Heterozygote Effects in Mice with Partial Truncations in the Growth Hormone Receptor Cytoplasmic Domain: Assessment of Growth Parameters and Phenotype
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《内分泌学杂志》
Institute for Molecular Bioscience (L.M.K., M.J.W.) and School of Biomedical Sciences (J.E.R., M.W., P.G.N.), University of Queensland, St. Lucia, Queensland 4072, Australia
Abstract
The GH receptor (GHR) is essential for normal postnatal growth and development, and the molecular basis of GHR action has been studied intensively. Clinical case studies and more recently mouse models have revealed the extensive phenotype of impaired GH action. We recently reported two new mouse models, possessing cytoplasmic truncations at position 569 (plus Y539/545-F) and 391, which were created to identify functional subdomains within the cytoplasmic signaling domain. In the homozygous state, these animals show progressively impaired postnatal growth coupled with complex changes in gene expression. We describe here an extended phenotype analysis encompassing the heterozygote state to identify whether single copies of these mutant receptors bring about partial or dominant-negative phenotypes. It appears that the retention of the ubiquitin-dependent endocytosis motif in the N-terminal cytoplasmic domain permits turnover of these mutant receptors because no dominant-negative phenotype is seen. Nonetheless, we do observe partial impairment of postnatal growth in heterozygotes supporting limited haploinsufficiency. Reproductive function is impaired in these models in a progressive manner, in parallel with loss of signal transducer and activator of transcription-5 activation ability. In summary, we describe a more comprehensive phenotypic analysis of these mouse models, encompassing overall and longitudinal body growth, reproductive function, and hormonal status in both the heterozygote and homozygote state. Our results suggest that patients expressing single copies of similarly mutated GHRs would not display an obvious clinical phenotype.
Introduction
FIRST DESCRIBED BY Laron et al. (1) in 1966, GH insensitivity syndrome (GHIS) is a rare disease characterized by resistance to GH. Although molecular abnormalities have been reported for GH receptor (GHR) signaling pathways, and in the IGF-I gene in GHIS, defects in the GHR account for most of the reported cases to date (2). To date, nearly 50 distinct mutations responsible for GH resistance have been identified including nonsense, missense, splice, or frameshift mutations (3, 4, 5, 6). Although a complete GHR knockout mouse model has been available since 1997 (7), we have more recently described two transgenic lines carrying knock-in (KI) mutations within the GHR cytoplasmic domain (8). These mice, carrying either a truncation at mouse GHR position 569 (with two Y-F mutations at 539 and 545) or a truncation at 391, show a progressive impairment of postnatal growth in proportion to the severity of the truncation. We have shown this (8) to correlate with loss of signaling factor recruitment to the cytoplasmic domain upon GHR activation in the homozygous state, particularly of hepatic signal transducer and activator of transcription (Stat)5 [30% of wild type for 569 mice, 0% for 391 mice], despite normal Janus kinase (JAK)2 activation (8). This study concluded that the distal cytoplasmic domain of the GHR is critical for Stat5 recruitment, and its loss leads to loss of sexual dimorphism in the rodent as shown by changes in liver transcripts for cytochrome P450 enzymes and loss of major urinary proteins (MUPs) (8). However, the membrane proximal sequence is still able to generate 10% of postnatal growth, activate the MAPK pathway, and regulate the majority of GH-responsive genes (8).
Clinically, most GHIS cases are attributable to mutations in the extracellular domain of the GHR, which result in mutations causing protein misfolding and intracellular degradation; therefore, heterozygotes are clinically undetected and merely carry the mutation (9). However, it has been suggested that heterozygous or less deleterious mutations in the cytoplasmic domain of the GHR gene could cause partial GHIS and growth retardation in the heterozygote state if the mutants are able to act as dominant negatives (10). The first case of partial cytoplasmic domain truncation effecting a Laron phenotype was recently described by Milward et al. (11), who reported a homozygous truncation at position 450, resulting in a GHIS phenotype. In vitro coexpression of this mutant GHR with full-length GHR resulted in a dominant-negative phenotype, which impaired full-length GHR signaling (12). This was evident despite the UbiE internalization motif (DSWVEFIELD) remaining N terminal to the truncation site. No clinical heterozygotes have been reported for this truncation mutant, and the parents of these siblings were unavailable for study (11). However, a dominant-negative phenotype is seen for patients expressing heterozygous truncated mutant GHRs, which lack all but five cytoplasmic transmembrane proximal residues when these are coexpressed at the cell surface because they impede full-length receptor dimer formation (13, 14, 15).
Here we aimed to resolve whether less deleterious heterozygous truncations that still possess the internalization motif may have an impact on postnatal growth. Using the models described previously (8), we compared the physiological parameters of homozygous and heterozygote mutants 569 and 391 with reference to the classic Laron mouse model (GHR–/–). Additionally we describe more fully the 569 and 391 mutants that we reported recently (8).
Materials and Methods
Animals
The GHR–/– and truncation mutant lines used in this study have been described previously (7, 8). Animals were housed in an approved facility and treated according to University of Queensland Animal Ethics Committee and the Australian Office of the Gene Technology Regulator guidelines. Water and feed pellets were available ad libitum during a 12-h light, 12-h dark cycle at 20–22 C. All animals passed standard virus screens throughout.
Postnatal growth measurement
Litters from heterozygous crosses were used for this comparative study. Body weight, body length, and tail length measurements were taken at regular intervals from 10 to 60 d postnatally. Comparisons were made between animals of the same sex. Organ weights are expressed as actual values, as percent body weight and percent of WT weight, which expresses disproportionate growth changes as a percentage value (16).
IGF-I and GH measurements
Acid ethanol extracted serum IGF-I was measured from sera samples using an IGF-I RIA kit (Bioclone, Sydney, Australia). Serum GH was quantified with a RIA kit (Linco, St. Charles, MO; RGH-45HK). Transcripts were detected as described in Rowland et al. (8) using rat IGF-I cDNA for exon 3, kindly provided by Dr. Adrian Herington (Queensland University of Technology, Brisbane, Australia).
Major urinary protein
Urine samples were collected before dissection of mice at 42 and 60 d postnatal and frozen at –20 C. For visualization of MUPs, 1 μl of female urine or 0.5 μl of male urine was electrophoresed on 12% SDS-PAGE followed by Coomassie blue staining as described by Norstedt and Palmiter (17). For quantification of urine MUP samples, total protein values were estimated using BCA assay (Sigma Aldrich, St. Louis, MO).
Bone length
Hind legs were routinely dissected and x-rayed. X-ray films were scanned into gray-scale TIFF-formatted files, and morphometric analysis of femoral and tibial dimensions was performed using National Institutes of Health image software for Macintosh.
Statistics
Data are expressed as mean ± SEM throughout. Populations were compared by Tukey-Kramer multiple comparisons ANOVA where applicable. Annotations are made to indicate P values: , P < 0.05; , P < 0.01; , P < 0.001.
Results
Postnatal growth
The mouse models used for this study expressed either 569stop Y539/545-F or 391stop mutant GHRs (8) (Fig. 1). To elucidate the physiological impact of the 569 and 391 mutations in the homozygous and heterozygous state, we measured overall body weight (Fig. 2), nose-anus length (see supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org), and tail length (see supplemental data) for both mutants with comparison with wild-type (WT) and GHR–/– mouse lines. A difference in these measures of growth was not expected before weaning because differences are not seen in the GHR–/– mice (7, 16) or Stat5 knockout mice (18, 19) before weaning. Both mutant 569 and 391 showed growth rates diverging from sex-matched WT controls at approximately 21 d postnatal, which correlated with time of growth rate divergence seen in GHR–/– mice (7, 16). A difference between the sexes for the weights of WT controls was evident from d 20, as expected (16). This gender difference is due to the emerging actions of gonadal steroids, which induce a distinct pattern of GH secretion in males vs. females.
Significant reductions in body weight were seen for heterozygotes and homozygotes with 569 and 391 mice and GHR–/– mice (Fig. 2 and Table 1; see supplemental data). These reductions were significant for both sexes at both 42 and 60 d postpartum (P < 0.001). Ratios of WT to homozygous KI body weights remain relatively unchanged between 42–60 d of age for both males and females, indicating similar reduction in growth rates over this period when compared with WT littermates. Interestingly, the growth rate of the homozygous 569 male was similar to that seen for WT females, suggesting that full Stat5 generation is necessary for normal sexually dimorphic growth. The weights of male and female 391 KI mice was not significantly different, again supporting a lack of sexually dimorphic growth in the absence of GH-dependent Stat5 generation, as seen for GHR–/– mice (Fig. 2 and Table 1) (7, 16). For GHR–/– mice, a significant haploinsufficiency effect was seen in heterozygotes carrying only one copy of GHR as has been previously reported (7, 20). Body weights of GHR+/– mice at 60 d were approximately 85% of WT for males and 93–95% for females.
Organ weights
Organ weights were measured at 42 d (see supplemental data) and 60 d (Table 1) postnatal for 391, 569, and GHR–/– mice for both male and female heterozygotes and homozygotes. Kidney and spleen growth has been reported to be disproportionately reduced in GHR–/– mice, particularly in older animals (16). We also observed a disproportionate reduction in spleen growth for 391 and GHR–/– animals (Table 1; supplemental data). This effect was less clear in 569 mice in which spleen weight was only significantly different in 569 females at 42 d.
At 60 d progressive reduction in organ weights was seen for homozygotes in proportion to severity of mutation, as reported previously (Table 1) (8). Disproportionately reduced bladder size was evident in male homozygotes and 391 and knockout (KO) heterozygotes, together with a disproportionate increase in brain size in homozygotes resulting from decreased body weight. A significant decrease in proportional growth of liver was seen for all homozygous 391 and KO mice and heterozygous 391 and KO mice. Additionally, a proportionately decreased epididymal fat pad weight was seen for both heterozygous 391 and KO male mice.
Longitudinal growth
GHR–/– mouse investigations revealed a dramatic effect on linear bone growth and skeletal development in the absence of GHR function (16, 21), which is in agreement with clinical observations (1, 22). Therefore, the relative changes in femoral and tibial dimensions were investigated for our KI models. Femoral and tibial bones from all 42- and 60-d-old mice studied were x-rayed and scanned into TIFF-formatted picture files, and then lengths and widths were measured using National Institutes of Health Image software. The data comparisons of these measurements are recorded in Table 2 (60 d) and supplemental data (published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org) (42 d).
These data show that whereas homozygote effects are evident for both mice lines with truncated GHR, there is minimal impact on heterozygote bone lengths at these time points. There was no significant difference between males and females for femoral or tibial length at either 42 or 60 d postnatal, in agreement with previous reports (21). At 42 d, femoral length is not significantly reduced in 569 females, compared with WT, but is reduced in 569 KI males (92%), 391 KI males (82%), 391 KI females (80%), GHR–/– males (73%), and GHR–/– females (74%). At 60 d, femoral length is significantly reduced, compared with WT in 569 KI males (92%), 569 KI females (94%), 391 KI males (80%), 391 KI females (78%), GHR–/– males (71%), and GHR–/– females (73%). This suggests an increasing divergence in length due to the impaired growth rate of the KI/KO models, in agreement with the significant size increase observed in femoral length for both male and female WTs from 42 to 60 d of age. Femoral midpoint width was reduced in all homozygotes at 60 d age but not in heterozygotes, except for 391 males.
Tibial lengths are significantly reduced for all homozygous models at both 42 and 60 d of age. At 42 d of age, 569 males (95%), 569 females (94%), 391 males (87%), 391 females (86%), GHR–/– females (78%), and GHR–/– (78%) show progressive reduction in tibial lengths relative to the severity of the mutation/truncation. These differences are almost identical for 60-d comparisons, which agrees with the observation that there is no significant change in relative tibial length for WT males or females between 42 and 60 d of age (Table 2; support data 4). A significant reduction in tibial length was also seen for GHR+/– mice at 60 d (94% males, 96% for females); however, no significant change was found for either of the GHR truncated heterozygotes. Diaphyseal and epiphyseal tibial widths showed small comparative reductions that were most striking in the GHR–/– mice. These differences were not as clear as those measured for bone length but do agree with the observed trend of progressive impairment of bone growth with severity of mutation.
IGF-I and GH
The transcriptional regulation of IGF-I is somewhat divergent between tissues (16), however this study has focused on endocrine IGF-I by measuring liver transcript and serum IGF-I (Fig. 3). Liver is the major endocrine source of IGF-I as demonstrated by liver-specific IGF-I KO mice (lid) mice, which display only 25% of WT serum IGF-I levels (23, 24, 25, 26). The reduced serum IGF-I levels observed in GHR/binding protein KO mice (less than 10% of WT) were in agreement with estimates already reported to be between 7 and 10% of normal (7, 20, 27). Stat5B KO male mice are reported to display between 50 and 70% of WT serum IGF-I levels (18, 19), whereas Stat5A/B KO mice reportedly have 50 and 44% of WT serum IGF-I levels for males and females, respectively (19). The relative serum IGF-I levels observed for 569 mutants (16–21% of WT) and 391 mutants (<10% of WT) were far lower than that reported for any of the Stat5 KO models or lid mice. Additionally, the transcript levels for mutant 391 (9–11% of WT) were in agreement with the serum IGF-I estimates, whereas transcript levels observed for mutant 569 (36–67% of WT for Igf1b and Igf1a transcripts, respectively) were substantially higher than levels of serum IGF-I protein recorded. This may be attributed to the reduction or absence of IGF binding protein (IGFBP)-3 and acid labile subunit (ALS) seen in these transgenics, as reported by Rowland et al. (8). Importantly, significant reductions in both IGF-I transcript and protein were also seen for 569 and 391 heterozygotes with the exception of 391 females, which were reduced but not quite significantly (Fig. 3).
Assay of serum GH in male mice revealed a significant elevation of GH (P < 0.01) only in the case of the 391 mice (WT –9.2 ± 2.0, 569 –8.9 ± 3.3, 391 –28.8 ± 1.7, mean ± SEM, n = 3).
Fertility and sexual dimorphism
GHR deletion has been shown to have an impact on reproductive fertility (7, 28); thus, this was examined in our transgenic models. Heterozygote matings yielded a reduced (non-Mendelian) proportion of KI or KO homozygotes in their progeny in proportion to the severity of the mutation (Fig. 4A). Additionally, when homozygotes were mated, litter sizes were reduced in proportion to severity of receptor truncation (Fig. 4B).
MUP is the most abundant liver mRNA and is easily detectable in male urine, and about a third as much is evident in female urine. MUP levels are decreased in the urine of little (lit/lit) mice (17), Stat5B KO (17), Stat5A/5B KO (19), cytokine-induced SH2-containing protein-overexpressing mice (29), and liver IGF-I KO males but not females (24). In most cases this corresponds with overall growth retardation and/or disruption to the pattern of GH exposure to the tissue. Hence, given the reduced growth rate and serum IGF-I levels observed for both 569 and 391 targeted KI mice, a corresponding reduction in urine MUP protein was expected. We already reported that several MUP transcripts are reduced in 569 and 391 transgenic homozygotes (8). Here we show that also at the protein level, a clear impact on urine MUP levels was observed for males, whereas the female MUP level remained unchanged in all populations (Fig. 4C). It is evident from Coomassie-stained sodium dodecyl sulfate gel electrophoresis of 569 and 391 homozygotes that the MUP proteins constitute the majority of urinary proteins. Although reductions appeared evident for male heterozygotes for all lines studied and for 569 KI, statistically significant reductions in MUP were observed only for homozygous 391 KI and KO mice. (Fig. 4D).
Discussion
Here we have reported an extended postnatal growth phenotype analysis for mice harboring GHRs truncated with different degrees of severity and have compared these with mice lacking GHR. We observe a progressive impairment of postnatal growth relating to the severity of cytoplasmic mutation, which is observed also in the heterozygote state. These truncated receptors are expressed and partially functional (8). In neither case do heterozygotes show a more severe phenotype than that seen for the GHR+/–. Furthermore, no data shown here identify any dominant-negative effects on growth in the heterozygote state when compared with the GHR+/– control, which cannot exert such effects. Thus, our truncation mutants do not display a dominant-negative effect as seen for patients expressing severely truncated mutant GHRs, which are overexpressed at the cell surface, thereby impeding full-length receptor dimer formation (13, 14, 15). Mutants 569 and 391 both retain their membrane proximal receptor internalization motif, which should permit the mutant receptors to turn over similarly to WT GHR, thus explaining the intermediate phenotype.
Interestingly, the IGF-I transcript levels reported for Stat5B KO mice (50% of WT) (30) are similar to that recorded for mutant 569, although serum IGF-I in Stat5B-deleted mice is approximately 3 times higher than that of 569 (30). This may be explained by the reduced level of serum IGFBP-3 observed for the 569 mutant (33–52% of WT) (8). Although the relative levels of IGFBP-3 or ALS have not been reported for the Stat5 KO mouse models, it is possible that this difference relates to a specific reduction in Stat5-independent up-regulation of IGFBP-3 and/or ALS by 569 and 391 mutant GHRs (8).
It is expected that these transgenic mice would display an increased plasma GH due to reduced negative feedback by IGF-I, and this was clearly the case for the more severely truncated homozygous 391 mice. A larger sample size or frequent sampling may reveal an increase in GH secretion in the less debilitated 569 mutants. Elevated plasma GH would be expected to increase the activation of JAK2 and pathways dependent on direct JAK2 action, potentially partially compensating for lack of recruitment of certain signaling factors to the distal cytoplasmic GHR. It is also possible that the heterodimeric receptor subunits are able to phosphorylate and recruit some Stat5 to the full-length GHR half of dimers in vivo, although distinguishing this from remaining full-length homodimer activation in vivo is not possible for heterozygotes.
Longitudinal bone growth is impaired in GHR/BP KO mice due to the lack of both direct and IGF-I driven actions of GH (16). It has recently been shown that this reduction may be largely attributed to reduced autocrine/paracrine stimulation of IGF-I because lid mice do not display large reductions in bone growth (31). Furthermore, bone growth is impaired in Stat5A/B KO mice (femoral length 90% of WT), although this is not as severe as that seen for GHR/BP KO mice (femoral length 75% of WT), suggesting that other GH-responsive factors also contribute to bone growth (21). Thus, it was not unexpected to see a progressive reduction in femoral and tibial lengths in our mutant models corresponding to a progressive decrease in IGF-I transcript expression. The relative femoral lengths of 569, 391, and GHR–/– mice (92–94, 78–80, and 71–73% of WT) and the relative tibial lengths (94–95, 86–87, and 78% of WT) at 60 d postnatal reflect the reduction seen for body weights. However, the much larger reduction in serum IGF-I for the 569 KI does not correlate with the relatively small reduction seen in longitudinal bone growth. As mentioned, the sharp decline in serum IGF-I is likely a result of increased degradation of circulating IGF-I in concordance with reduced ALS and IGFBP-3 levels. This supports the view that a substantial portion of GH-dependent bone growth is mediated via local IGF-I generation. It is also possible that IGF-I-independent GH effects are driving bone growth in the 569 model, i.e. induction of bone morphogenetic proteins (32, 33).
In contrast, the 391 model showed reduced bone lengths on par with those seen for the GHR–/– mouse, indicating that local GH-induced bone growth via both direct and IGF-I mediated actions is absent or not detectable in 391 mice. Taken together it would appear that the majority of GH-driven bone growth is dependent on the 391–569 portion of the growth hormone receptor,
GH impacts reproductive function in rodents. GHR–/– mice exhibit delayed puberty (male and female), prolonged pregnancy (1 d longer), and increased placental size (28, 34). For GHR–/– mice, litter size and weight of newborn pups is reduced, compared with normal mice. (Fig. 4) (28). They are fertile but experience a delayed age of first conception for KO matings, and their litter sizes are approximately 40% that of WT and heterozygotes, partly due to a reduced ovulation rate (7, 28, 35). Greatly decreased levels of IGF-I in GHR–/– females might also diminish the supply of nutrients available to the fetus resulting in fetal growth retardation (28). Our observations that 569 and 391 truncations substantially reduce litter sizes from homozygous matings indicates that GHR signaling in the distal cytoplasmic domain is critical for normal reproductive function in rodents. It is unclear at this time whether reduced litter sizes from homozygous matings are due to impaired male or female fertilities or both (35). It may be possible to resolve distinctive GHR pathways required for reproductive pathways in mice using this model in the future.
The sexually dimorphic expression of MUPs reported here supports our finding that Stat5 is activated submaximally in the native state for mutant 569 (8). We have shown that dimorphic cytochrome P450 gene expression is altered, whereas urine MUP levels were reduced for mutant 569 and absent in mutant 391 (Fig. 4) (8). Stat5B activity has been strongly linked with the sexually dimorphic response of the liver in relation to both MUP and cytochrome P450 expression (18, 36). Hence, it is reasonable to propose that the reduction in MUP for mutant 569 males and females relates simply to submaximal stimulation of Stat5 in vivo. Whether the altered sexually dimorphic response in mutant 569 relates to simply submaximal Stat5 activation or is due to the elevated GH troughs that would be predicted due to impaired IGF-I feedback to the pituitary is uncertain. Indeed, demasculinization of liver enzymes has been reported for lid mice, which display reduced circulating IGF-I levels, which are almost double those reported for either 569 or 391 transgenics (24).
The data presented here represent an extended in vivo phenotype analysis of mouse models with disruptions to the GHR cytoplasmic domain. The relatively minor impairment of body growth observed for heterozygotes bearing the mutations suggests that the predicted 25% full-length receptor homodimers are sufficient for most signaling or that mutant receptor subunits are forming heterodimeric complexes with full-length receptor subunits to form subactive signaling complexes. The heterozygote effect is not as severe as the dominant-negative effect seen for patients expressing truncated mutant GHRs in vivo, which are overexpressed at the cell surface, thereby impeding full-length receptor dimer formation (13, 14, 15). Mutants 569 and 391 both retain their membrane proximal receptor internalization motif, which should permit the mutant receptors to turn over similarly to WT GHR, thus explaining the intermediate phenotype. Therefore, whereas it is likely that patients with subnormal postnatal growth rates may carry heterozygous mutations to the cytoplasmic GHR, this is unlikely to be detected in the general population unless this mutation disables the internalization motif of the GHR.
Acknowledgments
We thank Professor J. J. Kopchick and Dr. K. T. Coshigano for generously supplying us with the GHR–/– mice used in this study. We also thank Paul Addison, Elisabetta d’Aniello, and Terry Daly for excellent technical assistance.
Footnotes
This work was supported by the National Health and Medical Research Council of Australia.
First Published Online September 15, 2005
Abbreviations: ALS, Acid-labile subunit; GHIS, GH insensitivity syndrome; GHR, GH receptor; IGFBP, IGF binding protein; JAK, Janus kinase; KI, knock-in; KO, knockout; MUP, major urinary protein; Stat, signal transducer and activator of transcription; WT, wild type.
Accepted for publication September 8, 2005.
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Davey HW, Wilkins RJ, Waxman DJ 1999 STAT5 signaling in sexually dimorphic gene expression and growth patterns. Am J Hum Genet 65:959–965(Jennifer E. Rowland, Linda M. Kerr, Mary)
Abstract
The GH receptor (GHR) is essential for normal postnatal growth and development, and the molecular basis of GHR action has been studied intensively. Clinical case studies and more recently mouse models have revealed the extensive phenotype of impaired GH action. We recently reported two new mouse models, possessing cytoplasmic truncations at position 569 (plus Y539/545-F) and 391, which were created to identify functional subdomains within the cytoplasmic signaling domain. In the homozygous state, these animals show progressively impaired postnatal growth coupled with complex changes in gene expression. We describe here an extended phenotype analysis encompassing the heterozygote state to identify whether single copies of these mutant receptors bring about partial or dominant-negative phenotypes. It appears that the retention of the ubiquitin-dependent endocytosis motif in the N-terminal cytoplasmic domain permits turnover of these mutant receptors because no dominant-negative phenotype is seen. Nonetheless, we do observe partial impairment of postnatal growth in heterozygotes supporting limited haploinsufficiency. Reproductive function is impaired in these models in a progressive manner, in parallel with loss of signal transducer and activator of transcription-5 activation ability. In summary, we describe a more comprehensive phenotypic analysis of these mouse models, encompassing overall and longitudinal body growth, reproductive function, and hormonal status in both the heterozygote and homozygote state. Our results suggest that patients expressing single copies of similarly mutated GHRs would not display an obvious clinical phenotype.
Introduction
FIRST DESCRIBED BY Laron et al. (1) in 1966, GH insensitivity syndrome (GHIS) is a rare disease characterized by resistance to GH. Although molecular abnormalities have been reported for GH receptor (GHR) signaling pathways, and in the IGF-I gene in GHIS, defects in the GHR account for most of the reported cases to date (2). To date, nearly 50 distinct mutations responsible for GH resistance have been identified including nonsense, missense, splice, or frameshift mutations (3, 4, 5, 6). Although a complete GHR knockout mouse model has been available since 1997 (7), we have more recently described two transgenic lines carrying knock-in (KI) mutations within the GHR cytoplasmic domain (8). These mice, carrying either a truncation at mouse GHR position 569 (with two Y-F mutations at 539 and 545) or a truncation at 391, show a progressive impairment of postnatal growth in proportion to the severity of the truncation. We have shown this (8) to correlate with loss of signaling factor recruitment to the cytoplasmic domain upon GHR activation in the homozygous state, particularly of hepatic signal transducer and activator of transcription (Stat)5 [30% of wild type for 569 mice, 0% for 391 mice], despite normal Janus kinase (JAK)2 activation (8). This study concluded that the distal cytoplasmic domain of the GHR is critical for Stat5 recruitment, and its loss leads to loss of sexual dimorphism in the rodent as shown by changes in liver transcripts for cytochrome P450 enzymes and loss of major urinary proteins (MUPs) (8). However, the membrane proximal sequence is still able to generate 10% of postnatal growth, activate the MAPK pathway, and regulate the majority of GH-responsive genes (8).
Clinically, most GHIS cases are attributable to mutations in the extracellular domain of the GHR, which result in mutations causing protein misfolding and intracellular degradation; therefore, heterozygotes are clinically undetected and merely carry the mutation (9). However, it has been suggested that heterozygous or less deleterious mutations in the cytoplasmic domain of the GHR gene could cause partial GHIS and growth retardation in the heterozygote state if the mutants are able to act as dominant negatives (10). The first case of partial cytoplasmic domain truncation effecting a Laron phenotype was recently described by Milward et al. (11), who reported a homozygous truncation at position 450, resulting in a GHIS phenotype. In vitro coexpression of this mutant GHR with full-length GHR resulted in a dominant-negative phenotype, which impaired full-length GHR signaling (12). This was evident despite the UbiE internalization motif (DSWVEFIELD) remaining N terminal to the truncation site. No clinical heterozygotes have been reported for this truncation mutant, and the parents of these siblings were unavailable for study (11). However, a dominant-negative phenotype is seen for patients expressing heterozygous truncated mutant GHRs, which lack all but five cytoplasmic transmembrane proximal residues when these are coexpressed at the cell surface because they impede full-length receptor dimer formation (13, 14, 15).
Here we aimed to resolve whether less deleterious heterozygous truncations that still possess the internalization motif may have an impact on postnatal growth. Using the models described previously (8), we compared the physiological parameters of homozygous and heterozygote mutants 569 and 391 with reference to the classic Laron mouse model (GHR–/–). Additionally we describe more fully the 569 and 391 mutants that we reported recently (8).
Materials and Methods
Animals
The GHR–/– and truncation mutant lines used in this study have been described previously (7, 8). Animals were housed in an approved facility and treated according to University of Queensland Animal Ethics Committee and the Australian Office of the Gene Technology Regulator guidelines. Water and feed pellets were available ad libitum during a 12-h light, 12-h dark cycle at 20–22 C. All animals passed standard virus screens throughout.
Postnatal growth measurement
Litters from heterozygous crosses were used for this comparative study. Body weight, body length, and tail length measurements were taken at regular intervals from 10 to 60 d postnatally. Comparisons were made between animals of the same sex. Organ weights are expressed as actual values, as percent body weight and percent of WT weight, which expresses disproportionate growth changes as a percentage value (16).
IGF-I and GH measurements
Acid ethanol extracted serum IGF-I was measured from sera samples using an IGF-I RIA kit (Bioclone, Sydney, Australia). Serum GH was quantified with a RIA kit (Linco, St. Charles, MO; RGH-45HK). Transcripts were detected as described in Rowland et al. (8) using rat IGF-I cDNA for exon 3, kindly provided by Dr. Adrian Herington (Queensland University of Technology, Brisbane, Australia).
Major urinary protein
Urine samples were collected before dissection of mice at 42 and 60 d postnatal and frozen at –20 C. For visualization of MUPs, 1 μl of female urine or 0.5 μl of male urine was electrophoresed on 12% SDS-PAGE followed by Coomassie blue staining as described by Norstedt and Palmiter (17). For quantification of urine MUP samples, total protein values were estimated using BCA assay (Sigma Aldrich, St. Louis, MO).
Bone length
Hind legs were routinely dissected and x-rayed. X-ray films were scanned into gray-scale TIFF-formatted files, and morphometric analysis of femoral and tibial dimensions was performed using National Institutes of Health image software for Macintosh.
Statistics
Data are expressed as mean ± SEM throughout. Populations were compared by Tukey-Kramer multiple comparisons ANOVA where applicable. Annotations are made to indicate P values: , P < 0.05; , P < 0.01; , P < 0.001.
Results
Postnatal growth
The mouse models used for this study expressed either 569stop Y539/545-F or 391stop mutant GHRs (8) (Fig. 1). To elucidate the physiological impact of the 569 and 391 mutations in the homozygous and heterozygous state, we measured overall body weight (Fig. 2), nose-anus length (see supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org), and tail length (see supplemental data) for both mutants with comparison with wild-type (WT) and GHR–/– mouse lines. A difference in these measures of growth was not expected before weaning because differences are not seen in the GHR–/– mice (7, 16) or Stat5 knockout mice (18, 19) before weaning. Both mutant 569 and 391 showed growth rates diverging from sex-matched WT controls at approximately 21 d postnatal, which correlated with time of growth rate divergence seen in GHR–/– mice (7, 16). A difference between the sexes for the weights of WT controls was evident from d 20, as expected (16). This gender difference is due to the emerging actions of gonadal steroids, which induce a distinct pattern of GH secretion in males vs. females.
Significant reductions in body weight were seen for heterozygotes and homozygotes with 569 and 391 mice and GHR–/– mice (Fig. 2 and Table 1; see supplemental data). These reductions were significant for both sexes at both 42 and 60 d postpartum (P < 0.001). Ratios of WT to homozygous KI body weights remain relatively unchanged between 42–60 d of age for both males and females, indicating similar reduction in growth rates over this period when compared with WT littermates. Interestingly, the growth rate of the homozygous 569 male was similar to that seen for WT females, suggesting that full Stat5 generation is necessary for normal sexually dimorphic growth. The weights of male and female 391 KI mice was not significantly different, again supporting a lack of sexually dimorphic growth in the absence of GH-dependent Stat5 generation, as seen for GHR–/– mice (Fig. 2 and Table 1) (7, 16). For GHR–/– mice, a significant haploinsufficiency effect was seen in heterozygotes carrying only one copy of GHR as has been previously reported (7, 20). Body weights of GHR+/– mice at 60 d were approximately 85% of WT for males and 93–95% for females.
Organ weights
Organ weights were measured at 42 d (see supplemental data) and 60 d (Table 1) postnatal for 391, 569, and GHR–/– mice for both male and female heterozygotes and homozygotes. Kidney and spleen growth has been reported to be disproportionately reduced in GHR–/– mice, particularly in older animals (16). We also observed a disproportionate reduction in spleen growth for 391 and GHR–/– animals (Table 1; supplemental data). This effect was less clear in 569 mice in which spleen weight was only significantly different in 569 females at 42 d.
At 60 d progressive reduction in organ weights was seen for homozygotes in proportion to severity of mutation, as reported previously (Table 1) (8). Disproportionately reduced bladder size was evident in male homozygotes and 391 and knockout (KO) heterozygotes, together with a disproportionate increase in brain size in homozygotes resulting from decreased body weight. A significant decrease in proportional growth of liver was seen for all homozygous 391 and KO mice and heterozygous 391 and KO mice. Additionally, a proportionately decreased epididymal fat pad weight was seen for both heterozygous 391 and KO male mice.
Longitudinal growth
GHR–/– mouse investigations revealed a dramatic effect on linear bone growth and skeletal development in the absence of GHR function (16, 21), which is in agreement with clinical observations (1, 22). Therefore, the relative changes in femoral and tibial dimensions were investigated for our KI models. Femoral and tibial bones from all 42- and 60-d-old mice studied were x-rayed and scanned into TIFF-formatted picture files, and then lengths and widths were measured using National Institutes of Health Image software. The data comparisons of these measurements are recorded in Table 2 (60 d) and supplemental data (published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org) (42 d).
These data show that whereas homozygote effects are evident for both mice lines with truncated GHR, there is minimal impact on heterozygote bone lengths at these time points. There was no significant difference between males and females for femoral or tibial length at either 42 or 60 d postnatal, in agreement with previous reports (21). At 42 d, femoral length is not significantly reduced in 569 females, compared with WT, but is reduced in 569 KI males (92%), 391 KI males (82%), 391 KI females (80%), GHR–/– males (73%), and GHR–/– females (74%). At 60 d, femoral length is significantly reduced, compared with WT in 569 KI males (92%), 569 KI females (94%), 391 KI males (80%), 391 KI females (78%), GHR–/– males (71%), and GHR–/– females (73%). This suggests an increasing divergence in length due to the impaired growth rate of the KI/KO models, in agreement with the significant size increase observed in femoral length for both male and female WTs from 42 to 60 d of age. Femoral midpoint width was reduced in all homozygotes at 60 d age but not in heterozygotes, except for 391 males.
Tibial lengths are significantly reduced for all homozygous models at both 42 and 60 d of age. At 42 d of age, 569 males (95%), 569 females (94%), 391 males (87%), 391 females (86%), GHR–/– females (78%), and GHR–/– (78%) show progressive reduction in tibial lengths relative to the severity of the mutation/truncation. These differences are almost identical for 60-d comparisons, which agrees with the observation that there is no significant change in relative tibial length for WT males or females between 42 and 60 d of age (Table 2; support data 4). A significant reduction in tibial length was also seen for GHR+/– mice at 60 d (94% males, 96% for females); however, no significant change was found for either of the GHR truncated heterozygotes. Diaphyseal and epiphyseal tibial widths showed small comparative reductions that were most striking in the GHR–/– mice. These differences were not as clear as those measured for bone length but do agree with the observed trend of progressive impairment of bone growth with severity of mutation.
IGF-I and GH
The transcriptional regulation of IGF-I is somewhat divergent between tissues (16), however this study has focused on endocrine IGF-I by measuring liver transcript and serum IGF-I (Fig. 3). Liver is the major endocrine source of IGF-I as demonstrated by liver-specific IGF-I KO mice (lid) mice, which display only 25% of WT serum IGF-I levels (23, 24, 25, 26). The reduced serum IGF-I levels observed in GHR/binding protein KO mice (less than 10% of WT) were in agreement with estimates already reported to be between 7 and 10% of normal (7, 20, 27). Stat5B KO male mice are reported to display between 50 and 70% of WT serum IGF-I levels (18, 19), whereas Stat5A/B KO mice reportedly have 50 and 44% of WT serum IGF-I levels for males and females, respectively (19). The relative serum IGF-I levels observed for 569 mutants (16–21% of WT) and 391 mutants (<10% of WT) were far lower than that reported for any of the Stat5 KO models or lid mice. Additionally, the transcript levels for mutant 391 (9–11% of WT) were in agreement with the serum IGF-I estimates, whereas transcript levels observed for mutant 569 (36–67% of WT for Igf1b and Igf1a transcripts, respectively) were substantially higher than levels of serum IGF-I protein recorded. This may be attributed to the reduction or absence of IGF binding protein (IGFBP)-3 and acid labile subunit (ALS) seen in these transgenics, as reported by Rowland et al. (8). Importantly, significant reductions in both IGF-I transcript and protein were also seen for 569 and 391 heterozygotes with the exception of 391 females, which were reduced but not quite significantly (Fig. 3).
Assay of serum GH in male mice revealed a significant elevation of GH (P < 0.01) only in the case of the 391 mice (WT –9.2 ± 2.0, 569 –8.9 ± 3.3, 391 –28.8 ± 1.7, mean ± SEM, n = 3).
Fertility and sexual dimorphism
GHR deletion has been shown to have an impact on reproductive fertility (7, 28); thus, this was examined in our transgenic models. Heterozygote matings yielded a reduced (non-Mendelian) proportion of KI or KO homozygotes in their progeny in proportion to the severity of the mutation (Fig. 4A). Additionally, when homozygotes were mated, litter sizes were reduced in proportion to severity of receptor truncation (Fig. 4B).
MUP is the most abundant liver mRNA and is easily detectable in male urine, and about a third as much is evident in female urine. MUP levels are decreased in the urine of little (lit/lit) mice (17), Stat5B KO (17), Stat5A/5B KO (19), cytokine-induced SH2-containing protein-overexpressing mice (29), and liver IGF-I KO males but not females (24). In most cases this corresponds with overall growth retardation and/or disruption to the pattern of GH exposure to the tissue. Hence, given the reduced growth rate and serum IGF-I levels observed for both 569 and 391 targeted KI mice, a corresponding reduction in urine MUP protein was expected. We already reported that several MUP transcripts are reduced in 569 and 391 transgenic homozygotes (8). Here we show that also at the protein level, a clear impact on urine MUP levels was observed for males, whereas the female MUP level remained unchanged in all populations (Fig. 4C). It is evident from Coomassie-stained sodium dodecyl sulfate gel electrophoresis of 569 and 391 homozygotes that the MUP proteins constitute the majority of urinary proteins. Although reductions appeared evident for male heterozygotes for all lines studied and for 569 KI, statistically significant reductions in MUP were observed only for homozygous 391 KI and KO mice. (Fig. 4D).
Discussion
Here we have reported an extended postnatal growth phenotype analysis for mice harboring GHRs truncated with different degrees of severity and have compared these with mice lacking GHR. We observe a progressive impairment of postnatal growth relating to the severity of cytoplasmic mutation, which is observed also in the heterozygote state. These truncated receptors are expressed and partially functional (8). In neither case do heterozygotes show a more severe phenotype than that seen for the GHR+/–. Furthermore, no data shown here identify any dominant-negative effects on growth in the heterozygote state when compared with the GHR+/– control, which cannot exert such effects. Thus, our truncation mutants do not display a dominant-negative effect as seen for patients expressing severely truncated mutant GHRs, which are overexpressed at the cell surface, thereby impeding full-length receptor dimer formation (13, 14, 15). Mutants 569 and 391 both retain their membrane proximal receptor internalization motif, which should permit the mutant receptors to turn over similarly to WT GHR, thus explaining the intermediate phenotype.
Interestingly, the IGF-I transcript levels reported for Stat5B KO mice (50% of WT) (30) are similar to that recorded for mutant 569, although serum IGF-I in Stat5B-deleted mice is approximately 3 times higher than that of 569 (30). This may be explained by the reduced level of serum IGFBP-3 observed for the 569 mutant (33–52% of WT) (8). Although the relative levels of IGFBP-3 or ALS have not been reported for the Stat5 KO mouse models, it is possible that this difference relates to a specific reduction in Stat5-independent up-regulation of IGFBP-3 and/or ALS by 569 and 391 mutant GHRs (8).
It is expected that these transgenic mice would display an increased plasma GH due to reduced negative feedback by IGF-I, and this was clearly the case for the more severely truncated homozygous 391 mice. A larger sample size or frequent sampling may reveal an increase in GH secretion in the less debilitated 569 mutants. Elevated plasma GH would be expected to increase the activation of JAK2 and pathways dependent on direct JAK2 action, potentially partially compensating for lack of recruitment of certain signaling factors to the distal cytoplasmic GHR. It is also possible that the heterodimeric receptor subunits are able to phosphorylate and recruit some Stat5 to the full-length GHR half of dimers in vivo, although distinguishing this from remaining full-length homodimer activation in vivo is not possible for heterozygotes.
Longitudinal bone growth is impaired in GHR/BP KO mice due to the lack of both direct and IGF-I driven actions of GH (16). It has recently been shown that this reduction may be largely attributed to reduced autocrine/paracrine stimulation of IGF-I because lid mice do not display large reductions in bone growth (31). Furthermore, bone growth is impaired in Stat5A/B KO mice (femoral length 90% of WT), although this is not as severe as that seen for GHR/BP KO mice (femoral length 75% of WT), suggesting that other GH-responsive factors also contribute to bone growth (21). Thus, it was not unexpected to see a progressive reduction in femoral and tibial lengths in our mutant models corresponding to a progressive decrease in IGF-I transcript expression. The relative femoral lengths of 569, 391, and GHR–/– mice (92–94, 78–80, and 71–73% of WT) and the relative tibial lengths (94–95, 86–87, and 78% of WT) at 60 d postnatal reflect the reduction seen for body weights. However, the much larger reduction in serum IGF-I for the 569 KI does not correlate with the relatively small reduction seen in longitudinal bone growth. As mentioned, the sharp decline in serum IGF-I is likely a result of increased degradation of circulating IGF-I in concordance with reduced ALS and IGFBP-3 levels. This supports the view that a substantial portion of GH-dependent bone growth is mediated via local IGF-I generation. It is also possible that IGF-I-independent GH effects are driving bone growth in the 569 model, i.e. induction of bone morphogenetic proteins (32, 33).
In contrast, the 391 model showed reduced bone lengths on par with those seen for the GHR–/– mouse, indicating that local GH-induced bone growth via both direct and IGF-I mediated actions is absent or not detectable in 391 mice. Taken together it would appear that the majority of GH-driven bone growth is dependent on the 391–569 portion of the growth hormone receptor,
GH impacts reproductive function in rodents. GHR–/– mice exhibit delayed puberty (male and female), prolonged pregnancy (1 d longer), and increased placental size (28, 34). For GHR–/– mice, litter size and weight of newborn pups is reduced, compared with normal mice. (Fig. 4) (28). They are fertile but experience a delayed age of first conception for KO matings, and their litter sizes are approximately 40% that of WT and heterozygotes, partly due to a reduced ovulation rate (7, 28, 35). Greatly decreased levels of IGF-I in GHR–/– females might also diminish the supply of nutrients available to the fetus resulting in fetal growth retardation (28). Our observations that 569 and 391 truncations substantially reduce litter sizes from homozygous matings indicates that GHR signaling in the distal cytoplasmic domain is critical for normal reproductive function in rodents. It is unclear at this time whether reduced litter sizes from homozygous matings are due to impaired male or female fertilities or both (35). It may be possible to resolve distinctive GHR pathways required for reproductive pathways in mice using this model in the future.
The sexually dimorphic expression of MUPs reported here supports our finding that Stat5 is activated submaximally in the native state for mutant 569 (8). We have shown that dimorphic cytochrome P450 gene expression is altered, whereas urine MUP levels were reduced for mutant 569 and absent in mutant 391 (Fig. 4) (8). Stat5B activity has been strongly linked with the sexually dimorphic response of the liver in relation to both MUP and cytochrome P450 expression (18, 36). Hence, it is reasonable to propose that the reduction in MUP for mutant 569 males and females relates simply to submaximal stimulation of Stat5 in vivo. Whether the altered sexually dimorphic response in mutant 569 relates to simply submaximal Stat5 activation or is due to the elevated GH troughs that would be predicted due to impaired IGF-I feedback to the pituitary is uncertain. Indeed, demasculinization of liver enzymes has been reported for lid mice, which display reduced circulating IGF-I levels, which are almost double those reported for either 569 or 391 transgenics (24).
The data presented here represent an extended in vivo phenotype analysis of mouse models with disruptions to the GHR cytoplasmic domain. The relatively minor impairment of body growth observed for heterozygotes bearing the mutations suggests that the predicted 25% full-length receptor homodimers are sufficient for most signaling or that mutant receptor subunits are forming heterodimeric complexes with full-length receptor subunits to form subactive signaling complexes. The heterozygote effect is not as severe as the dominant-negative effect seen for patients expressing truncated mutant GHRs in vivo, which are overexpressed at the cell surface, thereby impeding full-length receptor dimer formation (13, 14, 15). Mutants 569 and 391 both retain their membrane proximal receptor internalization motif, which should permit the mutant receptors to turn over similarly to WT GHR, thus explaining the intermediate phenotype. Therefore, whereas it is likely that patients with subnormal postnatal growth rates may carry heterozygous mutations to the cytoplasmic GHR, this is unlikely to be detected in the general population unless this mutation disables the internalization motif of the GHR.
Acknowledgments
We thank Professor J. J. Kopchick and Dr. K. T. Coshigano for generously supplying us with the GHR–/– mice used in this study. We also thank Paul Addison, Elisabetta d’Aniello, and Terry Daly for excellent technical assistance.
Footnotes
This work was supported by the National Health and Medical Research Council of Australia.
First Published Online September 15, 2005
Abbreviations: ALS, Acid-labile subunit; GHIS, GH insensitivity syndrome; GHR, GH receptor; IGFBP, IGF binding protein; JAK, Janus kinase; KI, knock-in; KO, knockout; MUP, major urinary protein; Stat, signal transducer and activator of transcription; WT, wild type.
Accepted for publication September 8, 2005.
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