Báo cáo khoa học: Growth hormone/JAK-STAT axis signal-transduction defect A novel treatable cause of growth failure pdf

High concentrations of human growth hormone 1000 ngÆmL1 added to the culture medium induced activation of STAT3 and reduced the levels of p21WAF⁄ CIPI in the fibro-blasts of the four idio

A novel treatable cause of growth failure

Andrea P Rojas-Gil1, Panos G Ziros2, Leonor Diaz2, Dimitris Kletsas3, Efthimia K Basdra4,

Theodore K Alexandrides5, Zvi Zadik6, Stuart J Frank7, Vassiliki Papathanassopoulou1,

Nicholas G Beratis1, Athanasios G Papavassiliou2and Bessie E Spiliotis1

1 Division of Pediatric Endocrinology, Department of Pediatrics, University of Patras School of Medicine, Greece

2 Department of Biochemistry, University of Patras School of Medicine, Greece

3 Institute of Biology, NCSR ‘Demokritos’, Athens, Greece

4 Department of Orthodontics, Aristotle University of Thessaloniki, Greece

5 Division of Endocrinology, Department of Internal Medicine, University of Patras School of Medicine, Greece

6 Division of Pediatric Endocrinology, Department of Pediatrics, Kaplan Medical Center, Rehovot, Israel

7 Department of Medicine, Division of Endocrinology, Diabetes and Metabolism, University of Alabama at Birmingham and Endocrinology Section, Medical Service, Veterans Affairs Medical Center, AL, USA

Idiopathic short stature (ISS) in children is

character-ized by a normal or retarded growth velocity, a height

more than two standard deviations below the mean, a

normal birth weight, an absence of endocrine abnor-malities and no evidence of physical or psychological disease [1] When growth velocity is retarded, however,

Keywords

growth hormone; growth hormone receptor;

insulin-like growth factor; idiopathic short

stature; signal transducer and activator of

transcription-3

Correspondence

B E Spiliotis, Division of Pediatric

Endocrinology, Department of Pediatrics,

University of Patras School of Medicine,

26504 Rio-Patras, Greece

Fax: +30 2610 910869

Tel: +30 2610 999544

E-mail: besspil@endo.gr

A G Papavassiliou, Department of

Biochemistry, School of Medicine,

University of Patras, 26110

Rio-Patras, Greece

Fax: +30 2610 996110

Tel: +30 2610 996144

E-mail: gpapavas@pat.forthnet.gr

(Received 13 March 2006, revised 30 April

2006, accepted 31 May 2006)

doi:10.1111/j.1742-4658.2006.05347.x

Primary cultured fibroblasts of four patients with idiopathic short stature and severe growth delay, which displayed normal growth hormone receptor expression presented a reduced ability for activation of signal transducer and activator of transcription-3 (STAT3) Impaired STAT3 activation was accompanied by cell-cycle arrest at the Go⁄ G1 phase Increased levels of the cyclin-dependent kinase inhibitor, p21WAF⁄ CIPI, and reduced levels of cyclins were also detected in these patients High concentrations of human growth hormone (1000 ngÆmL)1) added to the culture medium induced activation of STAT3 and reduced the levels of p21WAF⁄ CIPI in the fibro-blasts of the four idiopathic short stature children Treatment of these chil-dren with exogenous human growth hormone significantly augmented their growth velocity Overall, our study provides the first evidence linking the idiopathic short stature phenotype with a functional aberration in the growth hormone signal transduction cascade which can be successfully overcome by exposure to high doses of growth hormone

Abbreviations

BrdU, 5-bromo-2¢-deoxyuridine-5¢-monophosphate; CDK, cyclin-dependent kinase; FACS, fluorescence-activated cell sorter; GH, growth hormone; GHIS, growth hormone insensitivity syndrome; GHR, growth hormone receptor; IGF-I, insulin-like growth factor-I; IFN-b,

interferon-b; ISS, idiopathic short stature; JAK, Janus tyrosine kinase; STAT, signal transducer and activator of transcription.

it is important that growth hormone (GH) deficiency

has been completely ruled out before the child is

diag-nosed as having ISS This is because GH is crucial not

only for skeletal growth, but also for the homeostasis

of proteins, lipids and carbohydrates as well as for

water–electrolyte balance, and its deficiency can cause

metabolic problems [2] GH deficiency is diagnosed by

the use of pharmacologic agents that stimulate GH

release from the pituitary gland and by the evaluation

of spontaneous 24-h GH secretion [3,4]

GH insensitivity syndrome (GHIS) is another cause

of significant short stature with retarded growth

velo-city that may be mistakenly diagnosed as ISS In

GHIS, there are normal or elevated GH serum

concen-trations because of a GH receptor (GHR) or

postre-ceptor defect, and exogenous human growth hormone

(hGH) therapy fails to increase the abnormally low

insulin-like growth factor-I (IGF-I) concentrations that

are present Exogenous hGH therapy is also incapable

of increasing the retarded growth velocity of the GHIS

children The majority of patients with GHIS have low

GH-binding protein concentrations due to mutations

or deletions in the GHR gene [5–7], although

muta-tions in this gene can also be seen in GHIS patients

with normal or elevated GH-binding protein levels

[8–14] Recently, a patient with GHIS was found to

have a homozygous missense mutation in the gene for

the signal transducer and activator of transcription

(STAT) 5b [15], which plays a crucial role in the

GH-induced activation of IGF-I [16,17]

The growth-promoting and metabolic actions of GH

are mediated through activation of the GH-signal

transduction pathway When a GH molecule binds

to a dimer of the GHR, it stimulates the

receptor-associated Janus tyrosine kinase-2 (JAK2) [18] JAK2,

in turn, phosphorylates itself and the GHR and

subse-quently STAT1, -3 and -5, which dimerize and

translo-cate to the nucleus to activate the transcription of

target genes [19] STATs play an important role in

regulating cell-cycle progression [20] JAK2 also

phos-phorylates and potentiates the mitogen-activated

pro-tein kinase and phosphatidylinositol-3 kinase cascades,

which together with the STATs mediate the cellular

effects of GH [21]

This study was undertaken to explore the GH

cell-signaling axis in a group of ISS children with severe

growth failure, who had a normal GH response to

pharmacologic stimuli, normal spontaneous 24-h GH

secretion and a normal increase in their abnormally

low serum IGF-I concentrations after hGH

adminis-tration GHIS and bioinactive GH were further

excluded by sequencing the GHR and GH-1 genes

Our analyses suggest a novel molecular defect that

appears to be responsible for these children’s growth failure

Results Sequencing of the GHR and GH-1 genes Mutations of the GHR or GH-1 genes in the heterozy-gous state have been implicated in the pathogenesis of short stature [5,22] To exclude any abnormalities in the GHR or GH-1 genes, the affected children were screened for mutations in the above genes No abnor-mality was detected in any of the 10 exons of the GHR gene or the five exons and splice sites of the GH-1 gene, in any of the patients, by employing gene sequencing

Expression of GHR The fact that no mutations were found in the GHR or GH-1 genes prompted us to investigate the signal-transduction pathway of GH To this end, fibroblast cultures were established from original gingival biop-sies derived from four children with ISS (S) and three control children (C) To exclude any abnormalities of GHR at the transcriptional, post-transcriptional or translational level, we checked the expression of GHR

in terms of mRNA and protein produced

The S cells expressed GHR mRNA at levels similar

to those of the C cells (Fig 1A) Both the C and the S fibroblasts yielded similar amounts of GHR by immu-noprecipitation with an anti-GHR IgG Culturing the fibroblasts with 200 ngÆmL)1 hGH for 5 and 15 min, resulted in a reduction in GHR levels in both the C and the S fibroblasts [23] (Fig 1B)

Induction of the JAK/STAT pathway after cultivation with GH

The next question was whether and to what extent the GH-transduction pathway is functional To answer this, the relative levels of activation (i.e tyrosine phos-phorylation) of JAK2, STAT3 and STAT5 were examined

Activation of JAK2 and STAT5b, as monitored by western immunoblotting of total cell lysates (after culturing the cells in fetal bovine serum-free medium for 24 h and subsequently in medium containing

200 ngÆmL)1 hGH) employing specific anti-(phospho-Tyr) IgG, was similar in the C and S fibroblasts (Fig 2) Normal expression of JAK2 and STAT5 was verified with antibodies specific for the nonphosphoryl-ated form of each protein (data not shown) No defect

in the nonphosphorylated or phosphorylated STAT5b

was found in the S fibroblasts (Fig 2)

In contrast to the normal activation of JAK2 and

STAT5 after the addition of 200 ngÆmL)1 hGH into

the culture medium, hGH-induced Tyr

phosphoryla-tion of STAT3 was either absent or significantly

decreased in the fibroblast cultures derived from the

four S patients, when compared with the C fibroblasts

(Figs 3A and 4) Differences in the expression levels of

the nonphosphorylated STAT3 protein and unequal

loading of the samples were excluded (Fig 3B,C)

STAT3 activation by different concentrations

of hGH

From the clinical data it is evident that the S patients

respond to pharmacological doses of GH and display

increased growth velocities (Table 1) This led us to

use higher doses of GH in the cell cultures of S cells,

and to assess whether these high doses are capable of bypassing the block in STAT3 activation

The C fibroblasts displayed a net increase in STAT3 phosphorylation when hGH was added to the culture medium at 200 and 500 ngÆmL)1, whereas STAT3 phosphorylation was almost completely suppressed at

an hGH concentration of 1000 ngÆmL)1 By contrast, the S fibroblasts exhibited significantly low activation

of STAT3 when hGH was added to the culture med-ium at concentrations up to 500 ngÆmL)1 There was

a moderate increase, however, in the activation of STAT3 in all S fibroblasts after the addition of hGH

at a concentration of 1000 ngÆmL)1(Fig 4)

STAT3 activation by interferon-b

To confirm the specificity of STAT3 malfunctioning we sought to investigate the STAT3 activation in response

to another stimulus, interferon (IFN)-b, which is

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Fig 1 GHR expression in cultured fibroblasts from normal children (C) and children with idiopathic short stature (S) (A) PCR amplification of cDNA derived from four S (S1, S2, S3, S4) and two C (C1, C2) fibroblast cultures (In order to exclude genomic contamination PCR was also performed in the same RNA samples omitting the RT step.) Results are normalized according to the measurement of actin Data are the mean ± SD from three different experiments (B) GHR immunoprecipitation using an anti-GHR IgG in total cell lysates from four S (S1, S2, S3, S4) and three C (C1, C2, C3) fibroblast cultures starved for 24 h and subsequently stimulated with hGH (200 ngÆmL)1) for 0, 5 and

15 min In the histogram, the GHR levels of the nontreated cells normalized according to the measurement of actin are presented Data are the mean ± SD from three different experiments.

known to signal through the same JAK⁄ STAT pathway

as GH [24] STAT3 Tyr phosphorylation was reduced

in the four S fibroblast cultures in comparison with the

C fibroblasts after induction with 100 UÆmL)1 IFN-b

Culturing of the C fibroblasts for 30 min in the pres-ence of a wide range of IFN-b concentrations (0–

1000 UÆmL)1), elicited activation of STAT3 that was evident at 10 UÆmL)1 and progressively increased with

pTyr STAT3 A

B

STAT3

S1 S2 S3 S4 C1 C2 C3

0 30 0 30 0 30 0 30 0 30 0 30 0 30

Fig 3 STAT3 activation in S and C

fibro-blasts after stimulation with or without hGH

(200 ngÆmL)1) for 30 min Tyrosine

phos-phorylation of STAT3 (activation) was

detec-ted using specific anti-pTyr serum (A) To

verify equal loading of the samples, the

membrane was stripped out and reprobed

with an anti-STAT3 serum (B) and

subse-quently with an anti-actin serum (C).

Table 1 Growth velocities (GV) (mean ± SD) and height SDS of the S children with idiopathic short stature before and during hGH therapy.

GV ⁄

Difference from pretreatment values: * p ¼ 0.001, ** p ¼ 0.005.

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Fig 2 Phosphorylation of JAK2 and STAT5b Equal amounts of lysates from fibroblasts derived from four children with ISS (S) and two nor-mal children (C), cultured in the absence or presence of hGH (200 ngÆmL)1) for 30 min, were subjected to western immunoblotting using a p-Tyr-JAK2 or a p-Tyr-STAT5b antibody The same membrane was stripped out and reprobed with an anti-tubulin serum (lower) Activation

of JAK2 and STAT5 is depicted in the histogram Data were normalized according to the measurement of tubulin Data are the mean ± SD from three different experiments.

the increase of the IFN-b dose (Fig 5A,B) In contrast,

STAT3 Tyr phosphorylation was not apparent at lower

doses (0, 0.1, 1 and 10 UÆmL)1) in the S fibroblast

cul-tures in response to IFN-b, whereas it was slightly

detectable in response to IFN-b at doses of 100 and

1000 UÆmL)1(Fig 5A,B)

Sequencing of the STAT3 gene

In order to exclude any defects in the STAT3 gene,

children with aberrant STAT3 phosphorylation were

screened for mutations in the above gene No

abnor-mality was detected in any of the STAT3 coding

regions of the four S children

Effect of GH on p21WAF/CIP1expression

It has been shown that the cyclin-dependent kinase

(CDK) inhibitor p21WAF⁄ CIP1 impairs STAT3

tran-scriptional activation [25] It is also known that

impaired STAT3 activation is associated with elevated

p21 protein levels [26] Bearing that in mind, we

inves-tigated a putative link between the elevated p21 and

the decreased STAT3 activation in the fibroblasts of

the four S patients

Expression of p21 was studied by western blotting

after 6 h starvation and subsequent induction with

200 ngÆmL)1 GH for 24 h The S fibroblasts (S1, S2,

S3 and S4 cultures) displayed p21WAF⁄ CIPI protein expression without GH induction, which was augmen-ted after hGH stimulation (200 ngÆmL)1), compared with C fibroblasts (Fig 6A) Cultivation of the fibro-blasts in the presence of 1000 ngÆmL)1 hGH, which induces STAT3 activation in the four S fibroblasts, reduced the expression level of the p21WAF⁄ CIPIprotein

in the S cells (Fig 6A) Taking into account that the S fibroblasts had high p21WAF⁄ CIPI protein levels, we wanted to investigate whether the over-expression of p21WAF⁄ CIPI protein could induce the same phenotype

in the C fibroblasts To this end, C fibroblasts were transiently transfected with an expression vector bear-ing the p21 gene or an empty vector and were subse-quently stimulated with GH As shown in Fig 6B the over-expression of p21 does not influence the ability of

GH to induce STAT3 activation in the C fibroblasts because no difference was observed between the cells over-expressing the p21 gene (lanes 2 and 4) and those that did not (lanes 1 and 3)

Analysis of cell growth rates Increased amounts of p21 in the quaternary complex with cyclins, CDKs and proliferating cell nuclear anti-gen led to inhibition of DNA synthesis and cell-cycle arrest [27] STATs play an important role in control-ling cell-cycle progression and apoptosis STAT3

pTyr STAT3

ACTIN

200 500 1000 200 500 1000 200 500 1000 200 500 1000 200 500 1000 ng hGH

C S1 S2 S3 S4

Fig 4 Dose-dependent activation of STAT3 in fibroblasts from four S and one C stimulated with increasing doses of hGH (200, 500 and

1000 ngÆmL)1) To verify equal loading of the samples, the membrane was stripped out and reprobed with an anti-actin serum Activation of STAT3 normalized according to the measurement of actin is depicted in the histogram Data are the mean ± SD from three different experiments.

activation prevents apoptosis and promotes

prolifera-tive processes including cellular transformation [20]

The impaired STAT3 activation and the concomitant

increased p21 expression in the S cells prompted us

to investigate the growth and cell-cycle state of the

S cells

The S cells grew at a slower rate compared to the C

cells A large number of senescent cells were present in

the S cultures already after the first passage They were

characterized by an increased cell size and perinuclear

autofluorescent aggregates

The mean growth rate of the fibroblasts of the four

S patients, as reflected by the growth curves of the

cul-tures, was always lower than that of the C fibroblasts

6 days after plating the cells (Fig 7A) Addition of

increasing concentrations of hGH (200–500 ngÆmL)1)

enhanced the growth rate of both the C and S

fibro-blasts Although greatly enhanced by the addition of

hGH in the culture medium, the growth rate of the S fibroblasts always remained lower than that of the C cells (Fig 7B,C) The highest growth rate of the C fibroblasts was achieved with the addition of

200 ngÆmL)1hGH (Fig 7B), whereas the S fibroblasts achieved the highest growth rate with the addition of

500 ngÆmL)1hGH (Fig 7C) It is noteworthy that with the addition of 200 ngÆmL)1 hGH the growth curve plateau was reached after
5 days for both groups of cells, whereas with the addition of 500 ngÆmL)1 this plateau was not reached even after 6 days The addition of 1000 ngÆmL)1 hGH caused a slight decrease in the growth rate of both the C and the S fibroblasts (Fig 7D), whereas the addition of hGH at

5000 ngÆmL)1caused even greater suppression of both the C and the S fibroblasts (Fig 7E)

Cell population doubling time and fold prolifer-ation of the fibroblasts within 48 h were determined

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Fig 5 STAT3 activation after IFN-b stimulation in starved S and C fibroblasts (A) Detection of STAT3 phosphorylation after 30 min stimula-tion with IFN-b (100 UÆmL)1) Activation of STAT3 normalized according to the measurement of tubulin is depicted in the histogram Data are the mean ± SD from three different experiments (B) Detection of STAT3 phosphorylation in C and S fibroblasts stimulated for 30 min with increasing doses of IFN-b (0–1000 UÆmL)1) Activation of STAT3 normalized according to the measurement of tubulin is depicted in the histogram (1–6 are C1 fibroblasts and 7–12 are S2 fibroblasts.) The results are expressed as a relative difference as compared with untreated cells Data are the mean ± SD from three different experiments.

S fibroblasts displayed a longer population doubling

1.5 ± 0.12 h, respectively) They also showed a

lower fold proliferation than C fibroblasts within

the same period (4.2 ± 0.23 and 16.55 ± 0.77 h,

respectively)

S fibroblasts also had a lower bromouridine (BrdU)

incorporation into DNA than C fibroblasts A drastic

inhibition in growth, as reflected by the percentage of

DNA-synthesizing cells, was observed in all the S

cul-tures (Fig 8A) The addition of different doses of

hGH increased the percentage of DNA-synthesizing

cells with the same kinetics in the C and S cells, albeit

the differences between C and S cells were maintained

(Fig 8A) The reduced incorporation of BrdU, which

directly measures S-phase cells, suggests a defect in

the G1⁄ S transition of S fibroblasts The latter was

explored by fluorescence-activated cell sorting (FACS)

analysis

FACS analysis showed that 69 and 93% of the

C and the S fibroblasts, respectively, resided in the

Go⁄ G1 phase of the cell cycle Addition of hGH (200

and 1000 ngÆmL)1) decreased the percentage of S cells

in the Go⁄ G1 phase from 93 to 80 and 72%, respect-ively, whereas the percentage of the C cells was almost the same (from 69 to 76 and 70%, respectively) (Fig 8B)

Low expression of the cell-cycle proteins, cyclin A, cyclin D and CDT1 was found in the S fibroblasts after 6 h of fetal bovine serum starvation and 24-h sti-mulation with hGH (200 ngÆmL)1), when compared to the C cells (Fig 9)

Discussion This is the first identification of a functional defect in the activation of STAT3 in the signal transduction pathway of GH in fibroblasts from children with ISS This defect is associated with a cell-cycle arrest at the

Go⁄ G1phase, which is also reflected by increased levels

of p21WAF⁄ CIPI and reduced expression of cyclin A, cyclin D and CDT1 The defect does not appear to be specific to hGH stimulation, because IFN-b, a cyto-kine that also signals through the same JAK⁄ STAT

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fi-broblasts from children with ISS and defici-ent phosphorylation of STAT3 (S) (A) Expression of p21WAF⁄ CIP1in C (C1, C2) and

S (S1, S2, S3, S4) fibroblasts was monitored

by western immunoblotting of cell lysates, after 6 h of fetal bovine serum starvation and subsequent stimulation with hGH (0,

200 and 1000 ngÆmL)1) for 24 h Data were normalized according to the measurement

of actin The results are expressed as a rel-ative difference as compared with untreated cells Data are the mean ± SD from three different experiments (B) C fibroblasts were transfected with p21 WAF ⁄ CIP1 (lanes 2 and 4)

or with an empty parental plasmid (lanes 1 and 3) After 24 h, the medium was chan-ged and the cells were starved for 24 h and subsequently stimulated with 200 ngÆmL)1 hGH for 30 min STAT3 phosphorylation was monitored by western immunoblotting (first panel) Over-expression of p21 WAF ⁄ CIP1 was confirmed by reprobing the membrane with anti-(p21WAF⁄ CIP1)-specific IgG (second panel), and equal loading was verified by reprobing with an anti-STAT3 IgG.

axis, had similar defective activation of STAT3 in the

cultured fibroblasts of the affected children This result

further strengthens the notion that the observed defect

in these children resides at the postreceptor level

It is noteworthy that the defect in the activation of

STAT3 in cultured fibroblasts from the four ISS

patients was overcome by exposing the cells to high

concentrations of hGH Moreover, administration of

exogenous hGH to the ISS patients during the 5-day

IGF-I generation test as well as during the 4 years of

hGH therapy, increased the low serum IGF-I

concen-trations to normal levels Accordingly, the children’s

growth velocities increased substantially showing

signi-ficant ‘catch-up’ growth during the 4 years of hGH

therapy

The regulation of IGF-I expression by GH has

been documented in many tissues, including

hepato-cytes, chondrohepato-cytes, glioma cells and muscle cells

[28,29], whereas much less is known about the action

of hGH on cultured fibroblasts [30] The role of

STAT5 in IGF-I production is well established,

whereas that of STAT3 in GH-induced IGF-I expres-sion was just recently shown in C2C12 myoblasts, whereby STAT3 is involved in the induction of IGF-I mRNA via GH-ignited JAK3 signaling [28] The

although GH has been reported to evoke Tyr phos-phorylation of JAK1 and JAK3 as well [21,31] A possible explanation for the ability of high concentra-tions of hGH to increase STAT3 phosphorylation in the fibroblasts of the four ISS patients studied, could

be through the induction of alternative pathways engaging JAK1, JAK3 or the epidermal growth factor receptor [21,23,30,31]

Activation of STAT3 has been correlated with posit-ive regulation of cell growth, and is highly augmented

in cancer cells [20,32] STAT3 also plays a pivotal role

in the G1 to S-phase transition, through upregulation

of cyclins A and D and the cyclin-dependent kinase-25 (cdk25), and the concomitant downregulation of the CDK inhibitor p21WAF⁄ CIPI [33] In agreement with previous reports, the prospect that the primary defect

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hGH The growth of cells is during their exponential phase of growth and is expressed as the fold increase from the original number of cells (A) No hGH added, (B) hGH 200 ngÆmL)1, (C) hGH 500 ngÆmL)1, (D) hGH 1000 ngÆmL)1and 5000 ngÆmL)1 Each point is the mean ± SD from three different experiments.

in our patients resides at the level of STAT3 activation

is further supported by the fact that the S fibroblasts

displayed an elevated expression of p21WAF⁄ CIPI and

low levels of cyclins A and D In addition, as expected,

impaired STAT3 activation was accompanied by

cell-cycle arrest Notably, high concentrations of hGH in

the culture medium restored the activation of STAT3

and, at the same time, downregulated the expression

levels of p21WAF⁄ CIPI Because p21WAF⁄ CIPI has been

implicated in exerting a negative control on STAT3 [25], the hypothesis that the STAT3-impaired activa-tion might be a consequence of p21WAF⁄ CIPI upregula-tion was explored by over-expressing p21WAF⁄ CIPI in normal fibroblasts cultivated under high concentrations

of hGH and examining the effect on STAT3 activa-tion The fact that no defect in STAT3 activation after over-expression of p21WAF⁄ CIPI was observed, excludes this possibility

Collectively, our findings suggest a novel defect of impaired activation of STAT3 in cultured fibroblasts

of children with ISS that exhibit severe growth delay The impaired STAT3 activation was corrected in vitro

by culturing the fibroblasts in medium supplemented with high concentrations of hGH Moreover, the growth failure of these children was successfully trea-ted with exogenous hGH We propose that this new clinical entity be named ‘growth hormone transduc-tion defect’ (GHTD) The exact molecular ‘coordi-nates’ of the defect underlying the impaired STAT3 activation in GHTD remain uncertain at this stage Further studies are necessary to shed light on the possible impact of this defect of GH-mediated signal transduction on other GH-driven actions, besides growth, such as its effect on the metabolism of pro-teins, lipids and carbohydrates as well as on the immune system

Experimental procedures Subjects

The study group (S) comprised four prepubertal children (9.4–10.3 years of age), with severe growth failure (height standard deviation scores 2.96 ± 0.30), bone age retardation ()3.4 ± 0.5 years) and abnormally low serum IGF-I concen-trations (60 ± 9 ngÆmL)1) The control group (C) included three prepubertal age-matched children of normal stature All children were recruited from the outpatient clinic of the Pediatric Endocrine Division of the University Hospital of Patras, Greece The S children had normal peak GH concentrations after provocation with the pharmacologic agents clonidine (22.3 ± 4.3 ngÆmL)1) and levo-Dopa (13.7 ± 1.9 ngÆmL)1) They also had normal 24-h sponta-neous GH secretion (mean 24-h GH: 4.0 ± 0.13 ngÆmL)1 and 24-h GH secretion rate: 240 ± 12 lgÆ

L)1Æ24)1h), compared with 54 normal prepubertal control children (mean 24-h GH: 3.97 ± 0.18 ngÆmL)1and 24-h GH secretion rate: 231 ± 15 lgÆL)1Æ24)1h) from the Division of Pediatric Endocrinology, Department of Pediatrics, Kaplan Medical Center, Rehovot, Israel All four ISS children had a normal increase of their abnormally low IGF-I concentra-tions during a 5-day IGF-I generation test (baseline IGF-I:

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Fig 8 (A) DNA synthesis assay of S and C fibroblast Cultures of

the second passage of these cells were labeled by BrdU without

GH and monitored with immunofluorescence (B) BrDU colorimetric

ELISA of S and C fibroblasts induced with various doses of hGH

(200 ngÆmL)1, 500 ngÆmL)1, 1 lgÆmL)1and 5 lgÆmL)1) Values are

the mean of four patients and four controls, standard deviation was

calculated for each time point Each experiment was performed

three times (C) Cell-cycle analysis of four S and four C

fibro-blasts cultured in the absence or presence of hGH (200 and

1000 ngÆmL)1) was performed using FACS Open bars, G 0 ⁄ G 1 ;

black bars, G 2 ⁄ M; grey bars, S phase The values are the means of

each group of samples (S and C) The results are expressed as

mean ± SD from three different experiments.

60 ± 9 ngÆmL)1; peak IGF-I: 280 ± 20 ngÆmL)1),

com-pared with 15 normal prepubertal control children (baseline

IGF-I: 148 ± 5 ngÆmL)1; peak IGF-I: 287 ± 11 ngÆnL)1)

from the Pediatric Endocrine Division of the University

Hospital of Patras, Patras, Greece

Because of the vigorous increase in the low IGF-I

con-centrations during the IGF-I generation test, the S patients

were treated daily with exogenous hGH (0.03 mgÆkg)1Æ

day)1) for 4 years All four S patients showed a significant

increase in their growth velocities and height SDS following

the long-term exogenous hGH therapy (Table 1) and they

maintained their serum IGF-I concentrations at normal

levels over all 4 years of hGH therapy Informed parental

consent and children’s assent were obtained in all cases

The study was approved by the Ethics Committee of the

University Hospital of Patras

Analysis of genomic DNA for GH and GHR

mutations

Genomic DNA was isolated from peripheral blood

leuko-cytes for the analysis of the GH-1 gene and was amplified

using PCR, with three pairs of oligonucleotide primers, according to Takahashi et al [22] GHR individual exons 2–10 were amplified by PCR using primers complementary

to flanking intronic sequences, as described previously [11,12] PCR products were recovered from 1% agarose gel using the Macherey Nagel kit (Macherey-Nagel Inc., Easton, PA), and sequencing was performed by the MWG-Biotech AG sequencing service (Ebersberg, Germany)

Cell cultures

Fibroblast cultures were established from gingival biopsies obtained from the four S and three C children Tissue pieces (0.5–1.0 mm3; 2–3 pieces per dish) were plated onto 60-mm2 culture dishes in Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal bovine serum,

2 mm l-glutamine, 50 IUÆmL)1 penicillin and 50 lgÆmL)1 streptomycin (Gibco, Carlsbad, CA), and incubated at

37
C in 5% CO2 atmosphere Cultures were replenished with fresh medium every 3 days and then subcultivated in

a 1 : 3 split ratio upon reaching confluency by using a trypsin⁄ EDTA solution (Gibco) All experiments were

S 3 S 2 S 1 S 2 C 1

n i c y

N I T C A

S 3 S 2 S 1 S 2 C 1

n i c y

N I T C A

4 S 3 S 2 S 1 S 2 C 1 C 1 T D C N I T C A

Fig 9 Effect of hGH on the expression of

cyclin A, cyclin D and CDT1 in normal

fibro-blasts (C) and fibrofibro-blasts from children with

ISS and deficient phosphorylation of STAT3

(S), as monitored by western

immunoblot-ting of 6 h starved cells and subsequent

stimulated with hGH (200 ngÆmL)1) for 24 h.

Equal loading of the samples was verified

by stripping the membrane and reprobing

with a specific anti-actin serum Data were

normalized according to the measurement

of actin Data are the mean ± SD from three

different experiments.