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

Growth hormone/JAK-STAT axis signal-transduction
defect
A novel treatable cause of growth failure
Andrea P. Rojas-Gil
1
, Panos G. Ziros
2
, Leonor Diaz
2
, Dimitris Kletsas
3
, Efthimia K. Basdra
4
,
Theodore K. Alexandrides
5
, Zvi Zadik
6
, Stuart J. Frank
7
, Vassiliki Papathanassopoulou
1
,
Nicholas G. Beratis
1
, Athanasios G. Papavassiliou
2
and Bessie E. Spiliotis
1
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:
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:
(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 G
o
⁄ G
1
phase. Increased levels of
the cyclin-dependent kinase inhibitor, p21
WAF ⁄ 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 p21
WAF ⁄ 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.
3454 FEBS Journal 273 (2006) 3454–3466 ª 2006 The Authors Journal compilation ª 2006 FEBS
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
A. P. Rojas-Gil et al. GH ⁄ JAK-STAT axis signal-transduction defect
FEBS Journal 273 (2006) 3454–3466 ª 2006 The Authors Journal compilation ª 2006 FEBS 3455
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 ngÆ mL
)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
A
oNHGh
tsalborbiF
1S2S3S4S1C2C
T
R
RHG
on-RT
NITCA
B
HG
R
(D
K
61
1
)
N
IT
CA
1C
2C3C
055105510 51 5
b
orbiFtsal
H

G
h
mni
1
S2
S
4
S








3
S
055105510 51 50
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HGR
(DK611)
N
ITCA
HGhmni
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.
GH ⁄ JAK-STAT axis signal-transduction defect A. P. Rojas-Gil et al.
3456 FEBS Journal 273 (2006) 3454–3466 ª 2006 The Authors Journal compilation ª 2006 FEBS
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
C
ACTIN
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 ⁄
SDS n Before 1st Year 2nd Year 3rd Year 4th Year
GV (cmÆ year
)1
) 4 2.3 ± 0.6 9.5 ± 0.2* 8.8 ± 1.0* 6.5 ± 0.7** 5.8 ± 0.3**
SDS 4 3.1 ± 0.3 2.1 ± 0.3* 1.7 ± 0.2* 1.6 ± 0.1* 1.4 ± 0.2*
Difference from pretreatment values: * p ¼ 0.001, ** p ¼ 0.005.
2KAJp
5TATSp
NILUBUT
030030030030030030
2C1C1S2S4S3S
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.
A. P. Rojas-Gil et al. GH ⁄ JAK-STAT axis signal-transduction defect

FEBS Journal 273 (2006) 3454–3466 ª 2006 The Authors Journal compilation ª 2006 FEBS 3457
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 p21
WAF/CIP1
expression
It has been shown that the cyclin-dependent kinase
(CDK) inhibitor p21
WAF ⁄ 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 p21
WAF ⁄ 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 p21
WAF ⁄ CIPI
protein
in the S cells (Fig. 6A). Taking into account that the S
fibroblasts had high p21
WAF ⁄ CIPI
protein levels, we
wanted to investigate whether the over-expression of
p21
WAF ⁄ 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.
GH ⁄ JAK-STAT axis signal-transduction defect A. P. Rojas-Gil et al.
3458 FEBS Journal 273 (2006) 3454–3466 ª 2006 The Authors Journal compilation ª 2006 FEBS

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

hGH (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
)1
caused 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.
-FNI
lm/U 001 β
β
3S 2S 1S 1C
2
C4S
ry
T
p3TA
T

S
N
I
LUBU
T
1C
A
B
2S
-FNI lm/U
β
01.010100100011.0 01010010001
ryT
p3
TATS
N
I
LUB
UT
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.
A. P. Rojas-Gil et al. GH ⁄ JAK-STAT axis signal-transduction defect

FEBS Journal 273 (2006) 3454–3466 ª 2006 The Authors Journal compilation ª 2006 FEBS 3459
S fibroblasts displayed a longer population doubling
time than C fibroblasts (79.9 ± 11.0 and
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 G
1
⁄ 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
G
o
⁄ G
1
phase of the cell cycle. Addition of hGH (200
and 1000 ngÆmL

)1
) decreased the percentage of S cells
in the G
o
⁄ G
1
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
G
o
⁄ G
1
phase, which is also reflected by increased levels
of p21
WAF ⁄ 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
uohtiWh tGH
S 3
S 2S 1S 2C 1C4
12P
FA
W
/IPI
C
NI
TC
A
stsalborbiF
HGh
HGhlm/g
n
002
NI
T
C
A
1
2
P
FAW/ IPIC
s
t
salborbiF

H
A
B
Gh
S 2S 1S

2
C
1C3
4
S
HGhlm/gn 0001
S



3S



2
S




1S

2C


1C
4
NITCA
12
P
WA /FCI IP
s
t
s
a
lbor
b
iF
HG
h

++
ryTp3TATS
1
2p
A
WF/C
I
PI
3
T

ATS
HG
4 3 2 1
Fig. 6. Effect of hGH on the expression of
p21
WAF ⁄ CIP1
in normal (C) fibroblasts and fi-
broblasts from children with ISS and defici-
ent phosphorylation of STAT3 (S). (A)
Expression of p21
WAF ⁄ CIP1
in 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-(p21
WAF ⁄ CIP1
)-specific IgG (second
panel), and equal loading was verified by
reprobing with an anti-STAT3 IgG.
GH ⁄ JAK-STAT axis signal-transduction defect A. P. Rojas-Gil et al.
3460 FEBS Journal 273 (2006) 3454–3466 ª 2006 The Authors Journal compilation ª 2006 FEBS
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, chondrocytes, 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 pre-
dominant JAK associated with GHR is JAK2,
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 G
1
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 p21
WAF ⁄ CIPI
[33]. In agreement with
previous reports, the prospect that the primary defect

0 ng/ml hGH
A
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C
5000 ng/ml hGH
0
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Fold increas
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200 ng/ml hGH
Days
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6
5
.5
6.9
6
D
1000 ng/ml hGH
0

2
4
6
8
10
12
86420
Days
Fold increas
C
S
8
5.4
65.6
5.3
5.0
Fig. 7. Growth curves of fibroblast from four C children (¤) and 4 S children (n) cultured in the presence of increasing concentrations of
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
)1
and 5000 ngÆmL
)1
. Each point is the mean ± SD
from three different experiments.
A. P. Rojas-Gil et al. GH ⁄ JAK-STAT axis signal-transduction defect
FEBS Journal 273 (2006) 3454–3466 ª 2006 The Authors Journal compilation ª 2006 FEBS 3461

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 p21
WAF ⁄ 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 p21
WAF ⁄ CIPI
. Because p21
WAF ⁄ 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 p21
WAF ⁄ CIPI
upregula-
tion was explored by over-expressing p21
WAF ⁄ 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 p21
WAF ⁄ 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
)1
Æ24
)1
h), compared with 54 normal prepubertal control
children (mean 24-h GH: 3.97 ± 0.18 ngÆmL
)1
and 24-h GH
secretion rate: 231 ± 15 lgÆL
)1
Æ24
)1
h) 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:
CS

A
0
10
20
30
40
50
60
500010005002000
ng/ml hGH
C
ng/ml hGH
S
500010005002000
% BrdU incorporating cells
B
0
20
40
60
80
100
120
S
C
SCSC
1000200
hGH n
g
/ml

% of Cells
0
C
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
,1lgÆmL
)1
and 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.
GH ⁄ JAK-STAT axis signal-transduction defect A. P. Rojas-Gil et al.
3462 FEBS Journal 273 (2006) 3454–3466 ª 2006 The Authors Journal compilation ª 2006 FEBS
60±9ngÆmL
)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
)1
Æ
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 mm
3
; 2–3 pieces per dish) were plated onto
60-mm
2
culture dishes in Dulbecco’s modified Eagle’s
medium, supplemented with 10% fetal bovine serum,
2mml-glutamine, 50 IUÆmL
)1
penicillin and 50 lgÆmL
)1
streptomycin (Gibco, Carlsbad, CA), and incubated at
37 °Cin5%CO

2
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 3S 2S
1S 2
C 1C
4
nilcyC
A
NI
TCA
S 3S 2S 1S 2C 1C4
nilcyCD
N
I
TCA
4S


3
S


2
S

1S

2
C

1
C
1TDC
NITCA
Fig. 9. Effect of hGH on the expression of
cyclin A, cyclin D and CDT1 in normal fibro-
blasts (C) and fibroblasts 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.
A. P. Rojas-Gil et al. GH ⁄ JAK-STAT axis signal-transduction defect
FEBS Journal 273 (2006) 3454–3466 ª 2006 The Authors Journal compilation ª 2006 FEBS 3463
carried out with cultured fibroblasts between the second
to sixth passages.
GH induction
Cell cultures at ~ 80% confluency were washed twice with
NaCl ⁄ P

i
and maintained for 6 or 24 h in serum-free Dul-
becco’s modified Eagle’s medium and then treated with var-
ious concentrations of hGH (200, 500 and 1000 ngÆmL
)1
).
Subsequently, the cells were washed with ice-cold NaCl ⁄ P
i
,
harvested by scraping and centrifuged. The cell pellets were
snap-frozen and stored in liquid nitrogen until further use.
For the STAT3 activation experiments, the S and C fibro-
blasts were cultured in the absence of fetal bovine serum
for 24 h and subsequently stimulated with hGH (200, 500
and 1000 ngÆmL
)1
) for 30 min p21
WAF ⁄ CIPI
expression was
assessed in cultured fibroblasts that were fetal bovine
serum-starved for 6 h and then cultivated in the presence of
hGH (200 and 1000 ngÆmL
)1
) for 24 h.
IFN-b induction
The specificity of the defective Tyr phosphorylation of
STAT3 in response to hGH in the S fibroblasts was tested
by the induction of STAT3 phosphorylation with IFN-b.
The fibroblasts were cultured in the presence of a wide
range of IFN-b concentrations (0–1000 UÆmL

)1
) for
30 min.
RT-PCR
Total RNA was prepared with Trizol reagent (Invitrogen,
Grand Island, NY) according to the manufacturer’s instruc-
tions from near-confluent cultured fibroblasts. Preparation
of cDNA from total RNA was performed using a Reverse
Transcription System (Thermoscrip RT, Invitrogen).
Human GHR was amplified from cDNA with primers
ACACTCAAGAATGGACTCAAG and TGTAAATTGG
CTCATCTGAG under the following conditions: denatura-
tion at 95 °C for 1 min, annealing at 52 °C for 1 min,
extension at 72 °C for 1 min, for 40 cycles. All samples
were treated with DNAse A and the RT-PCR reaction was
checked for contaminating genomic DNA by omitting
the RT.
Western immunoblotting
Western blotting was performed as described previously
[14]. For the analysis of GHR expression in the lysis
buffer, 1% deoxycholate was added. The following
dilutions and incubation times were employed for each indi-
vidual IgG: anti-GHR (University of Alabama at Birming-
ham, AL; 1 : 800, overnight, 4 °C), anti-(pTyr-STAT5b)
(Cell Signalling Technology, Inc., Danvers, MA; 1 : 500,
overnight, 4 °C), anti-STAT3 (Santa Cruz Biotechnology,
Santa Cruz, CA; 1 : 1000, 2 h, room temperature), anti-p21
(Transduction Laboratories, Lexington, KY; 1 : 500, over-
night, 4 °C), anti-(cyclin A) (SantaCruz Biotechnology;
1 : 1000, overnight, 4 °C), anti-CDT1 (a gift from Z. Lyge-

rou, University of Patras, Patras, Greece; 1 : 500, over-
night, 4 °C). Two anti-(pTyr-STAT3) IgG were used; one
from Cell Signalling Technology, Inc. at a dilution of
1 : 500, overnight at 4 °C, and the other from Upstate
USA (Waltham, MA) at a dilution of 1 : 1000, overnight at
4 °C. Quantification of the western signals (complexed pro-
tein bands) was performed using the image analysis pro-
gram, image-pro plus (version 4.5, Media Cybernetics,
Inc., Silver Spring, MD).
Immunoprecipitation
Equal amounts of all cell extracts (200 lg of protein) from
fibroblasts cultured in the absence or presence of hGH
(200 ngÆmL
)1
) for 15 and 30 min, were subjected to immu-
noprecipitation with an anti-GHR IgG against an overlap-
ping cytoplasmic domain of hGHR (GHR AL-37) [23]. The
immunocomplexes were resolved by SDS ⁄ PAGE, electro-
transferred onto poly(vinylidene difluoride) membranes and
the blot was probed with an polyclonal anti-(GHR rabbit
IgG) serum. The same membrane was stripped out and
reprobed with an anti-actin IgG.
Determination of the fibroblasts’ proliferative
capacity
Cells were suspended in Dulbecco’s modified Eagle’s med-
ium supplemented with 10% fetal bovine serum and plated
at a density of 10 000 cellsÆcm
)2
; they were supplemented
with fresh medium every 3 days. Cells were detached, sus-

pended in culture medium and counted at 24 and 72 h after
plating, while cultures were still at the exponential phase of
growth. To determine the growth curves, 5 · 10
4
cells were
plated onto six-well plates, without hGH or at hGH con-
centrations of 200, 500, 1000 and 5000 ngÆmL
)1
. Over
6 days, the cells were trypsinized and counted using a
hematocytometer.
Cell-cycle analysis
Fibroblasts were plated at a density of 10
4
cells per plate
in Dulbecco’s modified Eagle’s medium with 10% fetal
bovine serum. Control fibroblasts were harvested after
culturing for 72 h. Alternatively, cells were fetal bovine
serum-starved for 6 h and then treated with hGH (200 or
1000 ngÆmL
)1
). Cells were collected by trypsinization,
fixed in 70% ice-cold ethanol and stained with an
RNAse-containing propidium iodide solution. Cell-cycle
analysis was performed on a FACS calibre flow cytome-
GH ⁄ JAK-STAT axis signal-transduction defect A. P. Rojas-Gil et al.
3464 FEBS Journal 273 (2006) 3454–3466 ª 2006 The Authors Journal compilation ª 2006 FEBS
ter (Becton Dickinson, San Jose, CA) employing the
modfit software (Verity Software House, Topsham, ME).
The data were analyzed by using the spss program (SPSS

Inc., Chicago, IL).
BrdU incorporation
To study the effect of GH on the proliferation of the fibro-
blasts, we measured the BrdU incorporation into the cells.
For BrdU labeling, 10
4
cells were plated on ELISA plates
and cultivated in Dulbecco’s modified Eagle’s medium sup-
plemented with 10% fetal bovine serum medium with or
without hGH (0, 200, 500, 1000 and 5000 ngÆmL
)1
) for
72 h. Subsequently, cells were incubated in the presence of
1 lgÆmL
)1
BrdU (Roche, Mannheim, Germany) at 37 °C
and after 8 h, ELISA colorimetric assay (Roche) was per-
formed.
Analysis of the cDNA for STAT3
Total RNA was prepared with the Trizol reagent from per-
ipheral blood leukocytes. Preparation of cDNA from total
RNA was performed using an RT System (Thermoscript
RT, Invitrogen). Human STAT3 was amplified from cDNA
with specific primers (STAT3 R: TAGGCGCCTCA
GTCGTATCT and STAT3 F: AGCATCGAGCAGCT
GACTAC), under the following conditions: denaturation at
95 °C for 1 min, annealing at 58 °C for 1 min and exten-
sion at 72 °C for 1 min, for 35 cycles.
Cloning of p21
WAF/CIPI

and transfections
Human p21
WAF ⁄ CIPI
cDNA derived from the C children
was amplified using the following primers: GGAAAT
CATGTCAGAACCGGC and CTAGCTAGCTTAGGGC
TTCCTCTTGGA. Subsequently, it was cloned into the
EcoRI ⁄ NheI sites of the pCMX-F plasmid. Transient trans-
fections were performed by using a Lipofectamine 2000 kit
(Invitrogen) and were repeated at least three times. The
amount of transfected DNA was kept constant by the addi-
tion of appropriate amounts of the parental, empty expres-
sion vector.
Acknowledgements
This work was supported by the NIH grant DK 46395
(to SJF) and in part by Novo Nordisk, Hellas.
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