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Insulin-Like Growth Factors (IGF-I and IGF-II) and IGF-Binding Protein-3 Production by Fibroblasts of Patients with Turner’s Syndrome in Culture¹

Department of Endocrinology and Metabolism (A.B., G.D., M.A., P.P., A.C., G.G., F.M.), University of Genova, Genova; and the Departments of Pediatrics (D.L., F.S.) and Biology (F.L.), University of Pavia, Pavia, Italy

Address all correspondence and requests for reprints to: Dr. A. Barreca, Department of Endocrinology and Metabolism, University of Genova, Genova, Italy.

	Abstract

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Reports indicate that in plasma insulin-like growth factor I (IGF-I) and IGF-binding protein-3 (IGFBP-3) are normal in patients with Turner’s syndrome (TS). The aim of our study was to evaluate both the spontaneous and the stimulated synthesis of these peptides by mesenchymal cells obtained from skin biopsies of patients affected with TS.

We compared the ability of fibroblasts from six TS patients with that of fibroblasts from six age-matched control (C) subjects to synthesize in vitro IGF-I, IGF-II, and IGFBP-3 under basal and GH-, estradiol (E₂)-, or GH- plus E₂-stimulated conditions. Furthermore, we evaluated IGF-I, IGF-II, and IGFBP-3 messenger ribonucleic acid (mRNA) expression in fibroblasts from TS and C subjects.

Fibroblasts obtained from TS patients release into the medium significantly lower amounts of IGF-I and IGF-II than C fibroblasts (P = 0.0435 and 0.0318, respectively). In TS fibroblasts, GH and E₂ are able to induce a similar increase, although not significant, of IGF-I secretion into the medium (163 ± 75% and 112 ± 41% of control values). On the contrary, in C fibroblasts, GH is more effective (275 ± 61%; P = 0.0277) than E₂ (75 ± 46%). In both cell lines, GH and E₂ do not significantly modify IGF-II release. Interestingly, the medium conditioned by fibroblasts from TS contains, under basal conditions, significantly higher amounts (273 ± 79 ng/1 x 10⁶ cells) of IGFBP-3 than that from control fibroblasts (67 ± 19 ng/1 x 10⁶ cells; P = 0.0191). GH exerts a stimulatory effect, although it is not statistically significant, on IGFBP-3 secretion, particularly in control fibroblasts. By contrast, the effect of E₂ is inhibitory in all TS fibroblast cell lines, although it does not reach statistical significance (P = 0.067). In agreement with these data, a reduced mRNA expression of the genes encoding for IGF peptides was evident in TS fibroblasts, whereas no significant difference could be demonstrated for IGFBP-3 mRNA.

The results suggest a reduced autocrine/paracrine action of IGFs in TS and indicate that skin fibroblast cultures can give information on the local responsiveness to the treatment.

	Introduction

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SHORT STATURE is a prominent feature of patients with Turner’s syndrome (TS) and its variants. Analysis of spontaneous growth in patients with TS shows that their reduced adult stature is the result of intrauterine growth retardation, impaired prepubertal growth, and lack of a pubertal growth spurt (1). The pathogenesis of growth failure is not yet clear, and several disorders, such as the complete or partial absence of one of the two X chromosomes, abnormal hormonal regulation, and altered end-organ sensitivity, have been considered as possible contributing factors. The correlation of clinical features with cytogenetic abnormalities for individuals showing deletions of the X short arm or the X long arm indicates the presence of gonadal and statural determinants on the X chromosome, although their locations are still uncertain (2). Moreover, it has been shown that 45,X or X deletion cell lines have a prolonged cell generation time, which could be responsible not only for embryonic lethality and intrauterine growth retardation, but also for somatic anomalies and short stature (3).

To answer the question of whether an impairment of the GH axis might play a role in growth failure, spontaneous GH secretion, the GH response to provocative stimuli, and insulin-like growth factor I (IGF-I) concentrations in TS have been studied. Although the preponderance of evidence states that the short stature of TS cannot be ascribed to deficient GH secretion, conflicting results are reported regarding spontaneous GH secretion and the GH response to secretagogues (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14). Some investigators showed that GH secretion is normal until the age of 9 yr (4), whereas in older girls with TS it is lower compared with that of girls in puberty, probably due to a lack of endogenous estrogen (7). Other studies demonstrated that TS individuals have a lower GH secretion compared with that of normal growing prepubertal girls, and the absence of ovarian steroids cannot be accounted for by the reduced GH secretion during infancy (8). Analysis of pulsatile GH release over 24 h indicates that TS patients have a decreased number and frequency of peaks during the night compared with short children, suggesting an altered hypothalamic function (9). GH responses to secretagogues have been reported to be normal (11, 12) or decreased (4, 13). A reduced pituitary GH reserve is supposed to contribute to growth impairment in TS. There is no evidence that girls with TS produce an abnormal GH molecule. No significant difference in the levels of serum GH-binding protein, which corresponds to the extracellular domain of the hormone receptor (15, 16), was found in TS patients compared with normal individuals (14). Plasma IGF-I levels were reported to be normal in the prepubertal age range, but the puberty-associated IGF-I increase seems to be missing (5, 6).

As most of the previous studies and our own experience (unpublished results) demonstrated normal GH and IGF-I levels in TS, and no peripheral resistance to IGF-I from cultured TS fibroblasts could be found (17), we evaluated the autocrine/paracrine pattern of IGF-I, IGF-II, and IGF-binding protein-3 (IGFBP-3) in TS. For this reason we studied the ability of fibroblasts obtained from six TS patients compared with that of fibroblasts from six age-matched control (C) subjects to synthesize in vitro all of these parameters, under basal as well as GH-, estradiol (E₂)-, or GH- plus E₂-stimulated conditions. Furthermore, IGF-I, IGF-II, and IGFBP-3 messenger ribonucleic acid (mRNA) expression in fibroblasts from TS or C subjects was evaluated.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents

BSA and ethinyl estradiol were purchased from Sigma Chemical Co. (St. Louis, MO); Sephadex G-50 and the chromatographic column were purchased from Pharmacia (Uppsala, Sweden). [¹²⁵I]IGF-II (2000 Ci/mmol) was purchased from Amersham (Aylesbury, UK). DMEM was purchased from Irvine Scientific (Santa Ana, CA). Nonessential amino acids, FCS, and trypsin-ethylenediamine tetraacetate were purchased from Flow (Irvine, Scotland). Flasks (75 cm²) were purchased from PBI International (Milan, Italy). Glutamine and Pen-Strepto-Amphotericin B solution were provided by ICN Biomedicals (Costa Mesa, CA). Spectra-Por membrane (mol wt cut-off, 1000) was purchased from Spectrum (Houston, TX). GH for in vitro studies was provided by the National Pituitary Agency (NIDDK).

Human IGF-I and IGF-II complementary DNA (cDNA) probes (18, 19) were provided by Dr. Martin Jansen (University of Utrecht, Utrecht, The Netherlands). Human IGFBP-3 cDNA probe (20) was provided by Dr. Holger Luthman (Karolinska Institute, Stockholm, Sweden). ß-Actin was a 1.8-kilobase HindIII cDNA fragment inserted in pEMBL8 vector.

Cell culture

Human fibroblasts were obtained by punch biopsy of the forearm in six patients affected with TS (TS fibroblasts, Table 1) and from residual fragments of skin obtained during surgical treatment of the forearm in six age-matched normal subjects (C fibroblasts, Table 1).

In a preliminary study of two TS fibroblast cell lines, 5 x 10⁵ cells were exposed to increasing concentrations of GH (0, 5, 10, 25, 50, and 100 ng/mL medium) and E₂ (0, 5, 10, 25, 50, and 100 pg/mL medium) or to the combination of increasing concentrations of GH plus 25 pg/mL E₂. On the basis of the results obtained (Fig. 1), other experiments were performed using 1 x 10⁶ cells and 10 ng/mL GH, 25 pg/mL E₂, or GH plus E₂ combined.

View larger version (21K): [in this window] [in a new window]   Figure 1. Evaluation of the in vitro effect of GH, E2, or GH plus E2 on IGF-I release by two TS fibroblast cell lines (subjects 2 and 6 in Table 1). TS cells were plated (5 x 105cells/flask) in 75-cm2 flasks and, after 24 h of culture, were washed three times in PBS and placed in serum-free medium containing 0.1% BSA. After an additional 24 h, cells were exposed to fresh medium with or without increasing concentrations of GH (0, 5, 10, 25, 50, and 100 ng/mL medium) and E2 (0, 5, 10, 25, 50, and 100 pg/mL medium) or to the combination of a fixed dose of E2 (25 pg/mL, also at 0 GH concentration) plus increasing concentrations of GH; 48 h later, the conditioned medium was collected and processed as detailed in Materials and Methods. Results are expressed as the percent change () from the values obtained under unstimulated conditions (4.75 ng/5 x 105 cells for the cell line shown in the left panel and 7.88 for that shown in the right panel).

The media conditioned by fibroblasts obtained from TS or C subjects were acidified by 18-h dialysis at 4 C against 1 mol/L acetic acid, lyophilized, and resuspended with 0.1 mol/L acetic acid-0.15 mol/L NaCl, (pH of the mixture <3). IGF-I and IGF-II were separated from IGFBPs by gel filtration on a Sephadex G-50 column (1.6 x 90 cm) equilibrated with 0.1 mol/L acetic acid-0.15 mol/L NaCl, pH 2.75. Fractions corresponding to 0–0.20 (corresponding to the IGFBP elution volume), 0.21–0.40, 0.41–0.70 (corresponding to the free IGF elution volume), and 0.71–1 K_av were pooled, lyophilized, reconstituted in PBS, and analyzed for IGF-I, IGF-II, and IGFBP-3 immunoreactivity.

Assay methods

IGFs were measured by RIA using an antibody and [¹²⁵I]IGF-I provided by Medgenix (Fleurus, Belgium) for IGF-I, and a monoclonal antibody provided by Sera-Lab (Technogenetics, Trezzano, Italy) and [¹²⁵I]IGF-II for IGF-II; the standard curves were performed using recombinant IGF-I and IGF-II. The sensitivity of the assay is 90 pg/mL for IGF-I and IGF-II, and the between-assay coefficients of variation are 7.5% and 9%, respectively. No cross-reactivity was observed between IGF-I and IGF-II with the respective antibodies used in the assays up to concentrations of 500 ng/mL of both peptides. The IGF-I and IGF-II contents of media were evaluated in the fractions eluted from the Sephadex G-50 column in the mol wt range of the free peptide.

IGFBP-3 was measured by immunoassay, using reagents and tracer provided by Diagnostic Systems Laboratories (Webster, TX). The IGFBP-3 contents of conditioned media were evaluated in the fractions eluted from the Sephadex G-50 column in the molecular mass range of more than 30 kDa.

RNA isolation and Northern blot analysis

Total RNA was prepared from fibroblasts (20 x 10⁶ cells from each subject) obtained from four C subjects (no. 1–4 in Table 1) and three TS patients (no. 2–4 in Table 1), using the guanidine thiocyanate-cesium chloride method, as modified by Lund et al. (21). Northern blot analysis was also performed on pooled RNA (3 x 10⁶ cells from each subject) obtained from four C fibroblasts (no. 1–4 in Table 1) or from five TS fibroblasts (no. 1 to 5 in Table 1). Twenty micrograms of total RNA were denatured in glyoxal and dimethylsulfoxide, subjected to electrophoresis on 1% agarose gel, and transferred to nylon membranes (GeneScreen, New England Nuclear, Boston, MA) by capillary blotting. Polyadenylated RNA from fetal and adult rat liver was isolated by oligo(deoxythymidine)-cellulose affinity chromatography and served as a control. Human IGF-I, IGF-II IGFBP-3, and ß-actin cDNA probes were labeled by random priming, using [³²P]deoxy-CTP (SA, >3000 Ci/mmol; Amersham). Hybridization was carried out for 48 h at 42 C in a solution containing 50% deionized formamide, 6 x SSC, 50 mmol/L Tris-HCl (pH 7), 5 x Denhardt’s solution, 0.1% SDS, and 100 µg/mL sonicated denatured salmon sperm DNA (20 x SSC = 3 mol/L NaCl and 0.3 mol/L sodium citrate; 10 x Denhardt’s solution = 0.2% each of BSA, Ficoll, and polyvinylpyrrolidone). After hybridization, blots were washed six times in 2 x SSC with 1% SDS at 65 C and twice in 0.1 x SSC at 60 C. Filters were then air-dried and exposed to x-ray film (XAR-5, Eastman Kodak, Rochester, NY) at -80 C using intensifying screens. The integrity of each RNA sample was verified by monitoring 28S and 18S ribosomal RNA in ethidium bromide-stained parallel gels. Hybridization of the same filters with ß-actin was performed as a control to estimate the amounts of RNA loaded in the various lanes.

Statistical analysis

Statistical analysis was performed by nonparametric test on paired (Wilcoxon signed rank test) and nonpaired (Mann-Whitney test) observations. P < 0.05 was considered significant. Results are expressed as the mean ± SEM.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
No significant differences in cell number of C and TS cell lines were observed at the end of 72 h of incubation in serum-free medium with or without the test substances.

In the preliminary study of two TS fibroblast cell lines (IGF-I concentration in the unstimulated condition, 4.75 ng/5 x 10⁵ cells and 7.88, respectively), the addition of increasing concentrations of GH caused a rise of IGF-I in the conditioned medium, beginning at a concentration of 10 ng/mL. E₂ also resulted in an increased production of IGF-I, with a maximal effect at 25 pg/mL E₂ (concentration corresponding to the free form present in the ovulatory phase of a normally ovulating woman and of a subject receiving high dose exogenous E replacement). The combination of increasing doses of GH with 25 pg/mL E₂ (present also in the point at 0 ng/mL GH) was additive only in the fibroblasts from one patient (Fig. 1).

Fibroblasts from TS patients released less IGF-I (9.13 ± 2.21 ng/1 x 10⁶ cells) into the medium than fibroblasts from C subjects (20.23 ± 3.46 ng/1 x 10⁶ cells; P = 0.0435). In agreement with this result is the finding that Northern blot analysis showed lower expression of IGF-I mRNA in fibroblasts obtained from patients with TS than in fibroblasts from C subjects (Figs. 2 and 5). In TS fibroblasts, GH and E₂ were able to cause a similar increase, although not significant, of IGF-I secretion into the medium (163 ± 75% and 112 ± 41% of control values). On the contrary, in C fibroblasts, GH was more effective (275 ± 61%; P = 0.0277) than E₂ (75 ± 46%). In both TS and C fibroblasts, the effect of the combination of the two hormones was not significantly different from the effect of GH or E₂ alone (Fig. 2).

View larger version (17K): [in this window] [in a new window]   Figure 2. Left side, Northern blot analysis of pooled RNA extracted from fibroblasts (3 x 106 cells from each subject) obtained from four C subjects (no. 1–4 in Table 1) and five TS patients (no. 1–5 in Table 1); the autoradiograms were obtained after hybridization with IGF-I cDNA probe. Right side, Evaluation of the in vitro effect of GH (10 ng/mL of medium), E2 (25 pg/mL), or GH plus E2 on IGF-I release by fibroblasts in culture. Human C and TS cells were plated (1 x 106 cells/flask) in 75-cm2 flasks and, after 24 h of culture, were washed three times in PBS and placed in serum-free medium containing 0.1% BSA. After an additional 24 h, the medium was replaced by fresh medium with or without the test substances, and 48 h later, the conditioned medium was collected and processed as detailed in Materials and Methods.

View larger version (33K): [in this window] [in a new window]   Figure 5. Northern blot analysis of total RNA extracted from fibroblasts (20 x 106 cells from each subject) obtained from four C subjects (no. 1–4 in Table 1) and three TS patients (no. 2–4 in Table 1); the autoradiograms were obtained after hybridization with IGF-I, IGF-II, and IGFBP-3. Hybridization of each filter with ß-actin was performed as a control so as to estimate the amounts of RNA loaded in the various lanes (the figure shows a representative autoradiogram performed as a control for the IGF-II filter).

View larger version (17K): [in this window] [in a new window]   Figure 3. Left side, Northern blot analysis of pooled RNA extracted from fibroblasts (3 x 106 cells from each subject) obtained from four C subjects (no. 1–4 in Table 1) and five TS patients (no. 1–5 in Table 1); the autoradiograms were obtained after hybridization with IGF-II cDNA probe. Right side, Evaluation of the in vitro effect of GH (10 ng/mL medium), E2 (25 pg/mL), or GH plus E2 on IGF-II release by fibroblasts in culture. Human C and TS cells were plated (1 x 106 cells/flask) in 75-cm2 flasks and, after 24 h of culture, were washed three times in PBS and placed in serum-free medium containing 0.1% BSA. After an additional 24 h, the medium was replaced by fresh medium with or without the test substances, and 48 h later, the conditioned medium was collected and processed as detailed in Materials and Methods.

View larger version (17K): [in this window] [in a new window]   Figure 4. Left side, Northern blot analysis of pooled RNA extracted from fibroblasts (3 x 106 cells from each subject) obtained from four C subjects (no. 1–4 in Table 1) and five TS patients (no. 1–5 in Table 1); the autoradiograms were obtained after hybridization with IGFBP-3 cDNA probe. Right side, Evaluation of the in vitro effect of GH (10 ng/mL of medium), E2 (25 pg/mL), or GH plus E2 on IGFBP-3 release by fibroblasts in culture. Human C and TS cells were plated (1 x 106 cells/flask) in 75-cm2 flasks and, after 24 h of culture, were washed three times in PBS and placed in serum-free medium containing 0.1% BSA. After an additional 24 h, the medium was replaced by fresh medium with or without the test substances, and 48 h later, the conditioned medium was collected and processed as detailed in Materials and Methods.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

As exogenous GH does promote growth, a peripheral resistance secondary to an abnormality in IGF-I binding or IGF-I stimulation of cell replication has been suggested (14). However, data obtained by Rosenfeld et al. (17) indicate that TS fibroblasts have normal IGF-I receptors and respond appropriately to IGF-I stimulation of DNA synthesis and cell replication. On the other hand, all data available for IGFs and IGFBP-3 in TS are related only to their circulating concentrations, which mainly reflect liver production (26, 27) and involve the classical endocrine mechanism of action. The fact that IGFs and IGFBPs can also act as paracrine/autocrine growth factors (28, 29) induced us to evaluate the ability of cells of mesenchymal origin obtained from patients with TS to synthesize IGF-I, IGF-II, and IGFBP-3 under basal and stimulated conditions.

Northern blot analysis showed a lower expression of both IGF mRNAs in fibroblasts obtained from patients with TS than that in fibroblasts from C subjects. In agreement with these results is the finding that TS fibroblasts release into the medium lower amounts of IGF-I and IGF-II than fibroblasts from C subjects. Therefore, despite normal IGF plasma levels, in TS cells there is less IGF paracrine and autocrine action, which is responsible for a slower replication time, as demonstrated in other studies (3). Our study of fibroblast cell lines demonstrated an increase in IGF-I production in both normal and TS cells treated with GH; this result is in agreement with the clinical finding of an increase in growth velocity in TS subjects receiving GH therapy.

Several groups reported that treatment with low doses of estrogen has a limited beneficial effect even when used in association with GH, although it may induce breast development at an early age and accelerate bone maturation (24, 30). For these reasons, estrogen therapy, which is given to induce puberty in TS girls, must not begin until a bone age of at least 11 yr is reached. Our in vitro studies reveal that E₂ causes an increase in IGF-I production in TS fibroblasts, whereas the combination of GH and E₂ is not effective generally; however, when evaluating the responsiveness of single cell lines, an additive effect was observed in one patient. This finding indicates that skin fibroblast culture can provide information concerning local responsiveness to the treatment.

The finding that IGFs associate with specific binding proteins that modulate their peripheral effects prompted us to analyze IGFBP-3 behavior, as IGFBP-3 is one of the binding proteins produced and secreted by human fibroblasts (31, 32). IGFBP-3 has proven to be able to reduce the IGF bioavailability and inhibit IGF metabolic and mitogenic effects by competing for binding with their specific receptors (31). Northern blot analysis showed no significant differences in the expression of IGFBP-3 mRNA in fibroblasts obtained from TS patients compared with cells from C subjects. However, the medium conditioned by fibroblasts from TS patients contained more IGFBP-3 than that from C fibroblasts. As it has been demonstrated that human dermal fibroblasts produce and secrete into the conditioned medium metallo-proteinases to degrade IGFBP-3 (33), thus enhancing IGF bioavailability, an explanation for the discrepant data obtained by the mRNA analysis and the IGFBP-3 content of conditioned medium could reside in a reduced ability of TS fibroblasts to produce and/or activate these enzymes. Alternatively, the lack of an inhibitory modulation of the processes downstream from the IGFBP-3 RNA translation in TS fibroblasts can be hypothesized. As TS fibroblasts secrete into the conditioned medium higher amounts of IGFBP-3, which can further reduce the IGF bioavailability by competing for binding with their specific receptor, the reduction of IGFBP-3 induced by E₂ in all TS fibroblasts in culture, although not statistically significant, may have clinical relevance.

In conclusion, our results show that TS fibroblasts release into the conditioned medium lower concentrations of IGF-I and -II, but higher amounts of IGFBP-3 compared with C fibroblasts. These results suggest a reduced autocrine/paracrine action of IGFs in TS.

	Acknowledgments

 
The authors are indebted to Dr. Martin Jansen (University of Utrecht, Utrecht, The Netherlands) for providing human IGF-I and IGF-II cDNA probes, to Dr. Holger Luthman (Karolinska Institute, Stockholm, Sweden) for providing human IGFBP-3 cDNA probe, and to Dr. Louis E. Underwood (University of North Carolina, Chapel Hill, NC) for critical review of the manuscript.

	Footnotes

 
¹ This work was supported by research grants from MURST (40% and 60%).

Received March 1, 1996.

Revised December 2, 1996.

Accepted January 10, 1997.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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