Full text of DOI:10.1359/jbmr.2002.17.8.1408

JOURNAL OF BONE AND MINERAL RESEARCH
Volume 17, Number 8, 2002
© 2002 American Society for Bone and Mineral Research
Localization and Regulation of the Growth Hormone
Receptor and Growth Hormone–Binding Protein in the Rat
Growth Plate
EVELIEN F. GEVERS,1,2 BRAM C.J. VAN DER EERDEN,1 MARCEL KARPERIEN,1,3 ANTON K. RAAP,4
IAIN C.A.F. ROBINSON,5 and JAN-MAARTEN WIT1
ABSTRACT
Growth hormone (GH) has direct effects on the growth plate to stimulate longitudinal growth, but it is not clear
which chondrocyte populations GH acts on. The dual effector theory suggests that GH would act primarily on the
“stem cells.” However, staining with a GH receptor (GHR) antibody is found in all layers of the growth plate in
rabbits and humans. We now have investigated the localization and regulation of GHR and the related GH binding
protein (GHBP) in the rat growth plate using a sensitive immunohistochemical method involving tyramide signal
amplification (TSA) and antibodies specific for GHR or GHBP. Both GHR and GHBP were shown in the germinal
and proliferative chondrocytes, but most clearly in early maturing chondrocytes at the interface between prolif-
erative and hypertrophic cells. Staining for GHR and GHBP was located in both the cytoplasm and the nucleus.
Expression of GHR mRNA and GHBP mRNA in the growth plate was confirmed by reverse-transcription
polymerase chain reaction (RT-PCR). Immunohistochemical staining for GHR and GHBP decreased with age; in
12-week-old normal rats, only the early maturing chondrocytes were stained. In GH-deficient dwarf rats, staining
seemed less than in normal rats, and in hypophysectomized (Hx) rats, staining for GHBP was clearly reduced.
Treatment of Hx rats with thyroid hormones (T3 ؉ T4), via subcutaneously (sc) implanted osmotic minipumps,
induced little growth and induced a small layer of GHR-positive and GHBP-positive early maturing chondrocytes.
Treatment with GH and thyroid hormones (TH) resulted in greater growth and a broader layer of GHR-positive
and GHBP-positive cells, indistinguishable from normal rats. In contrast, dexamethasone treatment of normal rats
inhibited their growth and reduced GHR and GHBP staining in the growth plate. These results show that GHR
and GHBP in the growth plate are under hormonal control. The localization of GHR/GHBP suggests that in
addition to actions on germinal and proliferative cells in young rats, GH also has effects on early maturing
chondrocytes and may be involved in their differentiation to a fully hypertrophic chondrocyte. (J Bone Miner Res
2002;17:1408–1419)
Key words: growth plate, chondrocyte, growth hormone receptor, growth hormone binding protein, tyra-
mide signal amplification
(maturing) hypertrophic chondrocytes.(1,2) The border be-
tween the zone of proliferating cells and differentiating/
INTRODUCTION
HE GROWTH plate is divided into distinct layers of chon-
drocytes, resting (also referred to as “reserve,” “germi-
nal,” or “stem”) cells, proliferating cells, and differentiating
maturing cells consists of a few cell layers, often called the
transition zone. The cells in this layer and the upper part of
the hypertrophic cell layer are considered as “early maturing
cells.” In addition to an indirect action via insulin-like
growth factor (IGF)-I generated at the liver, there is strong
T
The authors have no conflict of interest.
1Department of Pediatrics, Leiden University Medical Center, Leiden, The Netherlands.
2Present address: National Institute for Medical Research, Division of Molecular Neuroendocrinology, The Ridgeway, Mill Hill,
London, United Kingdom.
3Department of Endocrinology and Metabolic Diseases, Leiden University Medical Center, Leiden, The Netherlands.
4Laboratory of Cytochemistry and Cytology, Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The
Netherlands.
5Division of Molecular Neuroendocrinology, National Institute for Medical Research, Mill Hill, London, United Kingdom.
1408

GHR AND GHBP IN THE GROWTH PLATE
1409
evidence that growth hormone (GH) acts locally at the accompanied by changes of GHR expression or localization
growth plate to stimulate longitudinal growth.(3,4) This hy- in the growth plate.
pothesis has been strengthened recently by experiments
showing that growth of transgenic mice with a targeted
disruption of the IGF-I gene in the liver is not different from
MATERIALS AND METHODS
normal mice.(5,6) There also is direct evidence that GH acts
on the stem cells(7) resulting in differentiation to a prolifer-
ative cell type that produces IGF-I,(8) which then acts in an
autocrine or paracrine fashion to induce further proliferation
and clonal expansion of the chondrocytes.(9) Although this
theory implicates the presence of GH receptors (GHRs),
primarily in the stem cells in the rat, immunostaining has
shown the presence of the GHR in all cell layers of the
growth plate.(10) However, this study was performed in the
rabbit and used a monoclonal antibody (MAb 263) directed
against the extracellular domain of the GHR, which there-
fore does not discriminate between GHR and the GH bind-
ing protein (GHBP), which corresponds to the GHR extra-
cellular domain.(11,12)
Animal experiments
Unless stated otherwise, rats were kept in a light- and
temperature-controlled room (12 h of light and 23–25°C)
with food and water available ad libitum. Experiments were
approved by the local ethical committee for animal exper-
iments.
Experiment 1: Male and female normal rats (Lewis strain;
Harlan, Zeist, The Netherlands) and GH-deficient dwarf
rats(23) (Lewis strain; Harlan), 1, 4, 7, and 12 weeks of age
(n ϭ 5–6 per group), were killed by decapitation. The
proximal part of the tibia was isolated, cut in half longitu-
dinally, fixed overnight in buffered picric acid/formaldehyde,
and then decalcified for 3 weeks in 20% EDTA (pH 8.0) as
described by Barnard and colleagues.(10)
Experiment 2: Male rats were Hx at the age of 21–28 days
(n ϭ 6 per group; Iffa Credo, Brussels, Belgium). One week
later osmotic minipumps (Alzet; Broekman Institute, Som-
eren, The Netherlands), delivering either 100 ␮g/day of
recombinant human GH (hGH; Pharmacia-Upjohn, Stock-
holm, Sweden) or 1.0 ␮g/day of T4 plus 0.125 ␮g/day of
T3 (T4/T3) (Sigma, St. Louis, USA) or both GH and T4/T3,
were implanted subcutaneously (sc) under halothane/O2/
N2O anesthesia. Control Hx or normal rats were implanted
with teflon rods. Five percent glucose and 0.9% NaCl were
added to the drinking water. Treatment was for 2 weeks
after which tibias were isolated as described previously.
Body weight was recorded weekly.
Experiment 3: Four-week-old normal Lewis female rats
(n ϭ 5 per group) received dexamethasone in the drinking
water in a dose of 0.3 mg/liter or 2 mg/liter of tap water with
0.02% alcohol. Two control groups received tap water with
0.02% alcohol. One of these groups was pair fed to match
the food intake of the group that received the highest dose
of dexamethasone. Treatment was for 1 week after which
tibias were isolated as described previously. Body weight
and body length were recorded.
Although GHBP is produced mainly in the liver,(5,6) it
also is produced in other tissues expressing GHR.(13) Its
function is not clear because, on one hand, it prolongs the
half-life of GH in vivo,(14) whereas, on the other hand, it
competes with GHR for binding to GH in vitro and can
reduce the effect of GH.(15,16) In rats, GHBP is produced by
alternative splicing of the primary GHR RNA(17) and the
GHR/GHBP mRNA ratio differs between tissues.(13,18,19)
Furthermore, different GHR/GHBP transcripts exist with
multiple 5Ј untranslated exons and these might be respon-
sible for tissue-specific regulation of GHR and GHBP ex-
pression.(20,21) Until now, regulation of GHR expression has
been studied mostly in the liver and brain. However, the
more relevant target for the regulation of skeletal growth by
GH is the expression of GHR and GHBP in the growth plate
itself.
In this study, we present data on the expression of GHR
and GHBP and mRNA in the growth plate at different ages.
By using antibodies specific for GHR, GHBP, or both, we
discriminated between GHR and GHBP and compared their
localization in the rat growth plate to identify target cells for
GH action.
A sensitive immunohistochemical method, using tyra-
mide signal amplification (TSA) with biotin-labeled tyra-
mides, readily detected GHR and GHBP. In the rat, GHBP
is produced by alternative splicing of exon 8, and this
generates a GHBP product with a unique 17 residue hydro-
philic tail.(17) Thus, an antibody specific for this peptide tail
could be used to localize GHBP specifically(22) while an
antibody against the intracellular domain of the GHR could
be used to identify GHR specifically. Reverse-transcription
polymerase chain reaction (RT-PCR) was used to confirm
Results of growth are shown as mean Ϯ SEM. Data were
subjected to ANOVA, followed by the Student–Newman–
Keuls test. Statistical significant difference was accepted
below p ϭ 0.05.
Immunohistochemistry
Preparation of tibial growth plates and the immunohisto-
the presence of GHR and GHBP mRNA transcripts in the chemical procedures were modifications of the method used
growth plate. To investigate hormonal regulation of GHR by Barnard and colleagues(10) to visualize GHR/BP in rabbit
and GHBP in the growth plate, immunohistochemistry was growth plate. Decalcified tibias were stored frozen. One day
performed on sections of growth plates from normal rats, before immunohistochemical analysis, 10 ␮m of cryostat
dwarf rats with a specific GH deficiency,(23) and hypophy- sections were mounted on gelatin-KCr(SO4)-glutaraldehyde–
sectomized (Hx) rats with and without thyroid hormone coated slides. Endogenous peroxidase activity was blocked
(TH) and/or GH treatment. We also studied the effect of in 1% H2O2 in PBS/40% methanol. Sections then were
treatment with dexamethasone in normal-growing rats to subjected to pepsin degradation (0.05% pepsin [Sigma] in
investigate whether growth inhibition by dexamethasone is HCl [pH ϭ 2]) for 7 minutes at 37°C and subsequently to

1410
GEVERS ET AL.
FIG. 1. Immunohistochemistry with
PAb iGHR in the growth plate of a
12-week-old female rat performed
(A) without and (B) with the use of
TSA. The line indicates 100 ␮m. Im-
munohistochemistry was performed
with DAB as chromogen (hematoxy-
lin background staining).
1% hydroxyl ammonium chloride for 15 minutes at room transcribed into cDNA with 50 U of Moloney murine leu-
temperature. Next, sections were incubated in 0.5% Boehr-
inger milk powder (in 0.1M TrisHCl/0.15M NaCl/0.05%
Tween 20 (Sigma; 60 minutes at 37°C) to block nonspecific
binding, followed by incubation with the following antibod-
ies: MAb 263 (1:10; Sanver Tech, Boechout, Belgium),
raised to the extracellular part of the rat GHR(11); MAb 4.3,
raised to a peptide corresponding to the 17 amino acids tail
of GHBP (1:10)(22); or a polyclonal antibody raised to the
intracellular part of GHR (PAb iGHR; 1:20). The latter two
antibodies were kindly provided by Dr. W. Baumbach (Cy-
anamid, Princeton, NJ, USA). A nonrelevant MAb (mouse
antibromodeoxyuridine; Dako A/S, Glostrup, Denmark)
was used as a control. Incubations with the primary anti-
bodies was for 60 minutes at 37°C. Sections then were
incubated with peroxidase-labeled rabbit anti-mouse anti-
body or swine anti-rabbit antibody (1:100 for 60 minutes at
37°C; Dako). Next, sections were incubated with biotin-
labeled tyramides (a generous gift of Perkin Elmer, Boston,
MA, USA; 1:300 in 0.1 M of Tris/0.1 M of NaCl/10%
dextran sulfate/10 mM of imidazol/1:1000 H2O2, for 30
minutes at room temperature), followed by a peroxidase-
labeled antibiotin antibody (1:500 for 45 minutes at 37°C;
Dako). The last antibody was visualized with diaminoben-
zidine (DAB; Sigma; 0.05% DAB in 50 mM of TrisHCl/1
mM of imidazol/0.085% H2O2, for 14 minutes at room
temperature). Finally, sections were washed in tap water
and dried and mounted in Fluoromount (BDH, Poole, UK).
Only in some sections hematoxylin counterstain was used
because we found that counterstaining reduced the visibility
of the immunostaining. In control sections, the first antibody
was omitted. Also, in other sections, immunohistochemistry
kemia virus (MMLV) reverse transcriptase, 7.5 pmol of
deoxynucleoside triphosphate (dNTP), 50 ng of random
primer, 0.1 ␮mol of dithiothreitol (DTT), and 0.5 ␮l of
RNasin in a total volume of 20 ␮l of buffer. The reaction was
carried out at 37°C for 1 h and then heated to 70°C for 10
minutes. Fifty units of MMLV reverse transcriptase and 0.5 ␮l
of RNasin were added again and the mixture was incubated for
30 minutes at 37°C and heated to 70°C for 5 minutes. Finally,
180 ␮l of 10 mM Tris (pH ϭ 8.0) was added.
PCR was performed in a total volume of 50 ␮l, containing
buffer, 2.5 mM of MgCl2, 0.3 mM of dNTP, 10 pmol of
forward and reverse primer, and 0.25 U of Taq polymerase
(Eurogentec, Seraing, Belgium), using 30 cycles (Hybaid
Omnigene System [Biozyme, Landgraaf, The Nether-
lands]). Each cycle consisted of 30 s of denaturation at
96°C, 30 s annealing at 56°C, and 1 minute extension at
72°C, preceded by an initial denaturation at 96°C for 3
minutes and followed by a final step at 72°C for 10 minutes.
In each PCR experiment, negative controls included tubes
with distilled water instead of cDNA or with 25 ng of RNA
instead of cDNA for each tissue. PCR products were ana-
lyzed by gel electrophoresis.
Oligonucleotide primers were purchased from Isogen Bio-
science BV (Maarssen, The Netherlands). For the amplifica-
tion of GHR cDNA, a primer set was chosen from the intra-
cellular sequence of the GHR: the sense and antisense primers
were 5Ј-GAGGAGGTGAACACCATCTTGGGC-3Ј and 5Ј-
ACCACCTGCCTGGTGTAATGTC-3Ј, respectively. Sense
and antisense primers for the amplification of GHBP cDNA
were 5Ј-TGGTGATTTGTTGGACGAAA-3Јand 5Ј-GCTA-
was performed without TSA to show the level of enhance- GGGATGGCAGATCCTC-3Ј, respectively.
ment by tyramides, using only a biotin-labeled second an-
tibody followed by a peroxidase-labeled antibiotin anti-
body. When comparing staining between different groups,
RESULTS
immunohistochemistry of the sections of all animals in
those groups was performed at the same time. Representa-
tive sections are shown in the figures.
Enhancement of signal by biotin-labeled tyramides
Figure 1 shows immunohistochemistry with PAb iGHR
in the growth plate of a 12-week-old female rat, performed
with and without the use of TSA. Without the use of TSA,
the staining was very weak (Fig. 1A). When TSA was used,
specific staining was enhanced whereas background stain-
ing in the growth plate was still very low (Fig. 1B). Staining
mainly was in early maturing chondrocytes (transition zone/
upper hypertrophic zone) but with the use of TSA staining also
RT-PCR
Growth plates of 1, 4, 7, and 12-week-old male and
female rats and livers of 8-week-old female rats were dis-
sected carefully. The edges of the growth plate were cut off
to avoid contamination with other cell types. Total RNA
was extracted following the method of Chomczynski and
Sacchi.(24) Two hundred fifty nanograms of total RNA was could be visualized, using higher magnification, in germinal

GHR AND GHBP IN THE GROWTH PLATE
1411
FIG. 2. Immunohistochemical stain-
ing in the growth plate of a 12-week-
old female rat with three different
antibodies: (A) omission of first anti-
body; (B) MAb 263, recognizing spe-
cifically the extracellular part of
GHR/GHBP; (C and E) PAb iGHR,
recognizing the iGHR; and (D and F)
MAb 4.3, recognizing the 17 amino
acids tail specific for GHBP. Staining
is mainly in the zone of early hyper-
trophic chondrocytes (E and F). At
higher magnification, staining in cells
in the resting zone (arrows) and some
proliferative cells is revealed. The
line indicates 100 ␮m. Immunohisto-
chemistry was performed with DAB
as chromogen (hematoxylin back-
ground staining).
cells. There was no staining when the nonrelevant MAb was
used (antibromodeoxyuridine).
Comparison of MAb 263, PAb iGHR, and MAb 4.3
Figure 2 compares the staining obtained with the three
different antibodies recognizing either the common extra-
cellular part of the GHR/GHBP (MAb 263), the internal part
of the GHR (PAb iGHR), or the tail of GHBP (MAb 4.3) in
the growth plate of a 12-week-old female rat. Specificity of
the antibodies and the tyramides was shown by the disap-
pearance of staining when the first antibody was omitted
(Fig. 2A). As can be seen, background staining in bone
tissue (but not in the growth plate) was seen. This probably
was caused by cross-reaction with rat antigen of the rabbit
anti-mouse second antibody used to detect the monoclonal
primary antibodies, because this background was not ob-
served when a swine anti-rabbit second antibody was used
(not shown). The specificity of these antibodies for their
respective antigens has been shown in other experiments by
competition with excess antigen.(22,25)
All three antibodies showed staining mainly in the tran-
sition zone/upper hypertrophic chondrocyte layer (Figs. 2B,
2C, and 2D showing staining with MAb 263, PAb iGHR,
and MAb 4.3). Immunohistochemistry with MAb 263 and
PAb iGHR also revealed staining in proliferative and ger-
minal chondrocytes (Figs. 2E and 2F). Although not the
primary object of this study, we noted that immunoreactiv-
ity also was intense in bone and bone marrow proximal and
distal to the growth plate (data not shown).
FIG. 3. RT-PCR showing presence of GHR mRNA and GHBP
mRNA in growth plate tissue and liver extracts of male and female rats
at 1, 4, 7 and 12 weeks of age. Lane 1–8: growth plate tissue extracts
in the following order: lane 1: 1-week-old female, lane 2: 4-week-old
female, lane 3: 7-week-old female, lane 4: 12-week-old female and lane
5: 1-week-old male, lane 6: 4-week-old male, lane 7: 7-week-old male,
lane 8: 12-week-old male. Lane 9: control using water instead of DNA.
Lane 10: liver tissue extract of 8-week-old female. Lane 11–20: con-
trols: PCR reactions in same growth plate tissue of lane 1–10 but
without reverse transcription.

1412
GEVERS ET AL.
FIG. 4. (A–D) Immunohisto-
chemistry for GHR, using PAb
iGHR in normal male rat growth
plates at (A) 1week, (B) 4 weeks,
(C) 7 weeks, and (D) 12 weeks of
age. Staining in 1-, 4-, and
7-week-old rats is in cells of the
resting zone, proliferative cells,
and early hypertrophic chondro-
cytes. In 12-week-old rats, stain-
ing mainly is in early hyper-
trophic chondrocytes in the
transition zone between the pro-
liferative and hypertrophic cell
layer. The line indicates 100 ␮m.
(E–H) Immunohistochemistry for
GHBP, using a MAb recognizing
the 17 amino acids tail specific
for GHBP (MAb 4.3) in growth
plates of the same rats as used in
panels A–D. Staining for GHBP
is similar as for GHR. (I–L) Im-
munohistochemistry for GHBP,
using MAb 4.3, in growth plates
of GH-deficient dwarf rats of (I) 1
week, (J) 4 weeks, (K) 7 weeks,
and (L) 12 weeks of age. GHBP
staining seems less in dwarf rats
than in normal rats (cf. with pan-
els E–H). The line indicates 100
␮m. Immunohistochemistry was
performed with DAB as chromo-
gen (no background staining).
FIG. 5. Presence of GHR and
GHBP immunostaining in cells of
the resting zone (arrows) in
4-week-old male rats. (A) PAb
iGHR, recognizing GHR; (B)
MAb 4.3, recognizing GHBP.
The line indicates 25 ␮m. Immu-
nohistochemistry was performed
with DAB as chromogen (no
background staining).

GHR AND GHBP IN THE GROWTH PLATE
1413
FIG. 6. GHBP immunostaining using MAb 4.3 in a 7-week-old (A)
male and (B) female rat. The growth plate appears smaller and the staining
less in the female rat. The line indicates 100 ␮m. Immunohistochemistry
was performed with DAB as chromogen (no background staining).
FIG. 8. Induction of GHBP immunostaining in the growth plate after
T3/T4 and GH treatment for 2 weeks in 4- to 5-week-old Hx rats. (A)
Saline-treated Hx rat, (B) T3/T4-treated, (C) GH-treated, (D) GH ϩ
T3/T4–treated, and (E) normal intact rat. See text for more details. The
line indicates 100 ␮m. Immunohistochemistry was performed with
DAB as chromogen (no background staining).
FIG. 7. Nuclear localization of (A) GHR immunostaining (PAb
iGHR) and (B) GHBP immunostaining (MAb 4.3) in hypertrophic
chondrocytes in a 4-week-old normal rat; (C) GHBP immunostaining
(MAb 4.3) in hypertrophic chondrocytes in a 4-week-old GH-deficient
dwarf rat. Note the decrease in nuclear localization of GHBP in the
dwarf rat. The line indicates (A) 25 ␮m or (B and C) 100 ␮m.
Immunohistochemistry was performed with DAB as chromogen (no
background staining).

1414
GEVERS ET AL.
TABLE 1. WEIGHT GAIN IN NORMAL AND HORMONE-
TREATED 4- TO 5-WEEK-OLD RATS
Regulation of GHR and GHBP by GH and thyroid
hormones (experiment 2)
Group
Weight gain (g/week)
To gain insight in the regulation of GHR and GHBP in
the growth plate, Hx rats were treated with GH, T3/T4, or
both, and normal rats were treated with dexamethasone,
after which immunohistochemistry was performed on their
growth plates. Hx rats showed virtually no growth when
treated with saline for 2 weeks. T3/T4 induced a small
weight gain of 5.2 Ϯ 0.2 g/week, and GH was more effec-
tive and the weight gain was not increased further by
additional T3/T4 treatment (Table 1).
Figure 8 shows staining for GHBP in the growth plate of
rats from these different groups. Compared with normal rats
(Fig. 8E), staining with MAb 4.3 was greatly reduced in Hx
rats (Fig. 8A). T3/T4 treatment alone induced a small row of
positive cells in the transition zone between the proliferative
and hypertrophic zone (Fig. 8B). GH treatment resulted in a
wider growth plate and a broader band of GHBP-positive
cells (Fig. 8C). Staining was indistinguishable in tibias from
GH-treated, GH ϩ T3/T4–treated, and normal rats (Figs.
8C–8E).
Hx ϩ saline
Hx ϩ T3/T4
Hx ϩ GH
Hx ϩ GH ϩ T3/T4
Normal control
1.6 Ϯ 0.5
5.3 Ϯ 0.2
15.6 Ϯ 1.0*,†
15.1 Ϯ 0.8*,†
44.1 Ϯ 2.7*
†
* p Ͻ 0.01 versus Hx, p Ͻ 0.01 versus Hx ϩ T3/T4.
Normal and hypophysectomized (Hx), 4- to 5-week-old male
rats were treated with saline, T3/T4 (0.125 ␮g; T3 ϩ 1.0 ␮g of T4),
GH (100 ␮g/day), or combined GH ϩ T3/T4 treatment, adminis-
tered via sc implanted osmotic minipumps for 2 weeks.
Detection of GHR- and GHBP mRNA in the
growth plate
RT-PCR confirmed the presence of GHR mRNA and
GHBP mRNA in the growth plate (Fig. 3). GHR and GHBP
RNA were clearly present in growth plate and liver and no
band was observed in the appropriate controls. GHR and
GHBP RNA were shown in the growth plate of both male
and female rats at all ages studied.
Similar results were obtained for MAb 263, but differ-
ences between groups were less clear when PAb iGHR was
used, suggesting that GHBP and GHR are regulated sepa-
rately in the growth plate.
Regulation of GHR and GHBP by dexamethasone
(experiment 3)
Expression of GHR and GHBP in growth plates at
different ages (experiment 1)
Table 2 shows the effect of low-dose (0.3 mg/liter drink-
ing water) and high-dose (2 mg/liter drinking water) dexa-
methasone treatment on gain in weight and length, com-
pared with pair-fed and normal-fed rats. Dexamethasone-
In 1, 4, and 7-week-old rats, staining with all three anti-
bodies was seen in stem cells, proliferative chondrocytes,
and early hypertrophic chondrocytes with faint staining in treated rats gained less weight and length (p Ͻ 0.01) than
both the ad libitum fed group and the pair-fed group (paired
to the high-dose group only). Figure 9 shows that GHBP
staining was clearly reduced in proliferative chondrocytes
of the dexamethasone-treated rats and was hardly detectable
in the rats treated with the highest dose, compared with both
ad libitum and pair-fed rats. Staining in the early hypertro-
phic cell layer also was clearly diminished in the
dexamethasone-treated rats. Similar results were obtained
when PAb iGHR and MAb 263 were used (not shown).
some more mature chondrocytes. In 12-week-old rats, stain-
ing in stem cells and proliferative chondrocytes was reduced
greatly and staining was seen mostly in the transition zone and
the early hypertrophic chondrocytes. This is illustrated in Fig.
4, showing the antibody PAb iGHR, recognizing GHR only
(Figs. 4A–4D), and MAb 4.3, recognizing GHBP only (Figs.
4E–4H). At all ages, cells in the transition zone showed the
most intense staining, although cells in the stem cell zone also
stained positive for both GHR and GHBP, shown at a higher
magnification with PAb iGHR and MAb 4.3 in Fig. 5. In 1, 4,
and 12-week-old rats there were no differences between gen-
der but in 7-week-old rats, staining with MAb 4.3 in stem cells
and proliferative chondrocytes consistently appeared more
abundant in male rats than in female rats (Figs. 6A and 6B). In
dwarf rats, localization of GHR/GHBP staining was similar to
normal rats, but intensity seemed less when using MAb 4.3
(Figs. 4I–4L). This difference was less clear when using the
other two antibodies.
DISCUSSION
There is an increasing amount of evidence that, in addi-
tion to the hepatic effects on IGF-I generation, GH affects
the growth plate directly to stimulate longitudinal growth in
the rat.(3–6,26) However, its exact cellular target in the
growth plate is not clear. The dual effector theory(9) impli-
cates GH action on the resting cells, but GHR is not exclu-
sively limited to this zone in the growth plate, at least in the
rabbit, where GHR has been shown not only in resting cells
but also in proliferative chondrocytes and in hypertrophic
chondrocytes.(10) One problem with most earlier studies is
that the antibody used does not distinguish between GHR
and GHBP, the soluble extracellular part of the GHR, which
Staining with all three antibodies was present in both the
cytoplasm, and in many chondrocytes, the nucleus (Figs. 7A
and 7B). Nuclear staining was most prominent in the early
hypertrophic chondrocytes. Interestingly, GHBP staining in
growth plates of dwarf rats was almost exclusively cyto-
plasmic (Fig. 7C).

GHR AND GHBP IN THE GROWTH PLATE
1415
TABLE 2. WEIGHT GAIN AND BODY LENGTH GAIN DURING
DEXAMETHASONE TREATMENT FOR 1 WEEK IN
4-WEEK-OLD FEMALE RATS
Gain in
Weight gain
(g/week)
length
(mm/week)
Group
Low-dose dexamethasone
High-dose dexamethasone
Control, pair-fed
13.3 Ϯ 1.2*
4.3 Ϯ 0.9†
17.1 Ϯ 0.8
36.5 Ϯ 0.7
12.6 Ϯ 0.8*
5.7 Ϯ 1.5†
13.4 Ϯ 0.9
20.8 Ϯ 0.8
Control, ad libitum fed
Dexamethasone was mixed in the drinking water in 0.3-mg/L
(low dose) or 2-mg/L (high dose) concentrations. Control rats were
ad libitum fed or pair fed with the rats receiving the highest dose
of dexamethasone.
* p Ͻ 0.01 versus ad libitum–fed controls; † p Ͻ 0.01 versus
pair-fed and ad libitum–fed controls.
also might be present in chondrocytes. To readdress these
questions in the rat, we have used a sensitive immunohis-
tochemical method to localize GHR and GHBP specifically
in the rat growth plate, using antibodies directed to the
internal part of GHR and to the hydrophilic tail of GHBP,
which enabled us to recognize GHR and GHBP separately
in this species.
To enhance the sensitivity of immunostaining, we used
biotin-labeled tyramides for amplification.(27,28) Tyramides
precipitate on interaction with peroxidase and hydrogen
peroxide. We used a peroxidase-labeled second antibody
and biotin-labeled tyramides so that a large deposit of
biotins is formed, which we detected with a peroxidase-
labeled antibiotin antibody. Specificity of this TSA was
shown by a complete disappearance of staining when either
primary or secondary antibody was omitted or when a
nonrelevant first antibody was used. The increased sensitiv-
ity achieved may account for the fact that we could detect
staining with all three antibodies in rat growth plates at all
ages, up to 12 weeks, whereas Barnard and colleagues did
not detect GHR/BP in rabbits older than 50 days.(10)
Both GHR and GHBP protein were located in the rat
growth plate. GHBP is produced mainly in the liver, but
many extrahepatic tissues also synthesize GHBP.(13) It is
likely that GHBP is produced also locally in the chondro-
cytes, but the immunostaining done could not exclude the
possibility that the GHBP in chondrocytes was obtained
from the circulation and then internalized by chondrocytes.
However, RT-PCR of growth plate cDNA confirmed the
presence of GHR mRNA and GHBP mRNA transcripts in
the growth plate and thus suggests local production of GHR
and GHBP in the growth plate.
FIG. 9. Reduction of GHBP immunostaining after 1 week of dexa-
methasone treatment in two different doses in 4-week-old female rats
compared with both ad libitum–fed and pair-fed rats (pair fed with rats
receiving the highest dexamethasone dose). (A) Controls, ad libitum–
fed (B) low-dose dexamethasone treatment (0.3 mg/liter of drinking
water), (C) high-dose dexamethasone treatment (2 mg/liter of drinking
water), and (D) controls, pair-fed to rats receiving 2 mg/liter of dexa-
methasone. The line indicates 100 ␮m. Immunohistochemistry was
performed with DAB as chromogen (no background staining).
In young rats, immunoreactive GHR and GHBP were
The localization of both GHR and GHBP in the same cell present in cells of the resting zone, as has been found before
is not surprising because both proteins are transcribed from in rat embryos,(30) young rats,(31) and young rabbits.(10)
the same gene(17) but the functional implications of the local Staining was especially clear in the groove of Ranvier,
GH-GHR-GHBP interactions in the growth plate are in- where cells migrate from the resting zone.(32) However, as
triguing. GHBP could be produced locally to capture free also discussed by Lupu et al.,(33) it is difficult to demarcate
GH to enable it to bind to the receptor or, alternatively, the resting zone from epiphyseal chondrocytes involved in
protect cells continuously exposed to GH by preventing it to the growth of the secondary ossification center and it is
bind to the receptor. It could also participate in the dimer- unclear whether cells in the resting zone are stem cells/
ization process and affect signaling.(29)
precursors of proliferative chondrocytes. The localization of

1416
GEVERS ET AL.
CCAAT/enhancer-binding protein (C/EBP), an antimito- requires GH binding for translocation to the nucleus. Nu-
genic factor involved in differentiation of other mesenchy- clear staining was not abolished completely but this might
mal cells like preadipocytes,(34) in these cells(31) could be because of the residual GH in these dwarf rats,(23) be-
suggest that these cells are chondrocyte precursors differ- cause in Hx rats staining completely disappeared.
entiating into chondrocytes. Thus, the localization of GHR
Interestingly, nuclear staining was seen mainly in chon-
in these cells supports a direct action of GH on resting cells. drocytes in the transition zone between proliferation and
However, it does not exclude a role for IGF-I, either pro- hypertrophy and rarely in stem cells or proliferating chon-
duced in the liver or produced locally in these cells, in the drocytes. This raises the possibility that proliferative chon-
differentiation of germinal chondrocytes to proliferative drocytes use a different intracellular signal transduction
chondrocytes.(35,36)
pathway than hypertrophic chondrocytes (e.g., plasma
More faint immunostaining was seen in proliferating membrane–associated protein phosphorylation vs. nuclear
chondrocytes of rats up to the age of 7 weeks but not in the translocation).
12-week-old rats. This is in agreement with the original
Having established the localization of GHR and GHBP in
study of GHR/BP in rabbits, in which a similar localization the growth plate, we then wished to study its regulation. In
was found in 20- and 50-day-old rabbits but not in older the rat, hepatic GHR and plasma GHBP are developmen-
rabbits,(10) and with a recent report of GHR/BP in uremic tally regulated with a gender difference occurring at the age
rats(37) and with GHR mRNA in situ hybridization data.(33) of puberty.(45–47) However, plasma GHBP mainly reflects
In other studies in rat tibial growth plates, immunostaining hepatic GHBP production and little is known about GHR/
with MAb 263 was not found in proliferative chondro- GHBP regulation in other target tissues. In the growth plate,
cytes,(31) but this might reflect the lower sensitivity of GHR and GHBP showed clear developmental regulation.
standard immunochemistry. The localization of GHR in Throughout sexual maturation, staining was very strong and
proliferative chondrocytes suggests that GH also might act was reduced as animals became older (and grew more
directly on these cells in young animals to stimulate mito- slowly), by which time staining was only detected in the
genesis. In culture, GH stimulates growth of large chondro- early maturing chondrocytes. A mild sex difference was
cyte colonies thought to represent rapidly proliferating detected only at the end of puberty (7 weeks of age) with
cells.(38,39)
staining less intense in female animals than in male animals,
However, the most prominently stained layer was seen in but this suppression is difficult to quantitate. At 7 weeks of
a band of early maturing chondrocytes at the transition age, growth velocity is less in female animals than in male
between proliferative and hypertrophic zones. Staining in animals; therefore, although we cannot quantify it from
the most distal layer of fully mature hypertrophic chondro- these data, it is tempting to speculate that there may be a
cytes was absent or faint, similar to findings in rat fetal relation between growth velocity and GHR/BP expression
growth plates(30) and localization of GHR mRNA.(33) This in the growth plate in this period of sexually dimorphic
is the first time, maybe because of the ultrasensitive immu- growth velocity.
nostaining technique, that the localization in this transition
A logical candidate for the in vivo regulation of growth
zone is so apparent. In older rats, GHR and GHBP local- plate GHR expression is GH itself, because in vitro studies
ization in this zone remained visible whereas GHR and have shown regulation of chondrocyte GHR by GH(8) and
GHBP staining disappeared from proliferative chondro- GH is a main regulator of hepatic GHR and plasma GHBP
cytes. The difference in staining between the early maturing in vivo.(48) Like plasma GHBP,(47) GHBP staining appeared
and the fully mature chondrocyte might indicate that GH less intense in dwarf rats compared with normal rats and this
receptor signaling is involved in the maturation process of difference in staining disappeared in the older animals when
chondrocytes.
normals and dwarves were growing at a more similar rate.
Interestingly, we previously showed that GH treatment in However, the polyclonal antibody specific for GHR showed
dwarf rats results in a decrease in alkaline phosphatase less difference between normal and dwarf growth plates.
activity in those chondrocytes(40); thus, this could be a direct The relative expression of GHR and GHBP in the growth
effect of GH. In addition, in the fetal rat, these chondrocytes plate may vary, but this will require confirmation with more
express GHR/BP mRNA but not IGF-I mRNA,(41) also quantitative techniques.
suggesting that GH can act directly on these transitional
chondrocytes without IGF-I as mediator, at least in the tomy, in which growth has completely ceased, GHR and
fetus. GHBP were virtually undetectable in the growth plate. GHR
In the multiple hormone-deficiency state of hypophysec-
Although not the primary aim of this study, we noted that and GHBP expression in chondrocytes was induced again
all three antibodies stained GHR or GHBP in nuclei as well by T3/T4 and GH treatment, especially in the transition
as cytoplasm, as has been shown before for GHR(42) and zone. Lewinson et al.(49) concluded from experiments in
GHBP(43) and also for GH.(44) The significance of GHR and hypothyroid rats that condylar chondrocytes were compro-
GHBP in the nucleus is not clear. They might provide an mised in their response to GH although they could not show
additional signal transduction pathway or act as transcrip- differences in GHR staining using MAb 263.
tion factors,(42–44) although this is still controversial. Trans-
The early maturing chondrocytes on the transition from
location of the GHR to the nucleus requires the intracellular the proliferative zone to the hypertrophic zone may be a
part of the GHR but also GH binding.(42) We observed a pivotal zone in chondrocyte differentiation. For example,
reduction of nuclear staining in GH-deficient dwarf rats Indian hedgehog (Ihh), parathyroid hormone–related pro-
most clearly with MAb 4.3, which may suggest that GHBP tein (PTHrP), PTH/PTHrP receptors, and basic FGF

GHR AND GHBP IN THE GROWTH PLATE
1417
(bFGF), which are involved in chondrocyte maturation, are versity Medical Center) for their help in setting up the
localized to the same chondrocytes.(28,50–54) We now show immunohistochemistry. We are grateful to Pharmacia-
here that it is again these cells in which GHR/GHBP ex- Upjohn for their continuing supply of recombinant hGH,
pression is restored after GH/T3/T4 replacement therapy.
and we thank Dr. C.W.G.M. Lo¨wik (Department of Endo-
We found that dexamethasone acts as a negative regulator crinology, Leiden University Medical Center) for helpful
of GHR/GHBP in the growth plate. Staining for GHR and comments.
GHBP had almost disappeared not only from resting zone
cells and proliferative chondrocytes but also from early
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London NW7 1AA, UK
Received in original form October 8, 2001; in revised form March
6, 2002; accepted April 2, 2002.