoid regulatory element within intron 1 in the GH-1 gene. AtT-20 cells were transfected with wt-GH or mutated GH-1 variants (5'IVS-3 + 2-bp T->C; 5'IVS-3 + 6 bp T->C; ISEm1: IVS-3 + 28 G->A) known to cause clinical IGHD II of varying severity. Cells were stimulated with 1 and 10 µM dexamethasone (DEX) for 24 h, after which the relative amounts of GH-1 splice variants were determined by semiquantitative and quantitative (TaqMan) RT-PCR. In the absence of DEX, only around 1% wt-GH-1 transcripts were the 17.5-kDa isoform, whereas the three mutant GH-1 variants produced 29, 39, and 78% of the 17.5-kDa isoform. DEX stimulated total GH-1 gene transcription from all constructs. Notably, however, DEX increased the amount of 17.5-kDa GH isoform relative to the 22- and 20-kDa isoforms produced from the mutated GH-1 variants, but not from wt-GH-1. This DEX-induced enhancement of 17.5-kDa GH isoform production, up to 100% in the most severe case, was completely blocked by the addition of RU486. In other studies, we measured cell proliferation rates, annexin V staining, and DNA fragmentation in cells transfected with the same GH-1 constructs. The results showed that that the 5'IVS-3 + 2-bp GH-1 gene mutation had a more severe impact on those measures than the splice site mutations within 5'IVS-3 + 6 bp or ISE +28, in line with the clinical severity observed with these mutations. Our findings that the proportion of 17.5-kDa produced from mutant GH-1 alleles increases with increased drive for gene expression may help to explain the variable onset progression, and severity observed in IGHD II.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HUMAN GH-1 gene located on the long arm of chromosome 17 encodes a mature transcript of 22 kDa, derived from five exons and four introns; other products are derived from alternative splicing, particularly within and around exon 3, which is flanked with weak splice sites (1). Splicing of the nascent mRNA transcript is carried out in the cell nucleus by a large macromolecular spliceosome complex (2, 3), recognizing consensus sequences at intervening sequence (IVS) boundaries (5'and 3' splice sites, called 5' SS and 3' SS) as well as the branch site (4). In addition, multiple cis-acting elements [exon splice enhancers (ESE) and intron splice enhancers (ISE)] and trans-acting factors [e.g. uridine-rich small nuclear ribonucleoprotein particles (U snRNP); U2 auxiliary factor (U2AF)] interact to activate and support the use of potential alternative splice sites, e.g. during development, or to achieve tissue-specific expression of different mRNA isoforms (5, 6, 7, 8, 9). It is now recognized that many disorders are caused by mutations within these important elements that disrupt normal splicing and lead to abnormal products (10, 11).

A good example is the autosomal dominant form of IGHD, type II (IGHD II). This is most commonly caused by mutations within the first 6 bp of 5'IVS-3 in the GH-1 gene, which result in a missplicing of the mRNA and the subsequent loss of exon 3 sequences, producing a 17.5-kDa human GH (hGH) variant (12, 13, 14). Other alterations causing IGHD II include mutations in exon 3 splice enhancer ESE1 and ESE2, and intron splice enhancer (ISEm1 and ISEm2) sequences as well as in deletions within intron 3, underlining the importance of intron length on the splicing machinery (1, 15, 16, 17, 18, 19, 20, 21). The common feature of all these GH-1 gene alterations is the loss of exon 3 from the mRNA, yielding the 17.5-kDa GH form that exhibits a dominant-negative effect on the secretion of the 22-kDa wt-GH in both tissue culture and in transgenic animals (12, 13, 22, 23). However, despite this common feature, clinical phenotype can vary quite widely depending on the individual genotypes, and severe short stature (< –4.5 SD score) is not present in all affected individuals (24, 25). The reason for this variability is not clear. Specifically, both quantitative and qualitative differences in phenotype might be explained by different types of mutations around the splice site having a variable effect on splice site usage, that may involve interactions between U1snRNP and the pre-mRNA 5' splice site resulting in different proportions of exon 3 skipped vs. full-length GH forms generated by different mutant alleles (26). This could then generate threshold (onset) and dose-dependent (progression and severity) effects because of different amounts of 17.5-kDa relative to 22-/20-kDa hGH entering the secretory pathway and damaging the somatotroph at different rates (14, 23, 24, 27, 28, 29, 30, 31, 32, 33).

The situation is more complex in vivo because GH expression and secretion is regulated by two opposing hypothalamic regulatory peptides, GHRH and somatostatin, which in turn are subject to negative feedback control by GH and IGF-I. In IGHD, the lack of GH and IGF results in increased stimulatory drive to the somatotroph to produce more GH, both from the normal and mutant alleles, and we hypothesized that this could result in increased 17.5-kDa production accelerating the loss of somatotrophs in a vicious circle.

To study this, we set up an in vitro model, to test the impact of increasing gene transcription in a mouse pituitary cell line (AtT-20) transfected with constructs expressing wt-GH-1 or three different GH-1 splicing mutations (known to cause IGHD II of varying severity in patients), to see whether increasing expression with glucocorticoids would alter production of the different GH isoforms from 22- and 20-kDa toward the 17.5-kDa GH isoform. We also used fluorescence-activated cell sorting (FACS) analysis to compare the impact of the different mutations on proliferation, DNA damage, and apoptosis in these cell lines.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Mouse pituitary (AtT-20/D16v-F2) cells were purchased from American Type Culture Collection (Manassas, VA) and cultured in DMEM (4.5 g/liter glucose) supplemented with 10% heat-inactivated fetal calf serum (Life Technologies, Invitrogen AG, Basel, Switzerland) and 100 U/liter penicillin/streptomycin.

Expression vectors and transfection
Wild-type GH (wt-GH, full-length accession no. J03071) was cloned in pXGH5 (Nichols Institute Diagnostics, San Juan Capistrano, CA) as previously described (18). Mutations were produced using QuikChange Site Directed Mutagenesis kits (catalog no. 200519; Stratagene, La Jolla, CA) (Table 1Go). AtT-20 cells were transiently transfected with 1.5 µg DNA with vectors expressing wt-GH or GH-mutants (5'IVS-3 + 2-bp T->C; 5'IVS-3 + 6-bp T->C; ISEm1: IVS-3 + 28 G->A) using the FuGENE6 transfection reagent (Roche, Rotkreuz, Switzerland), according to the manufacturer’s protocol.


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  		TABLE 1. Mutagenesis primers sequences

 
Analyses of splice site activation and mRNA splicing pattern
Total RNA was extracted 24 h after transfection from AtT-20 cells using QIAGEN RNeasy kit (catalog no. 74104; QIAGEN AG Basel, Switzerland) including deoxyribonuclease treatment. First-strand cDNA was synthesized from 1 µg of total RNA in 20 µl RT (reverse transcription) reaction using oligo(deoxythymidine)18 primers, 20 mM of each deoxynucleotide triphosphate, 10x first strand buffer solution, Moloney murine leukemia virus reverse transcriptase (Roche Molecular Biochemicals, Mannheim, Germany) and diethyl pyrocarbonate. PCR amplification of the cDNA from the AtT-20 cells transfected with different constructs was performed using PCR Super Mix High Fidelity (catalog no. 10790-020; Invitrogen) in the presence of primers specific for GH-1 as previously reported (1). The samples were placed on a PCR thermal cycler amplified at 95 C for 5 min, annealing at required temperature for each primer for 30 sec and with an extension of 72 C for 1 min, for 25 cycles. The PCR-amplified products were separated on 6% polyacrylamide gels. Glyceraldehyde-3-phosphate-dehydrogenase was used as an internal control for loading differences and cDNA quality. Negative controls included no template control, RT control, RNA isolated from AtT-20 cells without any transfection and RNA control (same concentration of RNA obtained from transfected cells to check for genomic contamination). Plasmid del32–71 cDNA construct was used as a positive control for the expression of 17.5-kDa GH and PCR control. To study the effects of glucocorticoids, transiently transfected AtT-20 cells were stimulated with 1 or 10 µM dexamethasone (DEX) for 24 h (34, 35, 36). In other experiments, the effects of DEX (1 µM) were blocked by the addition of the antiglucocorticoid RU486/mifepristone (1 µM). Unstimulated transfected AtT-20 cells served as controls.

Real-time PCR (TaqMan)
Total RNA was reverse transcribed (2 µg of total RNA) in 25 µl RT reaction using oligo(deoxythymidine)18 primers, 20 mM of each deoxynucleotide triphosphate, 10x first strand buffer solution, Moloney murine leukemia virus reverse transcriptase (Roche Molecular Biochemicals), and diethyl pyrocarbonate. Primers and probes were chosen and purchased from the predeveloped primer and probe lists of PE Applied Biosystems (Rotkreuz, ZG, Switzerland) (22 kDa: Hs00236859-m1; 20 kDa, Hs00737953-g; 17.5 kDa, Hs00737954-g1 and 18S: Hs99999901-s1). The PCR universal master mix was also purchased from PE Applied Biosystems. The PCR mixture (25 µl total volume) consisted of primers (forward and reverse 900 nM), TaqMan probe (250 nM), and TaqMan universal master mix (12.5 µl of 2x reaction mixture). The gene sequences were amplified within the cycler (TaqMan; PerkinElmer, Rotkreuz, Switzerland) using the gene-specific oligonucleotide primers. Negative controls included no template control and RNA control to check for genomic contamination. The 18S ribosomal RNA was used to normalize cDNA concentrations. Furthermore, relative amounts of splice variants mRNA expression were calculated with the threshold cycle (Ct) and the comparative Ct method was used for relative quantification as described by the manufacturer (PE Applied Biosystems; http://www.vetmed.ucdavis.edu/vme/taqmanservice/res_service.html) after confirming that wt-GH, the mutant GH variants and 18S were amplified with the same efficiency. All experiments were performed at least in quadruplicate.

Nucleofection and AtT-20 cell proliferation assay
To study the impact of different GH mutants on cell viability, cell proliferation assays were performed after transient nucleofection (Nucleofector Amaxa Biosystems GmbH, Cologne, Germany) with wt- or mutant GH-1 plasmids according to the manufacturer’s protocol, using 1 x 106 AtT-20 cells with nucleofector solution R and program A-23. Viability was assessed using a CellTiter 96 Aqueous One Solution Cell Proliferation Assay (MTS assay; Promega Corp., Madison, WI) according to the manufacturer protocol. Cells were plated in 96-well plates at 5 x 104 cells per well in triplicate and incubated for 3 d. The absorbance at 490 nm was recorded using a Rainbow ELISA reader (Magelan software; Tecan Co., Gröding, Austria). The transfection efficiency was checked using EGFP-N1 (enhanced green fluorescent plasmid) provided by Amaxa Biosystems.

Determination of cell death and apoptosis
To determine the type of cell death, DNA fragmentation analysis and annexin V staining were performed. AtT-20 cells were transiently transfected with 1.5 µg DNA with empty vector as well as vectors expressing wt- or GH-mutants using the FuGENE6 transfection reagent and transferred into fresh prewarmed DMEM with 10% fetal calf serum, cultured in triplicates and incubated for 1–3 d after transfection. DNA fragmentation assay was performed using the method of Nicoletti et al. (37, 38). Pellets of 5 x 105 cells were resuspended in 0.3 ml of 50 µg/ml propidium iodide (PI) solution [Sigma (St. Louis, MO) P4170, 10 mg/ml in PBS, 0.1% sodium citrate and 0.1% Triton X-100]. Samples were further incubated at 4 C for 1 h and DNA fragmentation was measured after 24, 48, and 72 h using flow cytometric analysis (FACScan, Becton Dickinson, Franklin Lakes, NJ) . In addition, untransfected AtT-20 cells were used as controls.

To assess apoptosis, the redistribution of phosphatidylserine was measured in cells transfected with mutants compared with wt-GH using an Annexin V assay (catalog no. 556547; BD Biosciences, Frankin Lakes, NJ) (38) according to the manufacturer’s protocol. Cells were cultured in triplicate and incubated for 1–3 d. Apoptosis was measured after 24, 48, and 72 h and analyzed by flow cytometric analysis. Untransfected AtT-20 cells were used as controls, and for setting FACS gating conditions. The transfection efficiency was checked using EGFP-N1.

Statistics
Statistical analyses were performed using ANOVA one-way test plus Dunnet’s multiple comparison tests comparing each mutant to wt-GH. P < 0.05 was considered as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Production of splice variants in cells expressing wt-GH vs. mutant GH-1
In all experiments, transfection efficiencies were checked using EGFP-N1 alone or with various wt-GH constructs. The relative amounts of 22-kDa full-length GH, 20-kDa cryptic splice site and 17.5-kDa exon 3 skipped transcripts amplified from unstimulated cells transfected with three different GH-1 mutants or wt-GH-1 plasmids are shown in Fig. 1Go, panel I. As expected, wt-GH produced mostly 22-kDa GH transcripts, with only small (1%) but detectable amounts of 17.5-kDa, whereas the three splice mutant constructs all showed between 30–80% 17.5-kDa transcripts, with corresponding graded reductions in 22 kDa (from 60–10%). Note that the rank order of increasing 17.5-kDa in this series corresponded with increasing proximity of mutations to the IVS-3 splice site.


Figure 1
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  		FIG. 1. Stimulation of alternative splicing pattern of GH-1 gene. AtT-20 cells were transiently transfected with vectors containing either wt-GH (wt-pXGH5) or GH-1 mutations labeled with 5'IVS-3 + 2 bp, 5'IVS-3 + 6 bp and ISE + 28, G to A. 5'IVS-3 + 2 bp and 5'IVS-3 + 6 bp are intronic single base pair substitutions (5'IVS-3 + 2 bp T -> C and 5'IVS-3 + 6 bp T->C), whereas ISE + 28, G to A represents an intronic single base pair substitution. AtT-20 cells were stimulated with DEX concentrations of either 1 µM (Fig. 1Go-II) and 10 µM (Fig. 1Go-III) or the 1 µM DEX stimulation was blocked by the addition of 1 µM RU486 (Fig. 1Go-IV). After isolation of total RNA of the various transfected AtT-20 cells quantitative RT-PCR analysis of the splicing pattern according to the splice-site activation was performed. Individual bands represent the different spliced product encoding each of the major protein isoforms of GH and are indicated to the right side: full-length isoform, 22-kDa GH; the cryptic isoform, 20-kDa GH; or the exon 3-skipped isoform, 17.5-kDa GH. Below the depicted gels the absolute mean values of quantitative RT-PCR analyses are presented (five independent experiments in triplicate; SD values were between 0.2 and 0.4% of all the data shown). The gels were ethidium bromide stained.

 
Expression quantification by real-time PCR (TaqMan).
To quantify the increase in transcription by DEX stimulation, which could be blocked in the presence of RU486, real-time PCR was performed and the data were compared with unstimulated AtT-20 cells for wt-GH as well as for each mutant studied. Table 2Go shows the DEX-induced increase in transcription as well as the blocking effect of RU486. Because real-time PCR methodology amplifies all three products (22, 20, and 17.5 kDa) individually, this does not provide an assessment of the total increase of mRNA, and thus the variable percentage of the three different splice products cannot be correctly quantified in relation to the total, but there is a clear change in mutant GH and wt-GH expression depending on splice site with DEX stimulation (Table 2Go).


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  		TABLE 2. Relative values of DEX stimulated wt-GH and mutant GH mRNA expression compared with unstimulated values

 
Impact of stimulation of GH-1 gene transcription by glucocorticoids on splice-site selection/activation
Incubation of similar transfected cells with 1 µM or 10 µM DEX yielded a significant increase of GH transcripts (P < 0.001) but the patterns were markedly different for the different mutants and for wt-GH (Fig. 1Go, panels II and III). DEX treatment of cells expressing wt-GH showed a small dose-dependent shift from 22 to 20 kDa, whereas the amounts of 17.5 kDa remained unchanged (1%). In marked contrast, DEX-treated cell lines expressing the 5'IVS-3 + 2-bp and +6-bp mutants showed a dramatic, dose-dependent increase in 17.5-kDa transcripts (P < 0.01), and a corresponding suppression of 22- and 20-kDa transcripts. Interestingly, DEX treatment of cells expressing the intron splice enhancer mutant (ISEm1: +28) had no effect on the relative proportions of the transcripts produced. To test the specificity of the effect of DEX, additional DEX incubations were performed in the presence of RU486. As shown in Fig. 1Go, panel IV, RU486 blocked the effect of DEX, such that the proportion of transcripts of each size returned to those seen in untreated AtT-20 cells for each mutant studied.

Reduction of cell proliferation rate of splice variant mutants compared with wt-GH
Because earlier clinical imaging observations suggested that a 5'IVS-3 + 2-bp GH-1 gene mutation might have more impact on the somatotroph population growth and survival than those with splice site mutations within 5'IVS-3 + 6 bp or ISE +28 G->A, we performed proliferation, DNA fragmentation and apoptosis assays using FACS analysis on cell lines transfected with the same mutant GH-1 constructs, studied 24, 48, and 72 h after transient transfection. The overall proliferation rate of all the cells expressing mutants was lower than that of the cells expressing the wt-GH (Fig. 2Go) and cell proliferation began to slow after 3 d (not shown). Both 5'IVS-3 + 6-bp T->C and ISE +28 G->A exhibited a similar time course of proliferation, at a rate significantly greater than that of the cells transfected with splice site mutation 5'IVS-3 + 2-bp T->C (48 h, P < 0.01; 72 h, P < 0.005). These experiments supported the idea that the 5'IVS-3 + 2 bp T->C mutation has a significant negative effect on cell viability and/or cell proliferation rate under these conditions. It is interesting to note that the proliferation rate of the cells bearing wt-GH was constantly higher than the rate found in the cells containing the empty vector (PUC 18).


Figure 2
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  		FIG. 2. Cell proliferation assay. Cell proliferation was assessed using MTS assay. AtT-20 cells were transfected with either wt-GH, empty pcDNA vector as control (PUC 18) or with mutants as 5'IVS-3 + 2 bp, 5'IVS-3 + 6 bp and ISEm1 (ISE +28). Proliferation was estimated at 0, 24, 48, and 72 h after transfection. Results are expressed as mean OD of five independent experiments in triplicate (mean ± SE).

 
Impact of splice variants mutants on cell apoptosis compared with wt-GH
These findings encouraged us to assess the impact of the various mutants on cell viability, using phosphatidylserine redistribution in annexin V binding assays and FACS analysis (25, 34, 37, 38). Untransfected AtT-20 cells as well as AtT-20 cells transfected with wt-GH [wt-(pXGH5)] were used to assess cell death rates independent of any impact of the mutants. The results of four independent studies are depicted in Fig. 3AGo (mean ± SEM).


Figure 3
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  		FIG. 3. Annexin V assay, detection of apoptosis. A, The relative number of apoptotic cells (% of cell death) produced after 1, 2, and 3 d after transfection of AtT-20 cells with wt-GH or mutants: 5'IVS-3 + 2 bp; 5'IVS + 6 bp; IVS-3 + 28. Results are expressed as mean of four independent experiments (mean ± SE). In addition, untransfected AtT-20 cells were also studied as controls. B, Representative example of two flow cytometric analysis of phosphatidylserine expression on the surface of AtT-20 cells transfected with either wt-GH or mutant 5'IVS-3 + 2 bp. Cells were stained with fluorescein isothiocyanate-labeled annexin V and the level of phosphatidylserine redistribution was measured by FACS analysis. Black filled histogram indicates wt-GH and white filled histogram represents 5'IVS-3 + 2-bp mutant. PS, Phosphatidylserine.

 
First, comparing untransfected AtT-20 cells with AtT-20 cells transfected with wt-GH, mean % cell death appeared reduced in AtT-20 cells containing wt-GH at d 2, although the differences were not statistically significant, whereas at d 3 the cells transfected with wt-GH showed reduced redistribution of phosphatidylserine (P < 0.005) (Fig. 3Go, A and B). In contrast, at all time points examined, the redistribution of phosphatidylserine in cells transfected with 5'IVS-3 + 2 bp was significantly higher (d 1, P < 0.01; d 2, P < 0.001; d 3, P < 0.001) than in the cells with 5'IVS-3 + 6 bp. In addition, although not significantly different from AtT-20 cells with wt-GH during the first 2 d, the percentages of cell death were higher after 3 d in AtT-20 cells transfected with either 5'IVS-3 + 6 bp (P < 0.005) and ISEm1 (P < 0.001). These results suggest that expression of the mutants induced overall more apoptosis than wt-GH, and imply that wt-GH expression might be cell protective, which is consistent with the cell proliferation data in Fig. 2Go.

Increased DNA fragmentation of mutants compared with wt-GH
Finally, we investigated whether the decreased rate of growth in cells expressing mutant GHs is accompanied by increased DNA fragmentation, using PI staining. DNA of transfected AtT-20 cells were stained with PI for 1 h and then analyzed by FACS. The results of four independent experiments for a consecutive 3 d are presented in Fig. 4Go, A and B. The mean percentage of fragmented DNA is presented in Fig. 4AGo. Both IVS-3 + 2 bp and IVS-3 + 6 bp showed a significant increase in DNA fragmentation compared with the control wt-GH (P < 0.01). The highest DNA fragmentation (26.02%) was observed in the mutant bearing 5'IVS-3 + 2-bp mutation (Fig. 4Go, A and B) on the third day after transfection. In contrast to the phosphatidylserine redistribution assay, where less apoptosis was found in wt-GH transfected AtT-20 cells when compared with AtT-20 cells at d 3 (Fig. 3AGo), DNA fragmentation showed no evidence for a decreased apoptosis (Fig. 4Go, A and B). This may be explained by the increased proportion of cells in the G2 phase reflecting the doubling phase of the cell cycle (Fig. 4CGo), resulting in an increased cell number and not necessarily preventing the baseline apoptosis associated with the cell culture conditions. The positive effect of GH on the proliferation rate of AtT-20 cells is also shown Fig. 2Go.


Figure 4
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  		FIG. 4. DNA fragmentation assay. A, The absolute mean values (percentage) of fragmented DNA are indicated after 1, 2, and 3 d after transfection of AtT-20 cells with wt-GH or mutants: 5'IVS-3 + 2 bp; 5'IVS +6 bp; IVS-3 + 28 (ISE + 28). Samples were incubated for 1 h at 4 C and uptake of PI by DNA was quantitatively measured using FACS analysis. Results are expressed as mean of four independent experiments (mean ± SE). B, These panels show one representative experiment of four after first and third day after transfection of the cells with entire wt-GH or mutants, 5'IVS + 2 bp; The quantitative percentage of fragmented DNA is indicated above the brackets. C, This table shows the percentages (mean values) of the frequency distribution of DNA content stained with propidium iodide in AtT-20 cells (with and without transfected wt-GH) in relation to sub-G1, G1, and G2 phases in the cell cycle obtained by flow cytometry.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Variable phenotype of IGHDII
The autosomal dominant form of IGHD type II (IGHD II) is mainly caused by mutations within the first 6 bp of intervening sequences 3 (5'IVS-3) in the GH-1 gene, which result in RNA missplicing to lose exon 3 sequences, producing a 17.5-kDa hGH isoform (13, 14). This exerts a dominant-negative effect on the secretion of the 22-kDa isoform both in tissue culture as well as in transgenic animals (23, 27, 39, 40). Transgenic mice overexpressing the 17.5-kDa hGH exhibit a dose-dependent phenocopy of IGHD II, with defects in the maturation of GH secretory vesicles and anterior pituitary gland hypoplasia due to a loss of somatotrophs (1, 23). Furthermore, in humans different mutations in IGHD II have a variable impact on pituitary size, as well as on onset and severity of GHD and the most severe, rapid onset forms of GHD may even be associated with the evolution of other pituitary hormone deficiencies (22, 25). Quantitative and qualitative difference in phenotype could reflect a threshold and a dose dependency effect of the amount of 17.5-kDa relative to 22-kDa hGH (23, 27, 30), caused by differential effects on splice site choice around exon 3, that give rise to different proportions of the dominant-negative 17.5-kDa from a single allele (1, 11).

Impact of stimulation of transcription on GH-1 gene splicing, splice site selection
We decided to test whether another source of variability in splice products from different alleles might be that the relative amounts of 17.5-kDa could vary depending on the extent of transcriptional activation and hence spliceosome load. This makes physiological sense because the normal negative feedback regulation of GH expression by both GH and IGF-I is lost in IGHD II, with chronic up-regulation of GHRH (23) to activate more GH gene expression. After its gradual loss of GH feedback in IGHD II, which itself will vary depending on the onset and severity of the GHD, we hypothesized that the production of 17.5-kDa misspliced forms could be increased to different extents with different splicing mutations in the affected GH-1 alleles. We therefore sought a simple way to test in vitro, the effects of stimulating gene transcription rates on the ratios of splice products generated from different mutations in the same cell system.

Although Robins et al. (41) reported that glucocorticoid regulatory elements are located in the 5'-flanking region of the hGH-1 gene, it has also been reported while using receptor binding and gene transfer analyses that the structure bearing the greatest resemblance to the 16-nucleotide consensus sequence is located within the first intron (IVS1) (34), which is unchanged in all our GH-1 genomic constructs containing IVS-3 splicing mutations. Therefore, we compared the effects of activating gene expression using different doses of glucocorticoids, testing different genomic GH-1 constructs either wt or containing GH-1 gene mutations (5'IVS-3 + 2-bp T->C; 5'IVS-3 + 6-bp T->C; ISEm1: IVS-3 + 28 G->A) known to cause IGHD II of variable severity and onset, and then evaluated the relative proportions of transcripts for 22-, 20-, and 17.5-kDa GH produced. We used a mouse pituitary secretory cell line that does not normally express GH to avoid the confounding effects of dominant-negative activity on endogenous mGH gene expression; the only wt-GH produced would be that generated from the constructs themselves.

With wt-GH expression in this system, we observed the expected pattern of GH mRNA isoforms, with 22-kDa the majority, 20-kDa a significant minor form, and small but detectable amounts of 17.5-kDa, as in human pituitaries (42). Importantly, whereas glucocorticoid/DEX treatment increased, and antagonist reversed, transcriptional stimulation, as confirmed by real-time PCR, this had no effect on splice site choice from the normal GH-1 allele. With DEX exposure, stimulation of gene transcription from the mutant GH-1 constructs resulted, not only in an increase in the relative amounts of the 17.5-kDa isoform from all the mutant constructs, but which showed a greater effect, the more pronounced the missplicing was in the absence of stimulation, and which was greatest for the 5'IVS-3 + 2-bp mutant causing the severest phenotype among those studied. Furthermore, this was a specific effect of glucocorticoid stimulation because the addition of the antiglucocorticoid RU486/mifepristone was fully able to reverse this increase in missplicing for each of the mutants. These results clearly suggest that the extent of exon 3 deletion from a given allele mutant increases with gene transcription (unlike from the wt allele), and that the magnitude of this increase in dominant-negative forms is in proportion with the degree of missplicing in the unstimulated condition. These data were all obtained under identical conditions with the same cell line, so it is reasonable to draw comparisons between constructs, although the absolute values may not reflect the proportions obtained in somatotrophs. Furthermore, it is possible that the differences reflect some specific effect of glucocorticoid-regulated transcription or splicing, although in that case these effects would also have to vary in proportion with the severity of the missplicing under basal conditions (i.e. in the absence of glucocorticoids) and in the stimulated wt-GH transfected cells. Therefore, it is unlikely that these results reflect an effect of glucocorticoids on mRNA stability because the same three transcripts are produced from the mutant and wt-GH constructs, albeit in different proportions.

The simplest explanation for our results is that the different mutations tested weaken the splice site choice for inclusion of exon 3 to different extents, and that the amounts of exon 3 skipped transcripts become proportionately greater as the load on the splice machinery increases. Therefore, from our results, one can predict that with incipient GHD, the gradual increase in expression resulting from loss of GH feedback does not increase the proportions of exon 3 transcripts from the wt allele but will do so from a mutant allele, the extent of which is dependent on the severity of the mutation in the resting state. Even mutations that have a mild effect under resting conditions will have their impact amplified because GHD drives expression in a vicious cycle.

We believe this provides a basis for the clinical variability described in patients studied with the same mutations. Our patients with a splice site mutation within the first 2 bp of intervening sequence 3 (5'IVS +1/+2 bp) were found to be more likely earlier diagnosed and more severe in progression, presenting with short stature already at a younger age, than the those with mutations within 5'IVS-3 + 5/+6 bp (24). One possible underlying mechanism for the severity of the 5'IVS-3 + 2-bp mutation compared with the others (e.g. 5'IVS-3 + 5/+6 bp), although discussed contradictorily in literature, may be an altered U snRNP RNA pairing having an impact on the aberrant splicing process (26, 43, 44, 45).

Influence of splice variants mutants on increased cell apoptosis and DNA fragmentation compared with wt-GH
As is well known, the presence of del32–71GH in the somatotrophs causes a blockade of wt-GH secretion, whereas the partial missplicing variant del32–46GH (the 20-kDa GH variant), which also lacks part of the loop connecting helices 1 and 2 (46), accounts for approximately 5–10% of the GH found in serum of normal individuals and does not have any adverse effect on the secretion of wt-GH (47). Although various hypotheses for the dominant-negative nature of the 17.5-kDa isoform have been advanced (1, 11, 23, 27, 39, 40), the end result of the effect of the 17.5-kDa isoform is to completely block the ability of somatotrophs to store and secrete 22-kDa GH isoform. However, the effects on the GH-producing cells can be quite variable. Some GH cells survive in culture, whereas others have been reported to undergo cell death as an autolytic process; in vivo, however, there is a major loss of somatotrophs, probably accelerated by an invasion of activated macrophages, which destroy defective somatotroph cells, and probably other cell types by bystander killing (23).

We deliberately chose to study variable splicing in a non-GH-producing cell line, to minimize these effects. However, all of the constructs also produce some wt-GH to differing extents, so we took the opportunity to evaluate the consequence of these mutations on cell proliferation and apoptosis using three different techniques. First, using cell proliferation assay, we found that cells expressing either 5'IVS-3 + 6-bp T->C or ISE +28 G->A maintained a significantly greater proliferation rate than cells transfected with 5'IVS-3 + 2-bp T->C already at d 2. Furthermore, the proliferation time courses were very similar in both clones (both 5'IVS-3 + 6-bp T->C and ISE +28 G->A), increasing after 24 h, whereas proliferation rate of the 5'IVS-3 + 2-bp T->C transfected cells did not change at all (5'IVS-3 + 6 bp and 5'IVS-3 + 2 bp: 48 h: P < 0.01; 72 h: P < 0.005). Second, analysis of cell apoptosis showed the most significant increase with the 5'IVS-3 + 2-bp mutation. We also observed an increase in DNA fragmentation of the cells transfected with 5'IVS-3 + 6 bp and 5'IVS-3 + 2 bp, but not in the cells bearing the mutant ISE +28 G->A. The greatest effect was observed in the 5'IVS-3 + 2-bp mutant, as well as 5'IVS-3 + 6-bp mutations on the third day after the transfection. Because the 5'IVS-3 + 2 bp generates significant amounts of both exon 3 and wt-GH products, it is possible that this construct could generate sufficient dominant-negative interacting products to inhibit cell proliferation and trigger cell death, earlier than the other constructs. Furthermore, comparing untransfected AtT-20 cells with AtT-20 cells containing wt-GH, cells transfected with wt-GH showed higher proliferation rate (Fig. 2Go) and reduced cell death at d 3, measured by annexin V staining which is a marker for early apoptosis detection (Fig. 3Go). In contrast, analyzing DNA fragmentation on the third day, cells transfected with wt-GH presented similar pattern of DNA fragmentation levels as found in untransfected AtT-20 cells (Fig. 4Go).

In conclusion, we provide novel evidence that stimulation of GH-1 gene transcription has a differential impact on splice site activation/selection among mutations causing autosomal dominant GH deficiency. This could help explain how different amounts of 17.5-kDa hGH are produced in increasing amounts as GHD progresses, which contributes to the variability in onset severity and progression of the IGHD II phenotype. The increase in dominant-negative GH isoforms blocks secretion and leads to a reduced cell proliferation rate and increased apoptosis of somatotrophs. The most severe mutations can generate 100% 17.5-kDa isoform under stimulation, and it is already known from our studies in an animal model of IGHD II and in patients, that the most severe rapid onset phenotypes can be accompanied by the development of other pituitary hormone deficiencies. The recognition that GHD-induced stimulation of GH-1 gene expression can increase the proportion of 17.5-kDa hGH from alleles with mutations that yield only mild-to-moderate missplicing under resting conditions may be significant in the context of withdrawal of hGH treatment from these subjects at final height, and emphasizes the importance of monitoring them for eventual involvement of other pituitary axes if left untreated with GH in adulthood.


   Acknowledgments
 
This work was supported by a Swiss National Science Foundation grant (to P.E.M.) (3200BO-105853/1) and by United Kingdom Medical Research Council support (to I.C.A.F.R.).


   Footnotes
 
Disclosure Statement: The authors have nothing to disclose

First Published Online October 12, 2006

Abbreviations: DEX, Dexamethasone; EGFP-N1, enhanced green fluorescent plasmid; ESE, exon splice enhancers; FACS, fluorescence-activated cell sorting; hGH, human GH; IGHD II, isolated GH deficiency type II; ISE, intron splice enhancer; IVS, intervening sequence; PI, propidium iodide; RT, reverse transcription; wt, wild type.

Received June 8, 2006.

Accepted for publication October 5, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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