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Endocrinology Vol. 138, No. 1 55-61
Copyright © 1997 by The Endocrine Society

ARTICLES

Human Skin Fibroblasts as a Model of Growth Hormone (GH) Action in GH Receptor-Positive Laron’s Syndrome

James S. Freeth1, Ruth M. Ayling2, Andrew J. Whatmore, Paul Towner, D. Anthony Price, Michael R. Norman and Peter E. Clayton

Endocrine Sciences Research Group, Department of Medicine, University of Manchester, Manchester, United Kingdom M13 9PT; the Departments of Clinical Biochemistry (R.M.A.) and Molecular Medicine (P.T.), Kings College School of Medicine and Dentistry, London, United Kingdom SE5 9PJ; and the Department of Medicine, Bristol Royal Infirmary (M.R.N.), Bristol, United Kingdom BS2 8HW

Address all correspondence and requests for reprints to: Dr. P. E. Clayton, Endocrine Sciences Research Group, Department of Medicine, University of Manchester, Stopford Building, Oxford Road, Manchester, United Kingdom M13 9PT. E-mail: peter.clayton{at}man.ac.uk


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Congenital GH insensitivity (Laron’s syndrome, LS) is often associated with a dysfunctional GH receptor (GHR) causing complete insensitivity to GH and absent serum GH-binding protein (GHBP). However, a proportion of children with LS have normal GHBP levels. We have identified four girls from two families with this condition (height SD score, -3.4 to -6.8) and undertaken studies on 1) their GHR genes and 2) their GH responses in cultured skin fibroblasts to define the etiology of their GH insensitivities.

No GHR gene mutations were identified in one family. In the other family, the affected siblings, an unaffected brother, and the father were heterozygous for a point mutation within exon 6 (D152H). In addition, use of intron 9 haplotypes to determine linkage to the GHR gene implied inheritance of different maternal GHR alleles in the two affected girls of the latter family. It is unlikely, therefore, that the D152H mutation alone could account for the LS phenotype.

End points of GH action [DNA synthesis, insulin-like growth factor-binding protein-3 (IGFBP-3) messenger RNA (mRNA) and peptide production] in skin fibroblast cultures established from three of the LS subjects and four normal children were examined. Whereas normal fibroblasts incorporated [3H]thymidine dose dependently in response to 10–1000 ng/ml GH (increment at 1000 ng/ml, 77 ± 19%), LS fibroblasts failed to respond significantly above basal levels (P < 0.01). In normal fibroblasts, IGFBP-3 mRNA and peptide increased maximally at 48 h in response to 200 ng/ml GH, as determined by ribonuclease protection assay, Western ligand blotting, and RIA. In comparison, LS fibroblasts produced significantly less IGFBP-3 peptide than normal fibroblasts in response to GH, whereas IGFBP-3 mRNA failed to increase above basal levels.

These studies have shown that 1) cultured human skin fibroblasts can be used to define the end points of GH action; 2) fibroblast cultures from the LS children show absent or reduced responses to GH; and 3) GH insensitivity in these children does not appear to be caused exclusively by GHR mutations, but is probably due to dysfunctional GHR signalling. Such patients may prove particularly important to elucidation of the key events in GH signaling.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
LARON’S SYNDROME (LS) is a rare, autosomal recessive condition of complete GH insensitivity, and is characterized by extreme short stature with a phenotype similar to that of severe GH deficiency. Although basal concentrations of circulating GH are elevated, levels of its effector hormone, insulin-like growth factor I (IGF-I), and IGF-binding protein-3 (IGFBP-3) are markedly reduced. The molecular defects that account for LS include deletions and point mutations within the region of the GH receptor (GHR) gene encoding the extracellular domain, which disrupt normal GH binding (reviewed in Ref.1). The N-terminal regions of the GHR (extracellular domain) and the serum GH-binding protein (GHBP) share sequence homology (2), and in humans, the GHBP is probably derived by proteolysis of the transmembrane receptor (3). Thus, LS is classically associated with an absence of detectable serum GHBP activity.

However, a proportion of LS patients (25% of a European study) have been identified with normal levels of circulating GHBP (GHBP-positive LS), with similar affinity to GH as that in normal controls (4, 5, 6). Two such unrelated individuals were found to be homozygous for a mutation within exon 6 of the GHR gene, resulting in the substitution of an aspartate residue by histidine at codon 152 (D152H) (7). This mutation abolishes the receptor homodimerization step that is critical for the initiation of GH signal transduction. Two siblings from another family are homozygous for a point mutation in the last nucleotide of exon 8, causing this exon (which encodes the entire transmembrane domain) to be spliced out, resulting in elevated serum GHBP levels (8). As yet, no other GHR gene mutations in GHBP-positive LS have been reported.

The GHR has been detected on a wide variety of human peripheral cell types, including osteoblast-like cells (9), adipocytes (10), and white blood cells (11). Cultured human skin fibroblasts also express GHR messenger RNA (mRNA) and protein, albeit at low levels (12, 13, 14). Furthermore, GH induces a mitogenic response in cultured skin fibroblasts via local production of IGF-I (15). These cells produce IGFBP-3 to -6 (16, 17, 18), of which the concentrations of IGFBP-3 mRNA (19) and peptide (16, 19) increase in response to GH.

We now report four cases of GHBP-positive LS from two unrelated Pakistani families, in whom sequence analysis of the GHR gene has been undertaken. We have also established skin fibroblast cultures from three of the subjects to investigate cellular responses to GH. We propose that the GH insensitivity in these children is probably due to a post-GHR defect(s). Such patients should provide important insights into the mechanisms of normal GH signal transduction in humans.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Patients
The four girls with LS from two unrelated Pakistani families were identified after presentation to the Manchester Growth Clinic for evaluation of short stature. Basal and peak GH concentrations during stimulation tests were markedly elevated (basal, 16–45 mU/liter; peak, 62–120 mU/liter), whereas basal IGF-I and IGFBP-3 concentrations were all below the fifth percentile for age. All children had normal levels of GHBP (17–25% [125I]GH bound/ml serum). The sisters, HI and HII, have a classical LS phenotype and have previously been reported by McGraw et al. (20). HI has achieved final height at 121.4 cm (3 ft, 11.8 in.; height SD score, -6.8), whereas HII has a height SD score of -4.6 at age 13 yr. There is no reported parental consanguinity. The sisters, MI [final height, 138.2 cm (4 ft, 6.4 in.); SD score, -4] and MII (height SD score, -3.4), do not show a classical phenotype and are taller than HI and HII. Consanguinity exists in the parents of MI and MII. In both families, midparental height (target height SD score, -1.5 in family H and -1 in family M) and all sibling heights are within the normal range. The younger affected sibling in each family was evaluated as part of the Kabi Pharmacia International Study of GH Insensitivity Syndrome (patients 24 and 25 in Ref.6).

Cell culture
Fibroblast cultures were established, with approval from the local ethics committee, from skin biopsies taken from healthy children with normal stature and three of the four children with LS (HI, MI, and MII). Cells were maintained in 75-cm2 tissue culture flasks (Costar Corp., Cambridge, MA) in DMEM containing 10% FBS, 1 mM L-glutamine, 50 IU/ml penicillin, and 50 µg/ml streptomycin. Cultures were passaged (1:4 split) upon reaching monolayer confluence by treatment with 1 g/liter trypsin-0.4 g/liter EDTA solution, and the medium was changed every 3 days thereafter.

For experiments with human (h) GH or IGF-I, cells between passages 6 and 11 were seeded at a density of 2.5 x 104 cells/ml into 75-cm2 culture flasks for the purpose of mRNA extraction or into 24-well plates (Falcon, Becton Dickinson Labware, Oxnard, CA) for other experiments. Cells were cultured in DMEM containing 10% FBS for 48 h, followed by serum-free DMEM containing 0.1% BSA for an additional 24 h. Cells were subsequently cultured in DMEM containing 0.1% BSA and 0.01% human hypopituitary serum to provide competence factors (15) with or without hGH (Pharmacia, Stockholm, Sweden) or IGF-I (Boehringer Mannheim, Indianapolis, IN).

Analysis of genomic DNA for GHR
Genomic DNA was isolated from mouthwash samples (21). Individual exons (no. 2–9) of the GHR were amplified by PCR with primers complementary to flanking intronic sequences. Exon 10 was amplified as two overlapping fragments extending from intron 9 into the 3'-untranslated region. All primer sequences are available on request. Amplification of an area of intron 9 containing several polymorphic sites was also performed (22). PCR products were electrophoresed on 1.25% agarose gels to verify fragment size and exclude contamination. Single stranded conformational polymorphism (SSCP) analysis was carried out on 1 µl of each PCR product by mixing with formamide loading buffer, denaturing by heating to 95 C for 5 min, and plunging into a dry ice-ethanol mixture. The sample was electrophoresed overnight on an 8% acrylamide gel containing 5% glycerol at both 4 C and room temperature. The gel was then stained with 0.1% silver nitrate. Manual sequencing of each amplified fragment was also undertaken by the chain termination method, using a Sequenase kit (Amersham International, Aylesbury, UK). To screen for the D152H mutation in exon 6 that destroys the EcoRV restriction site, the amplified exon 6 sequence was incubated with the enzyme EcoRV at 37 C for 1 h, and the products were electrophoresed on a 2% agarose gel.

Analysis of complementary DNA (cDNA) for GHR
Fibroblast cell pellets were lysed with 0.5 ml RNAzol B (Tel-Test, Friendswood, TX), and total cellular RNA was isolated according to the manufacturer’s instructions. Total RNA was quantified by absorbance at 260 nm, and the purity was assessed by the ratio of optical densities at 260 and 280 nm. Preparation of cDNA from fibroblast total RNA (75 µg) was achieved using a Reverse Transcription System (Promega Corp., Madison, WI). Exon 6 of the GHR was amplified from cDNA by 30 cycles of PCR with primers complementary to the flanking exons 5 (5'-CGTTTACCTCCATCTGGATACC-3') and 7 (5'-CGTTGTTTGGATCTCACACGCAC-3'). Each reaction was performed in the presence of 50 µCi [{alpha}-32P]deoxy-CTP (3000 Ci/mmol; Amersham International, Aylesbury, UK). PCR products were incubated with the enzyme EcoRV at 37 C for 1 h before electrophoresis through a nondenaturing 5% polyacrylamide gel. The dried gel was exposed to a phosphorimaging plate for quantitation using a Fuji Bio-imaging analyzer (Fuji Photo Film Co., Ltd. Japan) or Fuji RX x-ray film at -70 C for 2 days. To assess whether GHR transcripts contained or lacked exon 3, the GHR was amplified from cDNA by 35 cycles of PCR using primers complementary to sequences within exons 2 (5'-CCTACAGGTATGGATCTCTGGC-3') and 5 (5'-ATCCACTGTACCACCATTGCT-3'), followed by a further 35 cycles using 1 µl of a 1:1000 dilution of this product and internal primers within exons 2 (5'-CAGCTGCTGTTGACCTTGGC-3') and 5 (5'-ACAGCTGTTTTCCCCAGCAG-3'). PCR products were electrophoresed on a 2% agarose gel.

Cellular responses to GH
[3H]Thymidine incorporation. [Methyl-3H]thymidine (83 Ci/mmol; Amersham International) was added 20 h after treatment with GH or IGF-I to give a final concentration of 0.25 µCi/ml. At 24 h, cells were washed twice with 1 ml PBS and once with 1 ml 10% (wt/vol) trichloroacetic acid. After treatment with an additional 1 ml 10% trichloroacetic acid for 2 h at 4 C, cell lysates were solubilized overnight at 4 C with 0.1 M NaOH. Duplicate aliquots of 100 µl were counted in a Wallac 1219 RackBeta Spectral liquid scintillation counter using Optiphase HiSafe liquid scintillant (Wallac, UK).

Ribonuclease (RNase) protection assay. The plasmid pHBP3-502 containing 475 bp of human IGFBP-3 cDNA was the generous gift of Dr. S. Shimasaki (The Scripps Research Institute, La Jolla, CA). Human glyceraldehyde 3-phosphate dehydrogenase (GAP) cDNA in pBluescript SK- was purchased from American Type Culture Collection (Rockville, MD). The IGFBP-3 riboprobe was prepared using HindIII-digested pHBP3-502, T3 RNA polymerase, and 3.2 µCi/µl [{alpha}-32P]UTP (800 Ci/mmol; DuPont, Wilmington, DE). The GAP riboprobe was prepared using XhoI-digested GAP plasmid, T3 RNA polymerase, and 1.6 µCi/µl [{alpha}-32P]UTP. In vitro transcription was achieved using a RNA transcription kit (Stratagene, La Jolla, CA). Specific activities of riboprobes were assessed by scintillation counting. Each riboprobe (1 x 105 cpm) was simultaneously hybridized with 2 µg total RNA from fibroblast cells in hybridization buffer (80% formamide, 0.04 M piperazine-N-N'-bis[2-ethane sulfonic acid] (PIPES), 1 mM EDTA, and 0.4 M NaCl) overnight at 55 C. The mixture was treated with 0.1 U/µl RNase ONE (Promega Corp., Madison, WI) for 90 min at 35 C, then 10% SDS (0.6% final concentration) was added to inactivate the RNase. The hybrids were ethanol precipitated with 20 µg carrier transfer RNA and washed in 70% ethanol. The dried precipitate was resuspended in loading buffer (80% formamide, 10 mM EDTA, 0.1% bromophenol blue, and 0.1% xylene cyanol), heated to 95 C for 5 min, and electrophoresed through a 5% polyacrylamide-8.3 M urea gel. The dried gels were quantitated by phosphorimaging analysis and exposed to Fuji RX x-ray film at -70 C for 1–3 days.

SDS-PAGE and Western ligand blotting (WLB). Fifty-microliter aliquots of conditioned medium from fibroblast cultures were prepared for electrophoresis by the addition of 50 µl gel loading buffer [0.0125 M Tris-Cl (pH 6.8), 4% SDS, 20% glycerol, and 0.025% bromophenol blue] and boiled for 5 min. Samples were fractionated through nonreducing 10% polyacrylamide gels at 175 V for about 4 h and transferred overnight onto nitrocellulose membranes. IGFBPs were detected based on the method of Hossenlopp et al. (23). Briefly, membranes were blocked in 0.15 M NaCl-1% BSA; incubated with 150,000 cpm/ml [125I]IGF-I for 4 h at 25 C in buffer containing 0.15 M NaCl, 1% BSA, and 0.1% Tween-20; and washed with 0.15 M NaCl. Proteins were visualized by autoradiography (5-day exposure) and quantitated by phoshorimaging analysis (as described above).

IGFBP-3 RIA. Immunoreactive IGFBP-3 levels in the conditioned medium used for WLB were measured by RIA (24). Radioligand was prepared by covalently cross-linking [125I]IGF-I to IGFBP-3 by a modification of the method of Baxter et al. (25). Standard or sample (undiluted conditioned medium) was incubated overnight at 25 C with polyclonal antihuman IGFBP-3 (Celtrix) and [125I]IGF-I/IGFBP-3. Separation of bound [125I]IGF-I/IGFBP-3 was achieved by the addition of antirabbit IgG coupled to cellulose (Sac-Cel, IDS, Tyne and Wear, UK). Pellets were counted on a {gamma}-counter, and results were interpolated from a standard curve. The inter- and intraassay coefficients of variation were 4.2–7.2% and 3.6–5.6%, respectively.

Statistical analysis
The Mann-Whitney U test for nonparametric data was used to compare responses between normal and Laron fibroblast cultures. The means of duplicate or triplicate measures from each technique in 2–10 independent experiments in each of the 3 normal and 3 Laron fibroblast cultures were used for comparison. The Wilcoxon matched pairs signed ranks test was used to compare variables within a group. The Kruskal-Wallis one-way ANOVA was used to test the significance of GH dose responses.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Analysis of the GHR gene
In the affected children (HI, HII, MI, and MII), the coding regions of the GHR gene were screened for mutations by SSCP analysis and direct sequencing. At 4 C, a SSCP band shift was seen in the exon 6 fragment from the two affected sisters (HI and HII), their father, and an unaffected brother (data not shown). No abnormality was detected in any of the other eight coding exons and splice sites in these patients or in any of the coding exons and splice sites of the affected sisters of family M.

A mutation within exon 6 of the GHR gene that has been identified previously in two other GHBP-positive LS patients results in the substitution of aspartate by histidine at position 152 and destroys the EcoRV restriction site (7). Restriction digestion with EcoRV of the exon 6 DNA fragments in family H (Fig. 1AGo) indicated that HI, HII, their father, and one unaffected brother were heterozygous for this restriction site. DNA sequencing confirmed the presence of the D152H mutation on one allele (Fig. 1BGo) and the absence of other mutations in the coding sequence or at the intron/exon boundaries.



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  		Figure 1. A, EcoRV digestion of GHR exon 6 amplified from genomic DNA in family H members (lanes 1–8) and a normal control (lane 9). The arrow indicates undigested exon 6 fragments (184 bp), and the arrowheads indicate digested exon 6 fragments (65 and 119 bp). The affected sisters (closed circles, lanes 1 and 7), the father (lane 6), and a brother (lane 2) show a pattern consistent with the presence of wild-type and mutant alleles. B, Sequence analysis of GHR exon 6 in HI, showing heterozygosity for a G->C transversion within codon 152 (Asp->His). C, EcoRV restriction digestion of GHR exon 6 amplified from fibroblast cDNA in HI (lane 4) and a normal control (N; lane 2). Full-length exon 6 fragments before treatment with EcoRV in HI (lane 3) and N (lane 1) are also shown. The arrow indicates the full-length fragment (355 bp), and the arrowheads indicate the digested fragments (161 and 194 bp).

 
To assess whether there was differential allele expression, cDNA was generated from HI and one normal child (NII), and the fragment including exon 6 was amplified. The enzyme EcoRV fully digested the NII product, but only digested 40% of the product from HI (the 161- and 194-bp products combined for quantitation; Fig. 1CGo), indicating that her normal and mutant (D152H) alleles are expressed at a ratio of approximately 40:60.

cDNA from HI, MI, MII, and two normal children was amplified with primers complementary to sequences within exons 2 and 5 (Fig. 2Go). HI expressed GHR transcripts containing exon 3 only, whereas MI, MII, and one normal individual (NI) expressed exclusively transcripts lacking exon 3. NII expressed both variants.



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  		Figure 2. PCR amplification from fibroblast cDNA using primers complementary to sequences within exons 2 and 5 of the GHR in normal subjects (NI and NII) and LS patients (HI, MI, and MII). The arrow indicates GHR transcripts containing exon 3 (324 bp), and the arrowhead indicates GHR transcripts lacking exon 3 (258 bp).

 
Linkage analysis to the GHR gene using intron 9 polymorphisms in family M was uninformative, because both parents shared identical haplotypes (I/I frameworks; data not shown) (22). The affected sisters, HI and HII, did not share identical haplotypes (HI, I/I framework; HII, I/VI framework; data not shown), suggesting that the disease is not associated exclusively with the GHR.

[3H]Thymidine incorporation (TI)
TI assays were performed over the GH dose range 10–1000 ng/ml in normal and GHBP-positive LS skin fibroblast cultures. A dose response to GH was observed in the normal fibroblast cultures with a mean 77 ± 19% increase over that in untreated cells at 1000 ng/ml GH (Fig. 3AGo). No significant differences were observed among the three normal fibroblast cultures at each GH dose. In contrast, LS fibroblasts failed to incorporate [3H]thymidine significantly above basal levels at all doses of GH. No significant difference in TI between the two affected families was observed (data not shown).



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  		Figure 3. A, [3H]Thymidine incorporation into three normal (n = 19 experiments) and three LS (n = 15 experiments) fibroblast cultures in response to hGH (10–1000 ng/ml), expressed as the percent increase over that in untreated cells (mean ± SEM). *, P < 0.01; **, P < 0.005 (between normal and LS cultures). B, [3H]Thymidine incorporation into four normal (n = 19 experiments) and three LS (n = 13 experiments) fibroblast cultures in response to 10 ng/ml IGF-I. *, P < 0.05 (between normal and LS cultures).

 
Treatment of normal fibroblast cultures with 10 ng/ml IGF-I resulted in an increase in TI of 251 ± 43% over that in untreated cells (P < 0.01; Fig. 3BGo). In contrast, treatment of the three LS cultures with the same dose of IGF-I resulted in an increase in TI by 445 ± 86% over that in untreated cells (P < 0.01): LS fibroblasts responded to 10 ng/ml IGF-I significantly more than normal fibroblasts (P < 0.05).

Expression of IGFBP-3 mRNA
The induction of IGFBP-3 mRNA in fibroblast cultures, treated with or without 200 ng/ml hGH for 8, 24, and 48 h, was assessed. Results obtained with normal fibroblasts are shown in Fig. 4AGo. A significant constitutive increase in IGFBP-3 mRNA levels was observed over 48 h in untreated normal and LS fibroblasts (Fig. 4BGo), suggesting normal IGFBP-3 promoter function in all cells. No significant induction of IGFBP-3 mRNA expression occurred after 8 h of GH treatment in normal fibroblasts (Fig. 4CGo), but an increase was observed at 24 h (23 ± 10% over untreated cells), reaching a maximal induction at 48 h (83 ± 17% over untreated cells; P < 0.02). In contrast, GH failed to induce IGFBP-3 mRNA expression in LS fibroblasts above basal levels at all time points. No differences were observed between the two affected families (data not shown).



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  		Figure 4. A, RNase protection assay showing IGFBP-3 and GAP mRNA in normal fibroblasts at time zero or after treatment with (+) or without (-) 200 ng/ml hGH for 8, 24, and 48 h. Total fibroblast RNA (5 µg) was simultaneously hybridized with riboprobes for IGFBP-3 and GAP. N indicates the negative lane (i.e. no cellular RNA added), and P denotes the probe lane. B, Four separate RNA experiments were carried out in two normal fibroblast cultures and seven experiments in the three LS fibroblast cultures. Bands were quantitated by phosphorimaging analysis. The graph indicates the constitutive change in the ratio of IGFBP-3/GAP mRNA, expressed as the percent increase over time zero, in untreated normal and LS fibroblast cultures (mean ± SEM). *, P < 0.05, denotes a significant increase with time. C, The ratio of IGFBP-3/GAP mRNA in GH-treated cells is expressed as the percent increase over that in untreated cells (mean ± SEM). **, P < 0.01 between normal and LS cultures.

 
IGFBP-3 peptide secretion
As GH failed to stimulate IGFBP-3 mRNA expression in the three LS fibroblast cultures, the effect of GH on IGFBP-3 peptide secretion was examined. Normal and LS cultures were treated with 200 ng/ml hGH for 24 and 48 h, and the IGFBP-3 secreted into the medium was analyzed by WLB and RIA. As measured by WLB, a constitutive increase in IGFBP-3 secretion was observed from 24–48 h in untreated normal (60 ± 23%) and LS (63 ± 12%) fibroblast cultures (Fig. 5Go, A and B). GH further increased IGFBP-3 secretion from normal fibroblasts by 33% at 24 h and by 77% at 48 h. In contrast, the increment in IGFBP-3 release from LS fibroblast cultures induced by GH was lower than that from normal cultures at both 24 and 48 h, although this did not achieve significance (15% and 48%, respectively; Fig. 5BGo).



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  		Figure 5. A, WLB of IGFBPs in normal fibroblast-conditioned medium. Normal fibroblasts were treated with (+) or without (-) 200 ng/ml hGH for 24 or 48 h. Each sample was assayed in duplicate. The IGFBP-3 doublet (38 and 42 kDa) is indicated. P indicates the IGFBP profile in human plasma. B, Four separate WLB experiments were performed on two normal fibroblast cultures and six experiments on the three LS fibroblast cultures. The IGFBP-3 doublets were quantitated by phoshorimaging analysis. The graph indicates the percent increase in IGFBP-3 secretion by cells treated with or without GH over that secreted by untreated cells at 24 h (mean ± SEM). C, IGFBP-3 in the conditioned medium was also measured by RIA, and results were expressed as described in B. *, P < 0.05 between the increments in response to GH in normal and LS cultures.

 
A correlation between values obtained by RIA and quantitation of the IGFBP-3 doublet on WLB was significant (r = 0.614; P < 0.001). As measured by RIA, a constitutive increase in IGFBP-3 secretion was again observed from 24–48 h in untreated normal (68 ± 9%) and LS (80 ± 15%) fibroblast cultures (Fig. 5CGo). GH further increased IGFBP-3 secretion from normal fibroblasts by 22% at 24 h and by 65% at 48 h. GH had a significantly reduced effect, however, in the LS fibroblast cultures at 48 h (N vs. LS, P < 0.05). No significant differences in GH responses were observed between the two affected families or between normal fibroblast cultures.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
We have identified four girls from two families with GHBP-positive LS. No mutations were found within the coding exons of the GHR gene in the two affected siblings (MI and MII) with the less severe phenotype. The two affected siblings, HI and HII, were found to be heterozygous for an exon 6 mutation, resulting in the replacement of a highly conserved aspartate residue with histidine at codon 152 (D152H). A homozygous D152H mutation in two other unrelated children has been shown to cause LS by preventing GHR dimerization (7). The heterozygous parents of these patients were unaffected, implying that the phenotype associated with this mutation is recessive. Although in vitro studies have shown that the D152H mutation prevents the formation of hybrid complexes consisting of wild-type and mutant receptors (7), the unaffected father and a brother in family H are also heterozygous for the D152H mutation, making it unlikely that this mutation alone is sufficient to fully account for the LS phenotype in HI and HII. Compound heterozygosity in HI and HII is unlikely, as SSCP and direct DNA sequencing failed to detect a second exonic mutation in the GHR gene.

Next, we sought to assess whether there was an imbalance in the representation of alleles at the RNA transcript level, such that in HI and HII, expression of the normal allele was reduced, and expression of the mutant allele predominated. This phenomenon could be caused by promoter mutations, splice mutations, or mutations affecting transcript stability. Such analysis has previously been performed using an allele-specific expression assay (26). However, as the D152H mutation destroys an EcoRV restriction site, we were able to employ RT-PCR of exon 6 using skin fibroblast RNA, followed by restriction digestion with EcoRV. As the normal and mutant alleles in subject HI were expressed at a ratio of 40:60, respectively (i.e. approximately 1:1), it is unlikely that this slight imbalance of allele expression could account for the phenotype. We were unable to assess the expression of normal and mutant alleles in the unaffected brother and father because we had not established skin fibroblast cultures from these individuals.

Human tissues express two isoforms of the GHR that either include or exclude exon 3; no functional differences between the two isoforms have been identified (27, 28). The distribution of these isoforms has been attributed to tissue-specific alternative splicing of exon 3 (29, 30). However, it has recently been proposed that the expression of the two isoforms is not tissue specific, but specific for each individual (31). This is consistent with our data. Genomic exon 3 deletions have been found in a number of LS children and their unaffected parents, and it has been suggested that an exon 3 genomic deletion may provide a background for other point mutations or deletions that completely inactivate the GHR (32). None of our four GHBP-positive LS children are homozygous for a genomic exon 3 deletion. In addition, we have shown that fibroblasts from subject HI, who is heterozygous for the D152H mutation, express exclusively GHR transcripts containing exon 3. Conversely, the affected sisters MI and MII expressed only the isoform lacking exon 3, as did one normal individual, but no GHR gene mutations were detected. Thus, the LS phenotype in these children is independent of GHR exon 3 status.

As we have demonstrated that heterozygosity for the D152H mutation in HI and HII is unlikely to account entirely for their LS, and we found no GHR gene mutations in family M, we hypothesize that GH insensitivity in these children could be due to abnormalities within the intracellular GH signaling pathway. Indeed, the intron 9 haplotypes in HI and HII were not identical, implying the inheritance of different maternal GHR alleles. This would suggest that the disease is not linked exclusively to the GHR.

GH resistance has previously been demonstrated in erythroid and lymphoid cell lines derived from two LS patients (33). We wished to investigate cellular responses to GH in skin fibroblast cultures established from HI, MI, MII, and normal controls. Cultured skin fibroblasts are a suitable cell model to use because they have previously been shown to retain their intrinsic characteristics even after they are removed from their in vivo hormonal environment (34). GH receptor mRNA and protein has been identified in normal fibroblasts (12, 13, 14) as well as in fibroblasts derived from some patients with LS (35). We have confirmed the expression of GH receptor mRNA in fibroblasts from HI, MI, and MII by RT-PCR.

We have previously demonstrated, by incubating cells with [125I]hGH with or without a 100-fold excess of unlabeled hGH for 6 h at 4 C, that mouse Swiss 3T3 fibroblasts and primary human fetal fibroblasts specifically bind GH (37 ± 5% and 19 ± 8% of the total GH bound, respectively) (36). Identical experiments performed on the normal and LS fibroblast cultures used in this study revealed no significant difference in specific GH binding between normal and LS fibroblasts; normal fibroblasts (n = 15 experiments) specifically bound 1629 ± 637 cpm [125I]GH (15 ± 4% of the total GH bound), and LS fibroblasts (n = 12 experiments) specifically bound 1870 ± 497 cpm [125I]GH (18 ± 6% of the total GH bound) (our unpublished data). These data confirm that the LS fibroblasts are capable of normal GH binding.

In normal fibroblast cultures, GH (10–1000 ng/ml) stimulated mitogenesis in a dose-dependent manner, as shown by thymidine incorporation, reaching a mean increment of 77% over that in untreated cells. In contrast, LS fibroblast cultures failed to respond with any significant increase over basal levels. Cook et al. (15) previously demonstrated a dose response to GH (10–1000 ng/ml) in a single normal fibroblast culture, achieving a much higher level of thymidine incorporation than that in our normal cultures. This discrepancy could be attributed to a variation in responsiveness to GH between fibroblast cultures.

Our observation that LS fibroblast cultures responded to 10 ng/ml IGF-I significantly better than normal cultures at the level of DNA synthesis is qualitatively consistent with a previous comparison of IGF-I-stimulated thymidine incorporation between two normal and four Ecuadorian GHBP-negative LS fibroblast cultures (37). This may reflect a cellular adaptation to low levels of IGF-I in vivo, such as an increase in the number of cell surface IGF-I receptors, as has been shown on erythrocytes from subjects with LS (38). In addition, these results verify that the LS fibroblast cultures are not generally unresponsive.

The effect of GH on specific gene expression was investigated in normal and LS fibroblasts. IGFBP-3 was examined because its expression is known to be up-regulated by GH in fibroblasts (19), and IGFBP-3 mRNA is relatively abundant in fibroblast cells (>50-fold more abundant than IGF-I mRNA; data not shown). Furthermore, LS is associated with low levels of serum IGFBP-3 concentrations in vivo, with a poor increment in IGFBP-3 after GH administration (39). We have shown that 200 ng/ml GH increased IGFBP-3 mRNA in normal fibroblasts maximally by 87% at 48 h. It is unlikely, however, that intermediate auto/paracrine IGF-I production in these cells could account for the long time lag for maximal induction of IGFBP-3 mRNA, because IGF-I has been shown not to influence IGFBP-3 transcript levels in human fibroblasts (40). A direct role for GH in the regulation of IGFBP-3 mRNA has been demonstrated in other in vitro cell systems (41, 42). Schmid et al. (19) demonstrated that a GH-dependent increase in IGFBP-3 mRNA in human skin fibroblasts is evident even after 96 h, indicating that, once transcribed, IGFBP-3 mRNA is relatively stable. No increase in IGFBP-3 transcripts occurred in response to GH in the LS cultures at 8, 24, or 48 h, confirming GH insensitivity. Maximal induction in IGFBP-3 peptide secretion in response to 200 ng/ml GH was also seen at 48 h in normal fibroblasts, as shown by WLB and RIA. In contrast to the IGFBP-3 mRNA data in the LS cultures, a small stimulation of IGFBP-3 peptide occurred, suggesting that there may be an independent mechanism for posttranscriptional regulation of IGFBP-3. Our finding that LS fibroblasts are less sensitive to GH than normal fibroblasts at the level of IGFBP-3 peptide secretion is consistent with a previous study of normal and GHBP-negative LS fibroblast cultures (37).

The mechanisms of GH signal transduction have been studied extensively in a number of animal and human cell lines, and in rat hepatic tissues in vivo (reviewed in Ref.43), although our understanding remains incomplete. JAK2 (Janus kinase 2) has been identified as the GHR-associated tyrosine kinase that is activated by ligand binding. The STAT (signal transducers and activators of transcription) proteins 1, 3, and 5; insulin receptor substrate-1, SHC proteins, and mitogen-activated protein kinases are then activated by JAK2-mediated phosphorylation events. We have identified four cases of GHBP-positive LS in which analysis of the GHR gene would suggest that GH insensitivity is probably due at least in part to abnormal intracellular GH signaling rather than to a classical GHR-inactivating defect. We are currently investigating GH second messenger activity in our GHBP-positive LS fibroblast cultures, which should determine the level(s) at which GH insensitivity has occurred and provide important insights into the process of normal GH signal transduction in humans.


   Acknowledgments
 
Dr. Mark Thomas (Department of Biological Anthropology, University of Cambridge, Cambridge, UK) kindly provided technical assistance for the EcoRV DNA digestion experiment.


   Footnotes
 
1 Supported by the North West Regional Health Authority UK, Pharmacia and Upjohn UK. Recipient of an ESPE Research Fellowship, sponsored by Novo Nordisk. Back

2 Recipient of a Wellcome Clinical Training Fellowship. Back

Received June 14, 1996.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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