http://www.100kang.com 2007-5-7 16:09:24 Autosomal

	Abstract

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Autosomal dominant GH deficiency type II (IGHDII) is often associated with mutations in the human GH gene (GH1) that give rise to products lacking exon-3 (^exon3hGH). In the heterozygous state, these act as dominant negative mutations that prevent the release of human pituitary GH (hGH). To determine the mechanisms of these dominant negative effects, we used a combination of transgenic and morphological approaches in both in vitro and in vivo models. Rat GC cell lines were generated expressing either wild-type GH1 (WT-hGH-GC) or a genomic GH1 sequence containing a G->A transition at the donor splice site of IVS3 (^exon3hGH-GC). WT-hGH-GC cells grew normally and produced equivalent amounts of human and rGH packaged in dense-cored secretory vesicles (SVs). In contrast, ^exon3hGH-GC cells showed few SVs but accumulated secretory product in amorphous cytoplasmic aggregates. They produced much less rGH and grew more slowly than WT-hGH-GC cells. When cotransfected with an enhanced green fluorescent protein construct (GH-eGFP), which copackages with GH in SVs, WT-hGH-GC cells showed normal electron microscopy morphology and SV ovements, tracked with total internal reflectance fluorescence microscopy. In contrast, coexpression of ^exon3hGH with GH-eGFP abolished the vesicular targeting of GH-eGFP, which instead accumulated in static aggregates. Transgenic mice expressing ^exon3hGH in somatotrophs showed an IGHD-II phenotype with mild to severe pituitary hypoplasia and dwarfism, evident at weaning in the most severely affected lines. Hypothalamic GHRH expression was up-regulated and somatostatin expression reduced in^exon3hGH transgenic mice, consistent with their profound GHD. Few SVs were detectable in the residual pituitary somatotrophs in ^exon3hGH transgenic mice, and these cells showed grossly abnormal morphology. A low copy number transgenic line showed a mild effect relatively specific for GH, whereas two severely affected lines with higher transgene copy numbers showed early onset, widespread pituitary damage, macrophage invasion, and multiple hormone deficiencies. These new in vitro and in vivo models shed new light on the cellular mechanisms involved in IGHDII, as well as its phenotypic consequences in vivo.

	Introduction

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HUMAN PITUITARY GH (hGH) is encoded by exons 1–5 of the GH1 gene. The major bioactive product is a 22-kDa form, but alternative splicing can give rise to minor forms, the most prominent of which is a bioactive 20-kDa hGH that results from use of a cryptic 3' splice site in exon 3, deleting amino acids 32–46 (1). Rarely, however, mutations in GH1 can give rise to alternative spliced products that induce a form of GH deficiency (IGHDII) inherited as an autosomal dominant trait. Such individuals show variably reduced plasma GH levels and growth rates but usually respond positively to exogenous GH therapy (2). This frequently occurs with mutations at, or close to, the splice sites around exon 3 (3, 4), which generate mRNAs lacking sequences coding for this exon. The resulting exon-3 skip product (^exon3hGH) generates a 17.5-kDa hGH that lacks amino acids 32–71 (2, 4), which includes the loop connecting the first two helices of the wild-type GH (WT-hGH) structure (5); it also disrupts an internal disulfide bridge, by deletion of ⁵³Cys in WT-hGH.

Although the 17.5-kDa protein produced by such IGHDII mutations is unlikely to activate the GH receptor, the dominant nature and severity of the phenotype, together with the lack of 22-kDa hGH in the circulation, suggests that the ^exon3hGH product from the mutant allele efficiently prevents the production, storage, or release of WT-hGH from the other, normal allele. Because donor and acceptor splice sites are well conserved throughout vertebrates (6), it is possible to reproduce the misplicing by expressing such human genomic sequences in rodent cell lines, and when coexpressed with WT-hGH, ^exon3hGH products exert a dominant negative effect on WT-hGH production (7, 8) and cause variety of morphological abnormalities in heterologous cell lines (9). Many possible intracellular mechanisms have been invoked to explain the dominant negative effects of misfolded protein hormones such as ^exon3hGH (10, 11, 12), frequently based on acute transfection studies in heterologous cell lines. The mechanisms by which ^exon3hGH suppresses WT-GH production and release, and lead to progressive dysfunction of somatotrophs in vivo, remain unclear.

To address some of these issues, we have expressed ^exon3hGH in a GH-producing rat cell line with or without a coexpressed and copackaged fluorescent secretory vesicle (SV) marker (13) to study the effects of ^exon3hGH on cell and SV morphology and movements, using confocal, total internal reflection fluorescence (TIRF) and immunoelectron microscopy. Secondly, we have generated several lines of transgenic mice expressing ^exon3hGH, to create and characterize a murine model of human IGHDII and to study the effects of ^exon3hGH in primary somatotrophs for the first time.

	Materials and Methods

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Constructs
The constructs used in this study are shown in Fig. 1. A SacI/BglII fragment containing the IVS3 (+1GA) mutation in a GH1 genomic clone (4) was exchanged into pKS-GH.M, a 2.6-kb genomic construct containing 5'-and 3' untranslated GH1 sequences flanked by MluI sites (13). The +1G->A mutation generates an additional NlaIII site, not present in WT-GH1 DNA sequences. Both the WT and mutant IVS3 + 1GA constructs were confirmed by restriction mapping and DNA sequencing. For transfection studies, a BamH1/Not1 fragment of pKS-GH.M was subcloned into pCDNA3.1 (Invitrogen, Paisley, UK) containing a cytomegalovirus (CMV) promoter and a zeocin selection cassette (Fig. 1A); a similar expression vector was generated for WT-GH1, but with a neomycin selection cassette. For transgenesis, the pKS-GH.M insert was subcloned into a 40-kb cosmid (14) containing the entire GH1 locus control region (LCR (Fig. 1B), using an Mlu1 site engineered as previously described (13). The full-length insert was released from the cosmid as a Not1 fragment and purified for microinjection into oocytes. For simplicity, the DNA sequence containing the GH1-IVS3 (+1GA) mutation will subsequently be termed ^exon3hGH. To generate GC cells with fluorescent GH SVs, they were also transfected with a CMV expression vector [p48hGH-enhanced green fluorescent protein (eGFP)] containing sequences coding for the signal peptide and first 22 residues of mature GH1 followed by eGFP (13). We have previously shown that this generates a fluorescent product which is copackaged with GH in SVs both in rat GC cells and in somatotrophs of transgenic mice (13).

fig.ommitteedfig.ommitteed Figure 1. Constructs used in this study. A, A CMV expression vector (pKS-GH-M) was constructed, containing a 2.6-kb GH1 genomic sequence with a single base +1G->A substitution in intron 3 (*), and flanked by Mlu1 restriction sites (M). B, The Mlu1 fragment of A was cloned into a 40-kb cosmid containing the GH1 LCR, via a Mlu1 cloning site to give LCR exon3hGH (13 ). The insert was released as a Not1 fragment (N) for oocyte microinjection.

 
Culture of rat GC cells
Rat GC cells (15, 16) were cultured at 37 C in complete medium [DMEM, 15% horse serum, 2.5% fetal calf serum (PAA, Weiner Strasse, Linz, Austria)], 2 mM L-glutamine, and 1x antibiotic-antimycotic solution [penicillin/streptomycin/amphotericin]) and transfected with plasmids expressing either p48 hGH-eGFP, WT-hGH or ^exon3hGH alone or in combination, using Lipofectamine (Life Technologies, Inc., Gaithersburg, MD). Stably transfected cell lines were selected for neomycin resistance (G-418, 250 µg/ml, Life Technologies, Inc.) and/or zeocin resistance (zeocin 200 µg/ml, Invitrogen BV, Breda, The Netherlands) for at least 21 d.

Generation of ^exon3hGH transgenic animals.
All animal work was carried out under UK Home Office guidelines. Transgenic mice were generated by microinjection of the LCR-^exon3hGH Not1 fragment (Fig. 1B) into the pronucleus of fertilized one-cell Cba/Ca x C57Bl/10 mouse oocytes followed by oviductal transfer into pseudopregnant recipients. Tail DNA from the resulting pups were screened by Southern blot for GH1 and by PCR using primers that spanned the first GH1 exon/intron pair, as previously described (13).

RT-PCR
RNA was extracted from GC cells (SNAP total RNA Isolation Kit (Invitrogen BV) and 500 ng reverse transcribed with 200 U of Moloney-murine leukemia virus reverse transcriptase (Roche Molecular Biochemicals, Lewes, UK) supplemented with 1 µg random primers (Invitrogen), 0.3 mM deoxynucleotide triphosphates (Amersham Pharmacia Biotech), 40 U ribonuclease inhibitor (Promega Corp., Southampton, UK), and 5 mM dithiothreitol. After incubation at 37 C for 2 h the resulting cDNAs were cloned, amplified (TOPO Cloning Kit, Invitrogen BV) and sequenced. To distinguish WT-hGH and ^exon3hGH transcripts, two sets of primer pairs were used, both of which spanned exon 3 and should give 120-bp larger products for WT-hGH than for ^exon3hGH transcripts.

Southern blotting
After restriction digestion, DNA (10 µg of genomic DNA or 0.5 µg of plasmid/cosmid DNA) was separated by electrophoresis in 0.6% agarose gels, transferred onto Hybond-N⁺ nylon membranes (Amersham Pharmacia Biotech) in 0.4 M NaOH by capillary transfer and hybridized with a random primed [³²P]-labeled full-length GH1 genomic DNA probe (Prime-a-Gene, Promega Corp.) for 65 C overnight. Blots were washed and exposed to phosphoimager screens or to Kodak BioMax film at -70 C.

RIA
Pituitaries were dissected and homogenized in 1 ml PBS. Protein contents were measured with the BCA Protein Assay Reagent (Pierce Chemical Co., Rockford, IL) using BSA as standard (Sigma, Poole, UK). Aliquots were taken for assay of GH, PRL, LH, and TSH using specific reagents kindly provided by Dr. A. L. Parlow and by the NIDDK (17, 18); hGH was assayed using an antibody that detects 22-kDa hGH but not mouse or rat GH (rGH) (18).

In situ hybridization
In situ hybridization was performed as previously described (19) on 12-µm cryostat coronal sections of mouse hypothalamus using [³⁵S]-uridine triphosphate-labeled sense and antisense riboprobes for GHRH and SRIF generated using an SP6/T7 transcription kit (Roche Diagnostics). The GHRH riboprobe represented a full-length mouse cDNA (Image clone 1496474), whereas the SRIF riboprobe corresponded to nucleotides 280–556 of rat SRIF cDNA. Following overnight hybridization, sections were washed, dried and exposed to autoradiographic film (BioMax MR, Kodak, Rochester, NY) for up to 7 d before measuring integrated densities, using NIH Image as previously described (19). For each transcript, comparisons were made with the same batch of labeled riboprobe on sections from all animals processed at the same time.

Electron microscopy (EM)
After initial fixation (2.5% glutaraldehyde in 0.1 M sodium phosphate buffer for 2 h at room temperature, then 0.25% overnight at 4 C) cells or pituitary segments were post-fixed in osmium tetroxide (1% wt/vol in 0.1 M sodium phosphate buffer), contrasted with uranyl acetate (2% wt/vol in distilled water), dehydrated through increasing concentrations of ethanol (70–100%) and embedded in Spurr’s resin (Agar Scientific UK, Stansted, UK). For immunogold detection of GH, ultrathin sections (50–80 nm) were incubated at room temperature with rabbit antimouse GH 1:2000 (NIDDK), for 2 h, followed by protein A 15 nm gold (British Biocell, Cardiff, UK) for 1 h at room temperature. In control sections, the primary antibody was replaced by nonimmune rabbit serum. Sections were counter-stained with lead citrate and uranyl acetate and examined with a transmission electron microscope (JEM-1010, JEOL, Peabody, MA).

TIRF microscopy
GC cells stably transfected with p48hGH-eGFP, with or without cotransfected WT-hGH or ^exon3hGH were cultured on glass slides, and examined with a TIRF microscope. Light from TIRF images were passed through a dichroic (505DRLP02, Omega Optical, Brattleboro, VT) and an emission filter (530DF30) and collected with an intensified CCD camera (Remote Head Darkstar, S25 Intensifier, Photonics Science, Robertsbridge, UK). Images were digitized and stored in memory at 25 frames/sec by a frame grabber (IC-PCI 4Mb (AMVS), Imaging Technology, Bedford, MA) and then saved to disk. Image processing was carried out using Optimas version 6.5 (Optimas Corp., Bothwell, WA).

Data analysis
Unless otherwise stated, data are shown as mean ± SEM. Differences between groups were analyzed by ANOVA followed by Student’s t test or Mann-Whitney U test as appropriate. Differences of P < 0.05 were considered significant.

	Results

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GC cell transfection
Wild-type GH1 (WT-hGH) and IVS-3 + 1 G->A mutant GH1 constructs (^exon3hGH) were transfected into rat GC cells and stable lines established. RNA was extracted, and RT-PCR performed with two sets of primers spanning exon 3 (Fig. 2A). As expected,cells transfected with the ^exon3-hGH plasmid (^exon3hGH-GC clls) generated a major transcript that yielded an RT-PCR product 120-bp smaller than in cells transfected with WT-hGH (WT-hGH-GC cells). No detectable WT-hGH product was amplified from ^exon3hGH-GC cells, and neither product was detected in control GC cells. Sequencing the cDNA products obtained by RT-PCR from four different ^exon3hGH-GC cell isolates confirmed an exact deletion of exon 3 sequences. Culture flasks were inoculated with 250,000 cells and their growth monitored by cell counting over 14 d. Untransfected GC cells and WT-hGH-GC cells accumulated at similar rates, whereas there were 80% fewer ^exon3hGH-GC cells remaining after 14 d.

fig.ommitteedfig.ommitteed Figure 2. pKS-GH-M generates a exon3hGH transcript after stable transfection in GC cells and suppresses endogenous rGH production. A, RT-PCR of RNA extracts from GC cells stably transfected with CMV vectors expressing WT hGH (first six lanes), exon3hGH (next four lanes), or untransfected GC cells (last three lanes). Two different primer pairs that spanned exon 3 sequences amplified major products 120 bp shorter in exon3hGH-transfected cells, which when cloned and sequenced proved to lack exon 3 sequences. B, rGH (open bars) and hGH (solid bars) were measured by RIA in extracts of GC cells wihout transfection (GC) or stably transfected with WT-hGH (WT-hGH-GC) or exon3hGH (exon3hGH-GC). ***, P < 0.001 vs. WT-hGH-GC or GC.

 
rGH and hGH contents were compared in extracts from cultures of GC, WT-hGH-GC, and ^exon3hGH-GC cells (Fig. 2B). rGH production was similar in both GC and WT-hGH-GC cells (15.2 ± 0.6 µg/10⁵ cells and 14.4 ± 0.4 µg/10⁵ cells, respectively) and WT-hGH-GC cells contained similar amounts of hGH (17.2 ± 1 µg/10⁵ cells). In contrast, no hGH immunoreactivity could be detected in ^exon3hGH-GC cells, and their rGH contents were markedly lower (0.56 ± 0.19 µg/10⁵ cells, P < 0.005).

No morphological differences were apparent between GC and WT-hGH-GC cells in culture. In contrast, many more ^exon3hGH-GC cells were detached and had a shriveled appearance, with more cell debris evident in these cultures. Both WT-hGH-GC and ^exon3hGH-GC cells were examined by EM. Abnormal morphology was obvious in the ^exon3hGH-GC cell cultures (Fig. 3, C–F) compared with GC or WT-hGH-GC cells (Fig. 3, A and B). The majority of ^exon3hGH-GC cells showed a grossly abnormal cytology, with a notable absence of dense-cored SVs, but instead a collection of amorphous electron dense cytoplasmic aggregates without any obvious vesicular structure (Fig. 3, E and F). Many ^exon3hGH-GC cells showed a highly vacuolated cytoplasm and lipid accumulations, fragmented or lobular nuclei, and swollen organellar structures, including ER, Golgi apparatus, and mitochondria (Fig. 3, C–F). Immunogold EM stained the dense cored SVs in GC cells and WT-hGH-GC cells, but gave only diffuse labeling of the cytoplasmic aggregates in the ^exon3hGH-GC cells (not shown).

fig.ommitteedfig.ommitteed Figure 3. exon3hGH-GC cells lack GH secretory vesicles and show gross morphological disruption. GC cells and WT-hGH-GC cells show well-developed ER and Golgi with numerous dense-cored secretory vesicles (SV) visible under EM (A, B). In contrast, exon3hGH-G cells show grossly disrupted morphology, often highly vacuolated (C, D) with lipid inclusions and swollen mitochondria (*). They show no obvious dense-cored SVs (E) but contain aggregates of amorphous secretory material (arrows) in the cytosol (F). Scale bars, 1 µm.

 
To study GH SVs dynamically in living cells, we exploited an eGFP construct (p48-hGH-eGFP) whose product is copackaged with GH in the same SVs, rendering them fluorescent (13). When this was coexpressed with WT-hGH or ^exon3hGH, bright eGFP fluorescence resulted but in markedly different patterns visible under confocal or TIRF microscopy. WT-hGH/eGFP GC cells showed the punctate distribution of eGFP fluorescence typical of SVs (Fig. 4A), whereas ^exon3hGH/eGFP-GC cells exhibited few fluorescent SVs, but the eGFP formed large diffuse fluorescent aggregates throughout the cytoplasm (Fig. 4B), corresponding to the amorphous secretory material seen by EM in fixed cells. Individual SVs in both eGFP-GC cells and WT-hGH/eGFP-GC cells exhibited a range of movements that could be tracked in three dimensions and included both fast-moving and tethered vesicles in the same cell (Fig. 4C). Occasional spontaneous exocytotic events were also observed in these cells (Manneville, J.-B., unpublished). In contrast, most of the ^exon3hGH/eGFP-GC cells, showed no moving eGFP-filled SVs, and the eGFP-tagged cytoplasmic aggregates remained motionless with no exocytotic events observed.

fig.ommitteedfig.ommitteed Figure 4. TIRF microscopy and secretory vesicle movements in WT-hGH-GC cells and exon3hGH-GC cells. WT-hGH-GC cells and exon3hGH-GC cells were transfected with p48hGH-eGFP, grown on glass slides, and living cells examined by TIRF microscopy. EGFP fluorescence could be resolved in many individual SVs in WT-hGH-GC cells (A), whereas it distributed mostly in large diffuse aggregates (B) in exon3hGH-GC cells. C, Dynamic imaging resolved individual SVs in WT-hGH-eGFP-GC cells. Left panel shows an accumulated thresholded image of 1000 frames collected over 25 sec from a single WT-hGH-eGFP-GC cell. Right panels show three-dimensional tracking of two individual SVs showing long-range directed motion or short-range tethered motion over this time period. The large fluorescent aggregates in exon3hGH-GC cells remained motionless. Bar, 10 µm.

 
Transgenic mice
Of 34 pups surviving from oocyte microinjection of the 40-kb Not1 fragment containing ^exon3hGH and oviductal transfer, three transgenic founders were identified by PCR and Southern analysis (Fig. 5). The ^exon3hGH transgene transmitted in the expected 1:1 ratio and three lines with different relative copy numbers were established (Fig. 5C), though the high copy number line nos. 1 and 12 were subfertile. All three lines were maintained hemizygous, and all subsequent phenotypic comparisons were made between hemizygous transgenics vs. nontransgenic (NT) littermates.

fig.ommitteedfig.ommitteed Figure 5. Generation of exon3hGH transgenic mice. Three lines of transgenic mice were established following oocyte microinjection with the 40-kb LCR exon3hGH Not1 insert. A, Transgenic mice (T) were distinguished from NT littermates by a PCR assay of tail DNA, which amplifies across the first hGH intron (see Materials and Methods) and gives a 50-bp larger product for human vs. mouse GH sequences. B, Southern blotting with a full-length hGH genomic probe comparing WT-GH1 DNA (cosmid K2B, left panel) and DNA from an exon3hGH transgenic mouse (T, right panel). The +1G->A mutation generates an additional NlaIII site not present in WT-GH1 DNA sequences, giving two smaller fragments after digestion of the transgenic mice DNA with NlaIII, compared with the 1.1-kb band in WT GH1 DNA. C, To estimate relative copy numbers in the three transgenic lines, DNA was extracted, adjusted to the same DNA concentration, and analyzed on a phosphoimager after gel electrophoresis and hybridizing with the hGH genomic probe. Line no. 23 had least 4- to 8-fold fewer transgene copies than line nos. 1 and 12.

 
Body weights of litters from all three lines were recorded weekly from weaning to adulthood (Fig. 6). Transgenic animals from line nos. 1 and 12 showed significant dwarfism from 3–4 wk of age, with proportionate reductions in weight and length in both sexes. They remained significantly smaller than their sex-matched NT littermates even after 20 wk. Some smaller individuals were noted in line no. 23, but the transgenic group in line no. 23 was not significantly lighter than the NT littermate group. Nose-anus and tibial lengths were recorded in males from all three lines every 2 wk, for 10 wk. Transgenic ^exon3hGH animals from line nos. 1 and 12 were shorter than their NT littermates (e.g. line no. 1 at 10 wk, nose-anus length 78.2 ± 1.5 mm vs. 101.2 ± 1.7 mm in NT; P < 0.001; tibia length 15 ± 0.4 mm vs. 18 ± 0.36 mm in NT; P < 0.01); there were no significant differences in these length parameters in animals from line no. 23.

fig.ommitteedfig.ommitteed Figure 6. Growth curves in three lines of exon3hGH transgenic mice. Body weights were recorded from males (M) and females (F) for transgenic (T) and NT littermates from all three lines of exon3hGH transgenic mice (n = 12 per group). Both T males and females became significantly smaller and lighter than their NT littermates in line nos. 1 and 12 around 3–4 wk of age, but not in line no. 23. The effects were larger in males than in females, and the reduction in body size was proportionate to body weight (inset figure, animals from line no. 1). ***, P < 0.001 vs. NT males; ##, P < 0.01 vs. NT females.

 
RIA of pituitary homogenates showed a profound reduction of GH contents in both male and female ^exon3hGH transgenic animals in line no. 1 (Fig. 7A) compared with their NT littermates as early as 3 wk of age. Transgenic animals in line no. 12 also showed profound GH deficiency (10 wk; males 0.02 ± 0.01 µg/pit, females 0.025 ± 0.01 µg/pit, n = 12). In line no. 23, a significant, though less marked, GH deficiency was evident in the ^exon3hGH transgenic mice at 4 wk of age, but this progressed to a 75% deficit by 10 wk (Fig. 7B). The milder GH deficiency, later in onset, would explain the relatively normal growth of the transgenic animals in line no. 23. As expected from their more severe GH deficiency, marked anterior pituitary hypoplasia was evident in the ^exon3hGH transgenic animals from lines nos. 1 and 12 (average pituitary weights were 0.3 mg vs. 0.65 mg in NT mice) with a profound reduction in cells staining for GH by immunocytochemistry (not shown). Pituitary size was unaffected in line no. 23 at 10 wk of age.

fig.ommitteedfig.ommitteed Figure 7. GH contents in exon3hGH transgenic mice. Pituitary GH contents were measured by RIA in pituitaries harvested from males (left panels) and females (right panels) at two different ages. Upper panels, Line no. 1; lower panels, line no. 23. In both lines, and for both sexes, transgenic animals (T, dark bars) had lower pituitary GH contents than their NT littermates (open bars). GH deficiency was evident earlier and was much more profound in line no. 1, but also developed with time in line no. 23. **, P < 0.01, ***, P < 0.001 vs. age-matched NT animals. #, P < 0.05, 3 wk vs. 10 wk in T animals.

 
Differences between the lines were also apparent for other pituitary hormone axes. PRL, TSH, and LH were all significantly reduced in adult ^exon3hGH transgenic males of line no. 1 (Table 1) and line no. 12 (not shown). The ^exon3hGH females showed a significant reduction in PRL and TSH, but not in LH. In contrast, ^exon3hGH transgenic animals from line no. 23 showed only a slight reduction in PRL and their TSH and LH levels were not significantly altered (Table 1). No hGH immunoreactivity was detected in any of the pituitary extracts.

fig.ommitteedfig.ommitteed Table 1. Pituitary hormone contents in exon3hGH transgenic mice

 
Morphology of somatotrophs in ^exon3hGH transgenic mice
EM with or without immunogold labeling was performed on pituitary sections from ^exon3hGH transgenic animals of line no. 1. Very few somatotrophs could be identified, and these showed morphological features similar to those described above for the ^exon3hGH-GC cells with enlarged ER, mitochondria and Golgi, and many lipid vesicles (Fig. 8, A and B). Few GH SVs could be identified by immunogold labeling in ^exon3hGH transgenic pituitaries, compared with the abundant dense cored SVs immunolabeled in the somatotrophs of NT mice (Fig. 8F), and some of these presented an EM appearance of irregular shaped SVs more typically resembling those in lactotrophs (Fig. 8E). Corticotrophs, gonadotrophs, and lactotrophs could be recognized by their cell and vesicular morphology under EM (20), but their numbers also appeared compromised compared with NT mice. Numerous macrophages were noted throughout the ^exon3hGH transgenic anterior pituitaries (Fig. 8D) but not in NT pituitaries, with many perivascular macrophages at the boundary of the anterior lobe with the intermediate lobe; Fig. 8C.

fig.ommitteedfig.ommitteed Figure 8. Somatotroph disruption in exon3hGH transgenic mice. Immunogold EM of pituitary sections from transgenic mice of line no. 1 showed a massive depletion of recognizable somatotrophs, and the remaining GH cells were grossly abnormal with few if any dense cored SVs compared with the abundant somatotrophs filled with many GH-containing SVs clearly labeled with immunogold (arrows) in NT mice (F). Many cells were clearly disintegrating (A) or had dilated mitochondria or vacuolated cytoplasm (B). Many macrophages were evident throughout the pituitary (C and D), many filled with lipid accumulations and immunostained remnants (D). A few cells contained dense vesicular structures that immunostained for mouse GH, but these were irregular and misshapen (E). n, Nucleus; L, lipid; arrows, anti-GH immunogold; M, macrophage; m, mitochondria; v, vacuole. Scale bar, 1 µm.

 
Hypothalamic GHRH and somatostatin expression
The profound reduction in pituitary GH in ^exon3hGH transgenic mice would be expected to remove GH feedback, thereby increasing hypothalamic GHRH expression and decreasing SRIF expression. To study this, in situ hybridization was performed for these transcripts in hypothalamic sections prepared from ^exon3hGH transgenic and NT animals. As expected, arcuate GHRH mRNA abundance was significantly higher in the ^exon3hGH transgenic mice, and SRIF mRNA abundance was significantly lower in their periventricular nuclei, compared with that seen in NT mice (Fig. 9).

fig.ommitteedfig.ommitteed Figure 9. GHRH and somatostatin expression in exon3hGH transgenic mice. In situ hybridization was carried out on hypothalamic sections from NT and exon3hGH transgenic mice (line no. 1), using radiolabeled antisense riboprobes for GHRH (left panels) and somatostatin (right panels). Individual examples are shown in the upper panel, and the results from image analysis on arcuate (ARC) and periventricular nuclei (PeN) on similar sections from groups of animals are shown in the lower panels. ARC GHRH expression was significantly up-regulated, whereas PeN somatostatin expression significantly reduced in the same group of animals. ZI, Zona incerta. **, P < 0.01 vs. NT controls (n = 6 per group).

 

	Discussion

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Familial IGHD-II is often associated with dominant-negative mutations of the third intron of the GH1 gene, either by direct interference at the 5' splice site, or by compromising splice enhancer sequences; these mutations cause exon-3 skipping, which generates a 17.5-kDa hGH protein variant (12, 21, 22). We made use of a human genomic GH1 construct containing the IVS3 + 1 GA donor splice site mutation which causes familial IGHDII (4). It gave rise to ^exon3hGH but not WT-hGH when transfected in rodent GC cells and exerted a dominant negative effect on endogenous rGH production in these cells. The same genomic sequence was then inserted into a human LCR transgene construct that drives copy number-, position-independent transgene expression specifically in somatotrophs in transgenic mice (13, 14). This generated the first murine model of IGHDII in which it was possible to examine both the cellular and physiological consequences of ^exon3hGH expression in vivo.

How the expression of a dominant negative protein hormone suppresses the secretion of the normal product from an unaffected allele is unclear. Such mechanisms could include general defects in protein folding or aggregation (11), accumulation of mutant protein in the ER, or mis-sorting to degradative compartments (23, 24), a toxic effect of the mutant protein per se on cell function (9) or to a more specific interference with the production of the normal allele product (25, 26).

Using metabolic labeling in nonsecretory cell lines, Hayashi et al. (7) showed that coexpression of ^exon3hGH did not inhibit WT-hGH production nor did it affect cell viability, whereas in secretory pituitary cell lines, it inhibited WT-hGH secretion in a concentration-dependent manner. Lee et al. (8) found similar results and showed some specificity for hGH, because coexpression of ^exon3hGH with PRL did not affect PRL production and secretion. However, in COS7 cells, expression of ^exon3hGH did disrupt PRL production and disrupted ER to Golgi trafficking (9). An Arg¹⁸³His hGH mutation that also causes IGHDII (26) gives rise to a protein that was efficiently secreted from a neuroendocrine cell line when expressed alone but greatly impaired secretion when coexpressed with WT-hGH. Taken together, these results suggest that mutant GHs do not have a general toxic effect per se but can exert a powerful dominant negative effect when able to interact with WT-hGH in a cell with a prominent regulated secretory pathway.

Our morphological data from both cell lines and primary somatotrophs shed some light on this because expression of ^exon3hGH prevented or destabilized SV formation in a dominant negative fashion and prevented the normal packaging of endogenous mouse or rGH into dense-cored SVs. Instead, endogenous GH gradually accumulated in aggregates in the cytosol, unavailable for exocytotic release. In addition to swollen and disrupted ER and Golgi structures, severely affected ^exon3hGH-GC cells showed an abundance of intracellular lipid, also suggesting a substantial increase in membrane recycling.

These observations were made on fixed cells, but by using TIRF microscopy and an eGFP construct that copackages with GH in SVs we could observe this process dynamically at the level of individual SVs in single GC cells. TIRF, or evanescent wave, microscopy, illuminates fluorophores in a 100-nM-thick optical slice between a glass surface and the basal membrane of an adhering cell (27) and can thus be used to image individual SV movements in living cells (28). When TIRF was used to image eGFP-transfected GC cells, individual SVs could be resolved and their range of fast or slow movements tracked in either GC or WT-hGH-GC cells. However, very few eGFP-containing SVs were present in ^exon3hGH-GC cells. Instead, most of the eGFP fluorescence was localized in amorphous cytoplasmic aggregates corresponding to the diffuse secretory material seen under EM. This material remained motionless in the cell and did not give rise to exocytotic events. Because the eGFP construct does not require WT-hGH for packaging into SVs in other secretory cells (Robinson, I. C. A. F., unpublished), the lack of fluorescent SVs in ^exon3hGH/GC-eGFP cells provides further evidence that the mutant hGH disrupts SV formation in these cells. We suggest that this is the key process by which ^exon3hGH eventually destroys the somatotroph.

Packaging of hGH into SVs represent the culmination of many protein assembly processes that proceed throughout the secretory pathway (29). Protein folding, dimerization, and oligomeric association of SV proteins begins in the ER (30) and occur before trafficking via the Golgi to form condensing SVs (31). Secretory proteins must also be folded correctly to exit the ER (29, 32). Because deletion of exon 3 sequences includes Cys⁵³, the resulting unpaired Cys¹⁶⁵ could result in aberrant intra- or inter-molecular disulfide-linked misfolded aggregates. However, this cannot be the only explanation because a dominant negative effect is still observed after mutating the unpaired Cys¹⁶⁵ partner (8). Furthermore, dominant negative suppression of WT-hGH production is also seen with a single point missense mutation in GH1 not affecting disulfide bridges (26), and large amounts of WT-hGH are secreted in a patient bearing a heterozygous Arg⁷⁷Cys mutation, which also generates an additional unpaired Cys in a mutant hGH that is secreted but which then antagonizes WT-hGH at the GH receptor (33).

Misfolded proteins detected in the ER are transported back to the cytosol where they are degraded by proteasomes (34, 35) and do not usually cause a dominant negative effect (36) unless the production rate exceeds the proteasome degradation capacity. For example, there are other GH1 frameshift mutations (25, 37) that also produce misfolded proteins, but these are phenotypically recessive. The proteasome pathway has been implicated in the degradation of ^exon3hGH because its accumulation is enhanced when expressed in AtT20 cells treated with proteasome inhibitors (8).

Concentration, oligomerization, and condensation to form dense SV protein cores is promoted by mild acidification in the presence of high concentrations of Ca²⁺ or Zn²⁺ during transit from ER through the Golgi (38, 39). WT-hGH contains well defined Zn²⁺ binding sites that induce cooperative dimerization of hGH (40), and these may facilitate the same oligomerization/insolubilization process for GH SV cores, as has been well established for insulin hexamers (41). The (Zn²⁺-hGH)₂ complex is stable and resistant to denaturation during storage (40), and all of the identified Zn²⁺ binding residues in hGH are present in ^exon3hGH, though recent work suggests these may not be a prerequisite for condensation and aggregation (42).

We propose the following hypothesis to explain the dominant negative effects of ^exon3hGH: the 17.5-kDa hGH product progresses through the regulated secretory pathway, where it can form heterodimers with WT-hGH. Some WT-hGH homodimers may also form, but further oligomerization with heterodimers occurs poorly so efficient packing and condensation cannot occur, blocking the formation of dense-cored GH SVs from the trans-Golgi. Because the ^exon3hGH:WT-hGH complexes are stable, but cannot exit efficiently via SVs, both WT-hGH and ^exon3hGH accumulate in the Golgi, and back up in the ER. This triggers the misfolded protein response and the complexes are transported into the cytosol. Once the production of ^exon3hGH:WT-hGH complexes exceeds the degradative capacity of the proteasome pathway, they begin to accumulate as aggregates in the cytosol, ER and Golgi, eventually proving toxic to the cell. For GH-producing cells in culture, cell death occurs as an autolytic process; in vivo the process is greatly accelerated by increased trophic drive from GHRH to increase expression of both hGH and ^exon3hGH, to replace and expand the (defective) somatotroph population, and by an invasion of activated macrophages to destroy defective cells.

This hypothesis would explain why there is no toxic effect of ^exon3hGH when expressed alone or with proteins other than GH in other cell types, and also why a dominant effect does not usually occur when coexpressed with WT-hGH in nonsecretory cells (7). Were ^exon3hGH unable to interact with WT-hGH, then the WT-hGH proteins should homo-oligomerize from the mixture and form a population of normal dense cored SVs containing WT-hGH. Furthermore, the rate or extent of cellular damage would be proportional to the amount and rate of traffic of both WT-hGH and ^exon3hGH for SV packaging, being milder in the less granular GC cells, and more severe in highly granulated somatotrophs. In the ^exon3hGH-GC cultures, it was possible to observe cells at intermediate stages of morphological disruption, and though their growth rates were compromised, stable ^exon3hGH cell lines could be obtained. In contrast, the same features of degranulation and morphological disruption caused by ^exon3hGH had catastrophic consequences for the somatotroph population in the transgenic animals.

Our hypothesis would also predict that the onset, severity and rate of progression would be proportional to the relative amounts of ^exon3-hGH vs. WT-hGH expressed. Analysis of the different lines of transgenic mice was consistent with this, because the onset, severity, and specificity of their IGHDII phenotype was proportionate to their transgene copy number. We could not establish the relative RNA or ^exon3hGH protein levels directly in pituitary extracts because many of the cells were destroyed, and we were unable to detect the 17.5-kDa protein. However, we can assume that there is more ^exon3hGH expression in the high copy lines because this transgene LCR shows reliable copy number-dependent and position-independent expression (14, 43). The most severely affected lines were already GH deficient at weaning and developed proportionate reductions in weight and length and bone growth, more marked in males than in females, as in other transgenic dominant dwarf animals (18). Although the low copy number line showed a milder phenotype, with relatively normal postweaning growth, all animals eventually developed pituitary GH deficiency with time.

It is interesting to compare this murine phenotype with human IGHDII, which also shows variability in onset, severity, and progression, even within the same family (44). In human IGHDII, the allele ratio of WT-hGH:mutant hGH is 1:1, and it is assumed that each allele is transcribed equivalently in hemizygous individuals, although it has not been established that they generate equivalently stable or translatable RNA products. Severe short stature was only present in one third of the affected individuals at diagnosis in the study by Binder et al. (44), although children with splice site mutations were on average younger and shorter at diagnosis than those with the missense mutations. This argues for a more pronounced effect of a dominant negative mutation than of haploinsufficiency per se, although the severity of phenotype will also clearly depend on the nature of the missense mutation and the degree to which it disrupts GH structure.

Variability in effects on pituitary size also appears in human subjects with IGHDII; MR imaging in four children showed a normal adenohypophysis in two cases and mild hypoplasia in two others (44). However, a variety of different mutations are associated with IGHDII (3, 4, 21, 22, 44), including alterations or deletions in splice enhancers, which can give rise to different ratios of WT-hGH and ^exon3hGH transcripts (Ryther, R., L. McGuinness, J. Phillips, C. Moseley, C. Magoulas, I. C. Robinson, and J. Patton, in preparation). Although the correlation between the various GH mRNA isoforms, the amounts of their protein products, and the extent of pituitary damage caused remains to be established, our results suggest that variability in the ratios and amounts of 17.5- to 22-kDa isoforms produced could well be an important contributor to the variability of individual phenotype in some forms of human IGHDII.

Unexpectedly, both lines 1 and 12 were subfertile. This is unlikely to be due to GH deficiency or body size per se, because other equally small, equally GH-deficient mice (expressing different transgenes on the same genetic background) are normally fertile (Robinson, I. C. A. F., unpublished). It is more likely due to the other pituitary hormone deficiencies that developed in the most severely affected lines. A fall in PRL was expected because most models of GH cell hypoplasia or ablation also show reduced PRL (18, 45). One or two surviving GH-immunopositive cells in the ^exon3hGH mice presented an appearance under EM more resembling lactotrophs, typified by irregularly shaped SVs. There is a small population of pituitary mammosomatotrophs that express both GH and PRL, and these may express less GH (and hence less ^exon3hGH) per cell than do somatotrophs. Because ^exon3hGH does not block PRL secretion, mammosomatotrophs might be able to package some PRL into SVs and with less material accumulating in the cytosol, could perhaps survive longer than somatotrophs.

More surprising was the loss of other pituitary hormones in the high copy lines. Snell and Jackson dwarf mice with mutations in Pit-1 (46) are deficient in TSH as well as GH and PRL, and it is possible that multiple copies of Pit-1 elements in the GH1 transgene promoters compete for the available Pit-1 and reduce the transcription of other endogenous Pit-1-dependent genes, such as TSH. However, this explanation would not explain the fall in LH and gonadotrope numbers visible by EM. Large numbers of activated macrophages were evident in the high copy lines, especially at the intermediate lobe/anterior pituitary boundary where new GH cells first appear during development (47) so these are well placed to destroy newly emerging defective somatotropes as they differentiate from progenitor cells. Because this transgene LCR reliably restricts transgene expression to the somatotrope (13, 14, 43), we suggest that the massive and rapid autodestruction of GH cells induced by high expression of ^exon3hGH activates an inflammatory macrophage response resulting in significant bystander endocrine cell killing in these high copy lines.

The phenotype in the line no. 23 is probably a closer model for human IGHDII, in which the hormone deficiency appears largely confined to the GH axis (4, 21, 26, 44). Most reports suggest normal thyroid and adrenal function and normal plasma PRL levels in IGHDII. However, because multiple pituitary hormone deficiencies may evolve in some children initially diagnosed with isolated GHD, it may be important to reinvestigate older subjects with severe IGHDII diagnosed and treated in childhood, to see whether further pituitary hormone deficits emerge with time.

Our in vivo model also allowed us to investigate hypothalamic changes in IGHDII for the first time. GH normally regulates its own production by both direct and indirect feedback, repressing GHRH and increasing somatostatin expression, respectively (16, 19, 48). As expected, lack of GH feedback in the ^exon3hGH transgenic mice was associated with increased arcuate GHRH and decreased periventricular somatostatin expression compared with their NT littermate controls. We believe this may be an important additional factor that accelerates the rate of progression of IGHDII in vivo. The increased GHRH drive that stimulates somatotroph proliferation and WT-GH transcription will also increase transcription of ^exon3hGH, compounding the cellular blockade. Progressive GH deficiency and GHRH up-regulation would then form a vicious cycle to accelerate the production and autodestruction of the GH cell population, rapidly exhausting the capacity to generate new GH cells.

A reduction in GH cell number may ultimately be more important than compromised GH cell function in the longer term. Early treatment of IGHDII with exogenous GH replacement therapy may be important in rescuing a degree of pituitary function by providing a feedback signal to reduce the GHRH drive, reducing somatotroph proliferation and rate of self-destruction. A secondary benefit could be to reduce rate of the pituitary damage and hence loss of other endocrine cell types in IGHDII. If so, precipitate withdrawal of GH treatment following attainment of adult height in IGHDII could be deleterious.

　

	Acknowledgments

 
We are very grateful to Dr. A. L. Parlow and to the NIDDK for the continued provision of assay reagents, and to Dr. Nancy Cooke for providing us with the original hGH LCR cosmid.

Received August 13, 2002.

Accepted for publication October 4, 2002.

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