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From the Departments of Pediatrics (C.J., K.L., U.H., O.M.), University Children’s Hospitals, Heidelberg, D-69120 Giessen (W.B.), D-35392, Marburg (G.K.), D-35033, Germany; and Research Center of Endocrinology and Metabolism (C.O.), Sahlgrenska Hospital, University of Göteborg, Sweden S-41345
Address all correspondence and requests for reprints to: Prof. Dr. O. Mehls, Division of Pediatric Nephrology, University Children’s Hospital Heidelberg, Im Neuenheimer Feld 150, D-69120 Germany.
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In an experimental model of primary cultured rat growth plate chondrocytes, basal as well as GH (40 ng/ml) or insulin-like growth factor (IGF)-I (60 ng/ml)-stimulated growth was suppressed dose dependently (10-12–10-7 M) by dexamethasone (Dexa). An IGF-I antibody specifically and dose dependently inhibited the GH- but not the basic fibroblast growth factor (bFGF)-stimulated cell proliferation. GH increased the IGF-I concentration in conditioned serum-free culture medium; this was reversed by concomitant Dexa. Dexa time dependently suppressed the transcription of GH receptor (GHR) messenger RNA (mRNA) and down-regulated the basal and GH-stimulated expression of GHR. Whereas no suppressive effect on basal type I IGF-receptor (IGFR) was observed, Dexa blocked the IGF-I induced increase of IGF binding. These results were confirmed by GHR and IGFR immunostaining.
We conclude that Dexa impairs the GH-induced stimulation of local secretion and paracrine action of IGF-I and reduces the homologous increase of IGFR and GHR expression. The above experiments give further insight on the interaction between GH and glucocorticoids on the cellular and molecular level of growth plate chondrocytes.
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Longitudinal bone and total body growth is based on chondrocyte proliferation and subsequent enchondral ossification in the epiphyseal growth plates. Dexa has been shown to act locally to inhibit longitudinal bone growth (10). The finding that some children with growth failure due to chronic glucocorticoid therapy have normal GH and IGF-I levels points at the possibility of end-organ insensitivity for GH and IGF-I. This is in line with the therapeutic strategy to overcome local somatotropic insensitivity with GH in supraphysiological doses as proven in animal experiments and defined clinical settings (2, 3, 8, 11, 12).
It is, however, difficult to investigate the cellular and molecular mechanisms of interaction between glucocorticoids and somatotropic hormones in vivo. The aim of this study was to describe and further investigate the phenomenon of glucocorticoid-induced growth depression and its underlying mechanisms at the cellular level of the growth cartilage using an in vitro approach with cultured rat growth plate chondrocytes.
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Materials and Methods |
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IR3) were purchased
from Immundiagnostik (Bensheim, Germany); [125I]-hGH was
a gift from Novo Nordisk (Gentofte, Denmark),
[3H]-thymidine (25 Ci/mmol) was obtained from Amersham
Buchler (Braunschweig, Germany); Dexa, BSA, and ovine PRL (oPRL) were
from Sigma Chemical Co. (Munich, Germany), clostridium collagenase (EC
3.4.24.3), Deoxyribonuclease (DNase) I (EC 3.1.21.1) and trypan blue
from Boehringer (Mannheim, Germany); the GHR-mAb (mAb 263) was
purchased from Amgen (Brisbaine, Australia); standard low and low gel
temperature (LGT) agarose were from Bio-Rad (Richmond, CA); FCS, PBS,
HEPES, penicillin-streptomycin, Ham’s F-12 and DMEM were obtained from
Seromed (Berlin, Germany), charcoal from Serva (Heidelberg, Germany),
and Ultroser G from Life Technologies (Paisley, UK). Ultroser is a
biochemically defined serum substitute containing various growth
factors, nucleosides, vitamins, and lipids in BSA. It contained neither
Dexa nor GH (Seromed, personal communication and own determinations).
It contained IGF-I in a low concentration leading to a final
concentration of 0.52 ng/ml in the incubation medium. Insulin was
measured using a human ELISA with high bovine cross-reactivity (no.
K6219, Dako A/S, Denmark) at concentrations of 0.55 mU/liter or less in
various preparations of culture medium.
IGFR antibody, IR3, and GHR antibody, mAb 263, are
monoclonal mouse antibodies reactive against rabbit, rat, human and
other species and characterized elsewhere (13, 14).
Cell culture
Isolation of chondrocytes.Epiphyseal chondrocytes
from 60- to 80-g Sprague Dawley rats (Charles River, Kieslegg, Germany)
were isolated and cultured as described previously (15). Pooled
microscopically dissected epiphyses of 5–10 animals were digested for
3 h at 37 C by clostridial collagenase (0.12% wt/vol) and 0.02%
(wt/vol) bacterial DNase in F-12 medium. Viability, determined after
isolation and at the end of each experiment by the trypan blue
exclusion technique, always exceeded 90%. Dissociated cells were
counted using a Neubauer chamber (Scheik, Hofheim, Germany).
Monolayer cultures.Cells were cultured as described earlier (15, 16, 17) in 35-mm plastic dishes (Nunc, Wiesbaden, Germany) for growth curves and determination of local IGF-I synthesis, in 96-well plates for proliferation assays and in 24-well plates (Nunc, Wiesbaden, Germany) for binding studies. The F-12/DMEM:1/1 medium contained a nominal calcium concentration of 1.2 mM, measured by ion-sensitive electrode (Fresenius EH-F, Oberursel, Germany); it was supplemented with 10 mM HEPES, 100 µg/ml streptomycin, and 10% FCS at 37 C, and was gassed in humidified air with 5% CO2. In previous studies using the same culture system, we demonstrated maintenance of cellular differentiation state under these conditions. With prolonged culture periods, cells differentiated and mineralization of the matrix occurred (18). Medium and added hormones or vehicles were changed every other day unless indicated otherwise. Peptide hormones were dissolved in PBS, Dexa in ethanol (0.05% final concentration).
Agarose-stabilized suspension cultures.Cells were cultured in agarose according to Benya and Schaffer (16) as described earlier (15, 16, 17) in 35-mm dishes (Falcon Plastics). Dishes precoated with 1% standard low agarose in water were filled with cell suspension (40,000 cells/ml in 0.5% low gel temperature agarose) and kept at 37 C for 10 min before gelation at 4 C (10 min). Subsequently, 1 ml of F-12/DMEM containing 0.2% BSA, 0.3% UltroserG, and hormones or solvents as indicated were added. The serum substitute Ultroser had to be used because no colony formation was achieved despite maintained vitality of the cells when chondrocytes were cultured in serum-free medium, i.e. in the absence of growth factors. Medium was changed every other day, and cells were cultured for 3 weeks. Cultures were screened for clusters of more than three cells. No such clusters were seen at the start of culture in any experiment.
Assays of chondrocyte growth and proliferation in monolayer
cultures
Growth curves.Cells were seeded at 5000
cells/cm2 in parallel cultures and starved in serum-free
medium (F-12/DMEM) for 24 h. On day 1, medium was changed to
F-12/DMEM containing 0.2% BSA and 0.3% UltroserG for experiments
employing GH or IGF-I. Cells were counted on day 1 and every 2–3 days
thereafter. Charcoal-stripped FCS (Ch-FCS) at a concentration of 10%
was used for dose-response experiments with Dexa. According to our
measurements, cortisol concentration in FCS (50 pg/ml) was reliably
reduced to below 10 pg/ml (detection limit) by charcoal absorption
(Ch-FCS).
Clonal assay.Suspension cultures were terminated after 21 days by fixation in 4% buffered formaldehyde and methanol. Colonies were counted under the microscope in 100 squares (2-mm grid) for each dish. A cell colony was defined as a cluster of four cells or more with matrix stained by alcian blue as previously described (15, 20).
[3H]-thymidine assay.Incorporation of [3H]-thymidine into DNA was determined in parallel cultures as uptake of radioactivity in trichloroacetic acid-precipitable material as described previously (15, 16). Before the experiment, cells were starved in serum-free F-12/DMEM for 24 h. Synchronization of cell cycle was proven by flow cytometric analysis (FACS) as described previously (15). Medium was changed to F-12/DMEM with 0.2% BSA, and hormones and solvents were added as indicated for 48 h. For the last 3 h, cultures were coincubated with 2 µCi of [3H]-thymidine.
IGF-I RIA. IGF-I concentrations were measured in conditioned medium of subconfluent, synchronized serum-free cultures (0.2% BSA) using a highly sensitive (0.02 ng/ml), specific IGF binding protein (IGFBP) blocked RIA (21). In brief, the culture medium (100 µl/tube) was acidified with 10 µl of 0.5 mol/liter phosphoric acid to dissociate IGFs from IGFBPs. The first antibody (rabbit-antihuman IGF-I) was dissolved in a 0.1 mol/liter sodium phosphate buffer, pH 7.8, capable of reneutralizing the acidified sample. In addition, this solution contained a large excess of hIGF-II to preoccupy the binding proteins. Thus, IGF-I can readily be measured by conventional RIA without interference from the IGFBPs.
Receptor binding studies
GH- and IGF-I-binding.rhGH and mAb IR3
were radiolabeled using an iodogen method (22) to specific activities
between 2.6 and 3.1 MBq/µg ([125I]-hGH) and 1.25
MBq/µg for [125I]-IR3, respectively,
purified by PAGE (23) immediately after iodination and again 1 day
before the experiments by gel filtration (G-25) on a PD-10 column
(Pharmacia). Confluent cultures were starved in serum-free medium for
24 h. Hormones or solvents were added in medium supplemented with
0.2% BSA for 24 or 48 h as indicated. Thereafter, cells were
washed with PBS containing 1% BSA (pH 7.38) and then incubated with
increasing amounts of [125I]-hGH (0.2 to 1.2 nmol/liter)
or [125I]-IR3 (0.05 to 1.5 nmol/liter) in
PBS containing 1% BSA for 4 h at 24 C (GHR) or 1 h at 4 C
(IGFR). After the incubation period, cells were washed three times
thoroughly with ice-cold PBS and then solubilized in 1 N
NaOH for determination of radioactivity. Saturation analysis was
performed according to Scatchard (24). Specific binding was calculated
as the difference between total binding in the absence and unspecific
binding in the presence of 800-fold molar excess of unlabeled GH or
50-fold molar excess of IR3, respectively. All
determinations were carried out in duplicate or triplicate as
indicated.
Immunocytochemistry.Cells were cultured on glass
slides in parallel and under the same conditions as for the binding
studies (25). After incubating for 48 h with hormones or solvent
as indicated, cultures were rinsed thoroughly three times with PBS and
fixed in 3.7% paraformaldehyd (10 min at 4 C), methanol (3 min at -20
C), and acetone/3% H2O2 (1 min at -20 C).
Slides were preincubated with diluted horse serum (1:100 in PBS for 30
min), followed by incubation after three washes in PBS with the primary
antibodies (IR3; mg/ml diluted 1:200 in PBS-1%BSA) or
mAb 263 (mg/ml diluted 1:200) for 24 h at 4 C in a humidified
chamber with or without 40-fold molar excess of unlabeled hormone. The
following steps were performed at room temperature. After rinsing three
times in PBS-1%BSA, the fixed cells were incubated for staining with a
biotinylated rabbit antimouse secondary antibody (1:400, no. E0413,
DAKO A/S, Denmark) for 30 min. After further washing steps, an
incubation for 1 h with streptavidine (1:400 in PBS-1%BSA, no.
P0397, DAKO A/S, Denmark) followed. Finally, after another wash DAB
0.05% (wt/vol) + 0.1% (vol/vol) hydrogen peroxidase in PBS (Sigma,
no. D5905, Deisenhofen, Germany) was added for 20 min and cell cultures
were mounted in aquatex (Merck, no. 8562, Darmstadt, Germany). In part
of the experiments, nuclei were counterstained with hematoxylin for 2
min. Negative controls showed no specific staining when the primary
antibody was omitted or replaced by unspecific mouse IgG.
Ribonuclease (RNase)-protection solution-hybridization assay. Total nucleic acid (TNA) for the RNase-protection solution-hybridization assay was prepared by briefly homogenizing harvested cells with a sonifier (Branson Cell Disrupter B 15, Danbury, CT) in a buffer containing 1% (wt/vol) SDS, 20 mM Tris-HCl (pH 7.5), and 4 mM EDTA. Then, the homogenate was digested by an overnight proteinase-K treatment, and the TNA was prepared by a subsequent phenol-chloroform extraction according to Durnam and Palmiter (26).
The RNase-protection solution-hybridization assay was carried out according to the protocol described by Methews et al. (27). An antisense GHR [35S]-UTP-labeled RNA was synthesized from an EcoRI linearized pT7T3 18U plasmid carrying a 560 bp BamHI fragment of the rat GHR complementary DNA (28). The GHR complementary DNA fragment corresponds to a part of the extracellular domain of the GHR. Protected hybrids were precipitated with trichloroacetic acid, collected on glassfiber filters, and counted in a scintillation counter. The signal was compared with a standard curve obtained by hybridization to known amounts of GHR mRNA. The intraassay coefficient of variation was less than 10% in the range of 50–2500 amol RNA standard. The results were correlated to the DNA content as measured according to the method of Labarca and Paigen (29).
Statistics
Data are given as mean ± SE unless stated
otherwise. In each analysis, the distribution mode was evaluated by the
Kolmogorov-Smirnov test. For comparison of two normally distributed
groups, two-tailed, unpaired Student’s t tests and for more
than two groups one-way ANOVA followed by pairwise multiple comparison
(Student-Newman-Keuls method) were performed. For nonparametrically
distributed data, Kruskal-Wallis tests, followed by all pairwise
comparisons (Dunn’s method) were used. P < 0.05 was
considered statistically significant.
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Results |
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and 2;
Table 1). Due to
baseline variations in the data representing 8 individual experiments,
results are expressed in percentage over solvent control in serum-free
medium. GH at 40 ng/ml and IGF-I at 60 ng/ml, respectively, were
determined as maximal stimulatory hormone concentrations in preliminary
dose-response studies (data not shown) and used in all subsequent
experiments if not indicated otherwise. While Dexa 10-7
M significantly reduced basal thymidine incorporation to
80 ± 5% (P < 0.02), GH and IGF-I stimulated DNA
synthesis to a similar extent. Given concomitantly, Dexa significantly
reduced the effects of GH and IGF-I in comparison to solvent control by
45 ± 3% and 54 ± 3%, respectively (P <
0.001) (Table 1A). When GH was added concomitantly to a constant
concentration of Dexa (10-7 M), the negative
effect of Dexa on DNA synthesis was dose dependently compensated
(Table 1B).
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When using serum-free BSA medium supplemented with 0.3% Ultroser, GH
(40 ng/ml) substantially increased the number of growth plate
chondrocytes (Fig. 1a
). Coincubation with Dexa (10-7
M) prevented this increase. Likewise, IGF-I (60 ng/ml)
increased significantly the number of cells after 2 weeks (Fig. 1b).
Again, coincubation with Dexa reduced this effect. In each set of
experiments, the extent of growth inhibition caused by Dexa was
comparable, regardless of whether it was added to a control or a group
stimulated by GH or IGF-I.
Experiments with suspension cultures confirmed the described interaction of Dexa with IGF-I and GH on chondrocyte proliferation. The number of colonies containing four or more cells was elevated after 3 weeks under GH (40 ng/ml) and IGF-I (60 ng/ml) to 332 ± 14% and 632 ± 30%, respectively, of the control group (100 ± 16%, P < 0.001) but was significantly lower when coincubated with Dexa (10-7 M): 212 ± 28% of solvent control (GH + Dexa) and 500 ± 8% (IGF-I + Dexa), respectively (P < 0.005 vs. GH or IGF-I alone).
To examine a possible role of local IGF-I production in the
GH-stimulated chondrocyte cultures, [3H]-thymidine
incorporation and cell proliferation was studied in the presence of a
polyclonal IGF-I antibody, Ab-1, which had no intrinsic influence on
basal [3H]-thymidine incorporation. Ab-1 (1.25 µg/ml)
completely blocked the GH stimulated DNA-synthesis (Fig. 2a), and when
added in concentrations of 0.3–3 µg/ml, the IGF-I antibody dose
dependently reduced GH-driven cell proliferation to control levels
(Fig. 2b). This effect was specific for both GH and the Ab-1 because an
unspecific mouse Ab did not interfere with GH driven cell
proliferation, nor did the Ab-1 inhibit the basic fibroblast growth
factor (bFGF) stimulated DNA synthesis (Fig. 2a).
Local IGF-I secretion (Fig. 3)
Local IGF-I concentration was measured in the supernatant of
serum-free culture medium using a specific IGFBP-blocked RIA (Fig. 3).
Incubation with GH was followed by an increase in local IGF-I
concentration to 140 ± 1% of baseline levels after 24 h.
While GH led to a further increase of 191 ± 13% after 48 h
(control 100 ± 18% = 2.5–3.5 ng/ml in various assays,
P < 0.05), this increase was totally blocked by Dexa
10-7 M (84 ± 5% vs.
control). Dexa also inhibited basal IGF-I production (77 ± 3%,
P < 0.001).
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, 5, and 6; Tables 2 and 3)
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). Furthermore, Dexa counteracted the GH stimulated
rise when added concomitantly and decreased the GH stimulated amount of
binding by 52% (Fig. 4). The apparent dissociation constants
(KD) were comparable in all groups. In contrast to an
excess of unlabeled GH, which depressed total radioligand binding to
around 18% (unspecific binding), oPRL in the same molar excess
displaced only less than 10% of GH binding.
IGF-I treatment of chondrocyte cultures resulted in a 3-fold increase
of [125I]-IR3-binding. As shown in Table 3, actinomycin D (2 µg/ml), which did not affect basal IGFR
expression, obliterated the increase in Nmax seen with
IGF-I treatment. Dexa (10-7 M) had no
influence on IGFR binding on its own but completely blocked the
IGF-I-stimulated rise in IGFR (Fig. 5).
Immunocytochemical staining confirmed these regulatory mechanisms. The
pattern of immunostaining between the groups indicated an increase of
the fraction of IGFR-positive cells (i.e. proportion of
receptor-positive cells per culture) by IGF-I and a rise of receptor
number per positive cell (i.e. higher intensity of staining
on cells). GH (40 ng/ml) induced higher intensitiy of GHR staining
compared with controls. Again, coincubation with Dexa decreased the
GH-induced GHR staining (Fig. 6).
Regulation of GH receptor gene transcription (Fig. 7)
The time dependence of the Dexa (10-7 M)
mediated decrease of GHR mRNA is shown in Fig. 7. A significant
reduction of GHR mRNA was observed after 4 h (68 ± 2%
vs. a control value of 100 ± 10% in controls,
P < 0.005) and decreased further after 24 h of
incubation (31 ± 1% of control). In a separate set of
experiments, GHR mRNA transcription increased nearly 4-fold in cultures
treated with GH for 24 h (5.14 ± 0.06 amol/µg DNA
vs. a control value of 1.33 ± 0.06 amol/µg DNA,
P < 0.001). Concomitant incubation with GH and Dexa
for the same period of time slowed down the GHR mRNA transcription rate
to 4.45 ± 0.11 amol/µg DNA (P < 0.002
vs. GH alone).
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In the present experiments, GH had a maximal effect on DNA synthesis and cell proliferation at a concentration of 40 ng/ml. GH induced a marked increase of local IGF-I production. Immunocytochemical staining for IGF-I exhibited clusters of pericellular IGF-I immunoreactivity, which was most intense in areas where chondrocytes grew particularly dense (unpublished observation). This observation provides evidence that GH, at least in our culture system, stimulates chondrocyte growth primarily via endogenous IGF-I synthesis. GH was shown to stimulate undifferentiated chondrocytes in the reserve zone of rat epiphyseal plates (30) and to induce IGF-I mRNA in the proliferative zone (31, 32). In our cell system, most chondrocytes correspond to proliferative chondrocytes (18), paralleling the effect in the above in vivo studies.
The production of IGF-I in growth plate chondrocytes has been questioned until recently (33, 34). As evidenced by both specific dose-dependent blockage of GH-driven cell proliferation with an IGF-I Ab and direct measurements of IGF-I secreted into the supernatant of GH conditioned serum-free cultures, our results clearly argue against these reports and correspond to findings in human fibroblasts (35), rat skeletal cells (36), and to work by Brenner et al. (37) in which the proliferative action of GH on cultured fetal costal and articular chondrocytes was completely blocked by a type I IGFR mAb. IGF-I concentrations in the intercellular space of growth plate cartilage have not yet been determined. Given the fact that the measured IGF-I concentrations in conditioned serum-free medium of chondrocyte monolayers corresponded to 5–20% of the usual IGF-I serum concentration of prepubertal children (38), the locally synthesized amounts may well be of physiological relevance.
Dexa dose dependently reduced DNA synthesis and proliferation as shown by short-term [3H]-thymidine incorporation, cell growth curves in monolayer cultures (12 days) and agarose suspension cultures (3 weeks), which preserve the morphological character and three-dimensional structure of cartilage particularly well (19, 21, 39). Furthermore, high-dose Dexa (10-7 M) reduced the GH or IGF-I stimulated cell proliferation. The observed marked inhibition of local IGF-I production by Dexa after GH stimulation in serum-free cultures provides a possible mechanism by which Dexa impairs GH stimulated cell proliferation. This view is in line with the observation that cortisol decreased IGF-I mRNA in human osteoblast-like cells (40).
GH stimulated the expression of the GHR within 48 h. We could largely exclude the possibility that the GH induced increase of GHR is mediated via IGF-I as reported by Leung et al. for osteoblasts (41) because incubation of cultures with IGF-I did not influence GH binding, nor did the Ab-1 prevent the GH stimulated increase of GHR (unpublished observation). Divergent results have also been reported regarding homologous GHR regulation. Short-term (hours) GH treatment resulted in an acute decrease of GH binding in both cell culture (42, 43, 44) and in vivo experiments (45) of various tissues, most likely through endocytotic receptor internalization (46, 47). In contrast, an up-regulating effect has been shown for mid- and long-term GH treatment in rats, pigs, sheep, and other species (for review see Ref. 48). This phenomenon can be mimicked in vitro as reported for cultured hepatocytes (49), adipocytes (50), and in the present study for chondrocytes.
Heterologous (across species) binding studies, like the ones presented in our study, must principally be interpreted with caution because human GH ([125I]-rhGH) binds to both somatogenic and lactogenic binding sites in subprimate species. Nondisplacibility (<10%) of GH binding with excess oPRL as seen in all above described GHR assays served as control for the somatogenic origin of binding sites. The fact that no changes in binding affinity constants (KD) were observed indicates a single class of high affinity receptors and suggests that changes in binding capacity are due to an alteration of the average receptor number per cell rather than to a change in receptor affinity. Scatchard analysis cannot discriminate between the changing expression of the number of binding sites per cell and the changing number of cells expressing the receptor. However, immunocytochemistry of the GHR suggests that the regulation of binding is primarily due to a change of binding sites per cell.
Dexa exerted a dose-response effect in our experimental model. Incubation with low-dose Dexa (10-12 M) tended to increase the GHR number, suggesting that low (physiological) doses of glucocorticoids are required to maintain basal GHR expression, whereas (pharmacological) concentrations above 10-10 M decreased them. Furthermore, Dexa (10-7 M) reduced the positive effect of GH on GHR expression, but the number of binding sites remained above untreated control levels.
The influence of Dexa and GH on GHR status is obviously cell type specific and dependent on the respective culture conditions (e.g. pretreatment of cells, incubation periods, temperature at binding assay). Dexa has been reported to increase GH binding in human osteoblast cells (51) and osteosarcoma cells (52). In the latter study, the cells underwent binding to [125I]GH at room temperature over 24 h, certainly leading to internalization of a considerable amount of tracer, making it difficult to distinguish whether binding to cell surface receptors or receptor turnover was actually measured. Using serum-free incubation medium, our results showing a reduced expression of GHR under high-dose Dexa are consistent with a recent study performed on cultured fibroblasts (53). Similarly, in in vivo studies, Dexa induced a decrease of GHR expression in rat liver tissue (5).
In the present study, homologous increase of GHR expression was preceded by an increase of the GHR mRNA expression. The high transcriptional level of GHR mRNA (= 386% of control) in comparison to GHR protein binding (= 179% of control) may partially be explained by the fact that in the rat contrary to human physiology not all mRNA is translated to GH receptor protein, but GH-binding protein (GHBP) is also derived from alternative splicing of a common GHR/GHBP gene and mRNA precursor (54).
Dexa (10-7 M) time dependently reduced GHR gene transcription. Heinrichs et al. (55) reported in their in vivo experiments a biphasic dose-response on GHR mRNA levels with Dexa treatment. They found an increase of GHR mRNA in the liver and growth plates of young rabbits with low-dose but not with high-dose Dexa. Because GHR was not determined on the protein level, it remains unclear why the high dose of glucocorticoids did not result in reduced GHR mRNA transcription. In a detailed animal study, Gabrielsson et al. (5) could show that glucocorticoids (methylprednisolone 0.4 mg/day or Dexa 0.01 and 0.1 mg/day) over 12 days clearly decreased GHR mRNA levels and GH binding in both intact rats and in animals where the endogenous source of glucocorticoid production was removed by adrenalectomy (a model perhaps more comparable to our serum-free cell culture conditions).
Finally, we described the IGFR as a target of opposing interaction between Dexa and somatotropic stimulation. Dexa, which had no significant intrinsic influence on the IGFR, completely abolished the IGF-I induced expression of IGFR. While the first finding parallels an observation made recently by Conover et al. (56) in human fibroblasts, the latter adds an important element toward the aim to clarify the complex mechanisms of cellular interaction between glucocorticoids and somatotropic hormones.
All discussed experiments demonstrate that the action of GH is impaired
by Dexa on the cellular level. It is of major interest to ask whether
GH may be vice versa able to counterbalance the
antiproliferative effects of Dexa. Most of the interaction experiments
were performed with maximally effective doses of GH and Dexa; the
results (for instance growth curves in Fig. 1) can be interpreted in
both ways. When added to a fixed concentration of Dexa
(10-7 M), GH dose dependently antagonized the
glucocorticoid induced suppression of DNA synthesis (Table 1B). On the
other hand, concomitant incubation with maximally effective doses of
Dexa and GH did not result in a major increase of IGF-I concentration
in the supernatant compared with incubation experiments with Dexa alone
(Fig. 3). However, when GH was added to lower concentrations of Dexa
(10-8 M–10-9 M), an
increase of IGF-I concentration of about 50% of Dexa control was
observed (data not given). These results are in line with observations
in in vivo studies. In healthy and in uremic animals (3),
the glucocorticoid induced growth failure was dose dependently and
fully compensated by concomitant treatment with GH. Furthermore, in
glucocorticoid-treated children with renal transplants and near normal
renal function, growth failure was alleviated by concomitant treatment
with rhGH (8).
In conclusion, we have described several mechanisms of interaction between GH and Dexa for growth cartilage cells. These mechanisms might explain how glucocorticoids impair the growth-stimulating effects of GH. The results may also stimulate further research on GH treatment of glucocorticoid induced growth failure.
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Acknowledgments |
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Footnotes |
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2 Recipient of a scholarship granted by the Deutsche
Forschungs-gemeinschaft (DFG).
Received November 18, 1997.
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 E. Laplantine, F. Rossi, M. Sahni, C. Basilico, and D. Cobrinik FGF signaling targets the pRb-related p107 and p130 proteins to induce chondrocyte growth arrest J. Cell Biol., August 19, 2002; 158(4): 741 - 750. [Abstract] [Full Text] [PDF]
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 T Mushtaq and S F Ahmed The impact of corticosteroids on growth and bone health Arch. Dis. Child., August 1, 2002; 87(2): 93 - 96. [Full Text] [PDF]
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 K. R. Kritsch, S. Murali, M. L. Adamo, and D. M. Ney Dexamethasone decreases serum and liver IGF-I and maintains liver IGF-I mRNA in parenterally fed rats Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2002; 282(2): R528 - R536. [Abstract] [Full Text] [PDF]
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 D. KIEPE, D. L. ANDRESS, S. MOHAN, L. STANDKER, T. ULINSKI, R. HIMMELE, O. MEHLS, and B. TONSHOFF Intact IGF-Binding Protein-4 and -5 and Their Respective Fragments Isolated from Chronic Renal Failure Serum Differentially Modulate IGF-I Actions in Cultured Growth Plate Chondrocytes J. Am. Soc. Nephrol., November 1, 2001; 12(11): 2400 - 2410. [Abstract] [Full Text] [PDF]
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 G. K. Miller, M. G. Valerio, M. V. Pino, J. L. Larson, A. Viau, N. Hamelin, R. Labbe, and C. M. Banks Chronic Effects of the Novel Glucocorticosteroid RPR 106541 Administered to Beagle Dogs by Inhalation Toxicol Pathol, March 1, 2000; 28(2): 226 - 236. [Abstract] [PDF]
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K. Okazaki, S. Jingushi, T. Ikenoue, K. Urabe, H. Sakai, A. Ohtsuru, K. Akino, S. Yamashita, S. Nomura, and Y. Iwamoto Expression of Insulin-Like Growth Factor I Messenger Ribonucleic Acid in Developing Osteophytes in Murine Experimental Osteoarthritis and in Rats Inoculated with Growth Hormone-Secreting Tumor Endocrinology, October 1, 1999; 140(10): 4821 - 4830. [Abstract] [Full Text]
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H. Robson Bone growth mechanisms and the effects of cytotoxic drugs Arch. Dis. Child., October 1, 1999; 81(4): 360 - 364. [Full Text]
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Y. Kobayashi, C. K. Boyd, C. J. Bracken, W. R. Lamberson, D. H. Keisler, and M. C. Lucy Reduced Growth Hormone Receptor (GHR) Messenger Ribonucleic Acid in Liver of Periparturient Cattle Is Caused by a Specific Down-Regulation of GHR 1A That Is Associated with Decreased Insulin-Like Growth Factor I Endocrinology, September 1, 1999; 140(9): 3947 - 3954. [Abstract] [Full Text]
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P. Tollet-Egnell, A. Flores-Morales, A. Stavréus-Evers, L. Sahlin, and G. Norstedt Growth Hormone Regulation of SOCS-2, SOCS-3, and CIS Messenger Ribonucleic Acid Expression in the Rat Endocrinology, August 1, 1999; 140(8): 3693 - 3704. [Abstract] [Full Text]
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 Y. Umayahara, J. Billiard, C. Ji, M. Centrella, T. L. McCarthy, and P. Rotwein CCAAT/Enhancer-binding Protein delta Is a Critical Regulator of Insulin-like Growth Factor-I Gene Transcription in Osteoblasts J. Biol. Chem., April 9, 1999; 274(15): 10609 - 10617. [Abstract] [Full Text] [PDF]
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H. Jiang, C. S. Okamura, and M. C. Lucy Isolation and Characterization of a Novel Promoter for the Bovine Growth Hormone Receptor Gene J. Biol. Chem., March 19, 1999; 274(12): 7893 - 7900. [Abstract] [Full Text] [PDF]
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E. Canalis Editorial: Inhibitory Actions of Glucocorticoids on Skeletal Growth. Is Local Insulin-Like Growth Factor I to Blame? Endocrinology, July 1, 1998; 139(7): 3041 - 3042. [Full Text] [PDF]
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