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Division of Neuroendocrinology, Institute of Anatomy, University of Zurich (M.R., A.C.S.), 8057 Zurich, Switzerland; Division of Endocrinology and Diabetes, Department of Internal Medicine, University Hospital (J.Z.), 8091 Zurich, Switzerland; and M. E. Mueller Institute for Biomechanics (B.H.-M., E.B.H.), University of Bern, 3010 Bern, Switzerland
Address all correspondence and requests for reprints to: Manfred Reinecke, Ph.D., Division of Neuroendocrinology, Institute of Anatomy, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. E-mail: reinecke{at}anatom.unizh.ch
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Introduction |
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Because of these conflicting results, and because cellspecific localization of IGF-I in the growth plate is pertinent to the mode of GH action on longitudinal growth, we investigated this issue in normal rats and in hypox rats after 24-h treatment with GH or IGF-I. To determine whether differences exist between growth plate and articular cartilage, both regions were studied. To analyze the effects of our experimental conditions at the cellular level, both in situ hybridization for the detection of IGF-I mRNA and immunohistochemistry for the detection of the IGF-I peptide were used, and the results were morphometrically and statistically evaluated. We found that IGF-I is expressed in all chondrocyte layers of growth plate and articular cartilage, that hypophysectomy drastically reduces both IGF I mRNA expression and IGF-I immunoreactivity in growth plate but not in articular cartilage, and that GH, but not IGF-I, treatment restores both parameters to normal.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After 24 h of infusion, experimental and control animals were anesthetized with Innovar Vet (Pitman-Moore, Inc., Washington Crossing, NJ; 0.2 ml/100 g BW, im) and bled by aortic puncture. Blood samples were collected on ice and centrifuged for 15 min at 4 C (1500 x g). Serum was stored at -20 C until analysis.
RIA
Immunoreactive IGF-I in the serum was measured by RIA after
Sep-Pak C18 chromatography using rhIGF-I
(determination of infused IGF-I) or recombinant rat IGF-I
(GroPep Pty. Ltd., Adelaide, Australia; determination of
endogenous rat IGF-I) as standards. The assay procedures were described
in detail recently (11).
Tissue preparation and fixation
The proximal tibiae were cut sagittally into three slices. The
central slice was fixed in buffered 37% formalin for 24 h and
embedded in methacrylate. Sections were cut and stained with van Kossa
dye solution for the determination of growth parameters (10). The other
two slices were fixed with 4% paraformaldehyde containing 3% dextran
for 24 h. Thereafter, the bones were demineralized in 15% EDTA,
0.5% paraformaldehyde, and 3% dextran (pH 8.0) for 4 weeks at 4 C.
After washing in distilled water, the specimens were dehydrated in an
ascending series of ethanol and routinely embedded in paraplast.
Immunohistochemical protocol
Sections were cut at 4 µm and mounted onto glass slides. To
reduce unspecific binding, sections were treated with PBS containing
2% (wt/vol) BSA and 2% (wt/vol) normal goat serum and processed for
immunofluorescence. Antiserum 116 (12, 13), raised in rabbits against
human IGF-I, was used at a 1:800 dilution. Sections were incubated with
the IGF-I antiserum for 12 h at 4 C. After repetitive washing in
PBS (pH 7.4), the primary antiserum was detected using biotinylated
goat antirabbit IgG (Bioscience Products, Emmenbrucke, Switzerland;
diluted 1:100) for 30 min at room temperature. Thereafter, sections
were washed in PBS and incubated with streptavidin-fluorescein
isothiocyanate (Bioscience Products, diluted 1:100) for 30 min at room
temperature in the dark.
The specificity of the reactions obtained was tested using the following controls: 1) replacement of the primary antiserum by nonimmune rabbit serum, and 2) preabsorption of the IGF-I antisera with rhIGF-I, rhIGF-II, or bovine insulin (40 µg; 400 µg peptide/ml diluted antiserum). Sections of rat pancreas known to contain IGF-I-immunoreactive islet cells (14) were also processed in every incubation series and served as positive controls. Preabsorption of the antisera with 40 µg rhIGF-I/ml completely blocked immunoreactions. IGF-I immunoreactions were not affected by preabsorption with IGF-II or insulin at concentrations up to 400 µg/ml.
Photomicrographs were taken with a Carl Zeiss Axiophot (Carl Zeiss, Zurich, Switzerland). The fluorochrome was visualized with a fluorescence module for fluorescein isothiocyanate (BP, 450–490 nm; FT, 510; LP, 515–565 nm).
RNA extraction and complementary DNA (cDNA) synthesis
Total RNA from rat liver was prepared by the phenol/chloroform
method with the Ultraspec Extraction Kit (ams, Lugano,
Switzerland) following the instructions of the manufacturer. For cDNA
synthesis, 5 µg RNA were annealed with 1 µM of a
poly(deoxythymidine) primer [5'-CCTGAATTCTAGAGCTCAT(dT17)-3'] for 3
min at 70 C. The RNA/primer mix was incubated for 1 h at 37 C with
15 mM deoxy-NTPs and 10 U AMV reverse transcriptase
(Pharmacia Biotech, Switzerland) in 1 x
reaction buffer [50 mM Tris-HCl (pH 8.3), 40
mM KCl, and 6 mM
MgCl2].
RT-PCR and cloning of the IGF-I fragment
RT-PCR was performed with primers specific for the B and E
domains of rat liver IGF-I (15) (sense primer, 5'-TGGACGCTCTTCA
GTTCGTG-3'; antisense primer, 5'-CTGCACTTCCTCTACTTGTG-3'). One
microliter of cDNA was incubated with 1 µM of the
appropriate sense and antisense primers, 200 µM
deoxy-NTPs, and 1 U Taq polymerase (Pharmacia Biotech) in 1 x incubation buffer [10
mM Tris-HCl (pH 8), 50 mM
KCl, 1.5 mM MgCl2, and
0.001% gelatin]. The amplification program was optimized for a
Stratagene RoboCycler Gradient 40 as follows: one cycle of
10 min at 94 C, 1 min at 59 C, and 1 min at 72 C; 30 cycles of 1 min at
94 C, 1 min at 59 C, and 1 min at 72 C; followed by a final extension
of 5 min at 72 C. The amplification product had a size of 260 bp and
was separated on a 2% agarose gel and eluted using the QIAquick gel
extraction kit (QIAGEN, Chatsworth, CA). The PCR product
was cloned in a pCR-Script SK(+) cloning vector using the cloning kit
of Stratagene (Stratagene, Heidelberg,
Germany).
Synthesis of digoxigenin-labeled RNA probes
The plasmids containing the specific IGF-I fragments served as
templates for synthesis of the digoxigenin (DIG)-labeled RNA probes.
The linearized plasmids were transcribed from the T3 and T7 promotors
using an in vitro transcription kit (Roche,
Mannheim, Germany) in the presence of DIG-UTP according to the
instructions of the manufacturer. No further purification was necessary
after synthesis of the DIG-labeled sense and antisense RNA probes. The
integrity of the probes and the efficiency of the labeling were
confirmed by gel electrophoresis and dot blot analysis.
In situ hybridization
Sections were cut at 4 µm, mounted on SuperFrost Plus slides
(Menzel-Gläser, Germany), and dried overnight at 42 C.
After dewaxing and rehydration, the sections were fixed with 4%
paraformaldehyde and 0.1% glutaraldehyde in 1 x PBS. The
following steps were carried out with diethylpyrocarbonate-treated
solutions in a humidified chamber. The sections were digested with
0.02% proteinase K in 20 mM Tris-HCl (pH 7.4) and 2
mM CaCl2 for 10 min at 37 C to
denature proteins. The background was reduced by treatment of the
sections with 1.5% triethanolamine and 0.25% acid anhydride for 10
min at room temperature. The slides were incubated with 100 µl
prehybridization solution (50% formamide, 1 x PBS, 2.5 x
Denhardt’s solutin, 25 mM EDTA, 275 µg/ml single strand
DNA, and 250 µg/ml yeast transfer RNA) per section for 3 h at 55
C. Hybridization was carried out overnight at the same temperature with
50 µl of the hybridization buffer containing 50% formamide, 1
x PBS, 2 x Denhardt’s, transfer RNA (1.5 µg/ml), single
strand DNA (200 µg/ml), 10 mM dithiothreitol, 20%
dextransulfate, and 200 ng sense (negative control) or antisense probe
(previously denatured for 5 min at 85 C).
Slides were washed for 15 min at room temperature in 2 x SSC, and for 30 min at the specific hybridization temperature at descending concentrations of SSC (2, 1, 0.5, and 0.2 x). DIG detection was performed according to the instructions of the manufacturer. The alkaline phosphatase-coupled antibody against DIG was diluted 1:4000 in 1% blocking reagent (Roche) in buffer 1, and sections were incubated for 1 h at room temperature in the dark. After washing twice in buffer 1 for 15 min each time, the sections were treated with buffer 3 containing 3.5 mM levamisole, 0.1% gelatin, nitro blue tetrazolium (84/µl), and 5-bromo-4-chloro-3-indolyl-phosphate (42 ng/µl). Color development was carried out overnight at room temperature. The reaction was stopped by rinsing the slides in tap water for at least 15 min. Sections were mounted with glycergel. Hybridization with the antisense DIG-labeled RNA probe specific for rat IGF-I revealed positive responses in chondrocytes, whereas the sense RNA probe (negative control) showed no signals.
Statistics
Four-micron sections of both tibiae of 4 normal control rats, 4
hypox rats treated with vehicle, 4 hypox rats treated with GH, and 3
hypox rats treated with IGF-I were cut. For quantitative evaluation of
chondrocytes containing IGF-I immunoreactivity or IGF-I mRNA, 5
sections each per tibia were processed for either immunohistochemistry
or in situ hybridization. Growth plate and articular
cartilage areas were photographed and printed at a final magnification
of x720. On these photographs, the total number of chondrocytes in 2
parallel columns was counted, and the percentage of labeled cells was
determined. This procedure was performed twice to obtain a mean value
for each of the 2 columns in each tibial section and each animal and
for each evaluation technique (immunohistochemistry, in situ
hybridization). Thus, altogether 80 columns were evaluated for each of
the 2 cartilage regions (growth plate and articular cartilage) and each
of the 2 techniques in normal, hypox, and GH-treated rats, and 60
columns were evaluated in IGF-I-treated rats. The chondrocyte layers
were identified using the criteria published previously (10).
Statistical analysis of the data was performed with a StatView 4.5
program (Abacus Concepts, Inc., Berkeley, CA). This included an
ANOVA t test (unpaired) for body weight, serum IGF-I, and
growth plate and articular cartilage height and Bonferroni/Dunn
analysis with a significance level of 5% for the evaluation of
IGF-I-labeled cells. All data are expressed as the mean ±
SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
Replacement with rhIGF-I or rhGH raised serum IGF-I to 674 ± 117
and 945 ± 48 ng/ml, respectively (Table 1).
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). After 24 h of IGF-I or
GH infusion, growth plate heights were 199 ± 12 µm
(P < 0.05) and 211 ± 9 µm (P
< 0.05), respectively (Table 1).
The mean total height of the proximal tibia articular cartilage was
161 ± 8 µm in normal control rats. Neither hypophysectomy
(159 ± 7 µm) nor treatment with GH (160 ± 6 µm) or
IGF-I (162 ± 7 µm) caused significant changes (Table 1).
Inmunohistochemistry and in situ hybridization
Under all experimental conditions, including the normal control,
both IGF-I immunoreactivity and IGF-I mRNA signals were detected in all
chondrocyte layers of the growth plate (Fig. 1) and the articular cartilage (Fig. 2).
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): 60.6 ± 4.3% of the
chondrocytes expressed IGF-I mRNA, but only 24.8 ± 3% showed
IGF-I immunoreactivity (P = 0.0001). In the hypox rats
treated with vehicle, IGF-I, or GH, the number of IGF-I-immunoreactive
chondrocytes was not statistically different from that of IGF-I mRNA
containing chondrocytes (Tables 2 and 3).
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). Treatment with GH raised the percentage of
IGF-I-immunoreactive and IGF-I mRNA-containing cells in all zones of
the growth plate to or above the level found in normal control rats.
IGF-I increased the number of labeled cells only in the proliferative
zone (Table 2).
Articular cartilage
In articular cartilage, the number of IGF-I-immunoreactive
chondrocytes was not statistically different from that of IGF-I
mRNA-containing chondrocytes under the different experimental
conditions (Table 3). The highest
percentage (
30%) of IGF-I-immunoreactive and IGF-I mRNA-containing
chondrocytes was found in the upper hypertrophic zone (Table 3).
Neither hypophysectomy nor GH or IGF-I treatment caused any significant
change in the numbers of labeled chondrocytes in the different
zones.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our findings confirm and extend earlier data on rat (6, 16), human (17), and bony fish (13) cartilage, but are at variance with data reported by Shinar et al. for normal growing rats (8) and by Wang et al. for normal mice (9). The authors of the latter two reports detected IGF-II, but not IGF-I, mRNA in chondrocytes of the rat and mouse epiphyseal growth plate. One possible reason for this discrepancy could be the different age of the experimental animals (25 and 35 days vs. 50 days in our study). However, this is unlikely, because Nilsson et al. (6) found IGF-I mRNA hybridization signals within growth plate chondrocytes of 10-, 28, and 35-day-old rats; the signals were weaker in 10-day-old than in 35-day-old animals. In our study, IGF-I mRNA and IGF-I immunoreactivity correlated well within each of the different chondrocyte layers, and both parameters responded concomitantly to GH treatment after hypophysectomy. This is consistent with the view that IGF-I is produced by chondrocytes, and it supports the concept that this locally produced IGF-I acts at the chondrocyte level in a paracrine/autocrine manner to stimulate longitudinal growth. Recent studies using the Cre/loxP recombination system to delete the IGF-I gene exclusively in the liver of mice (18, 19) underline the potential importance of local IGF-I production, particularly by chondrocytes. Despite abrogation of liver IGF-I mRNA expression and largely reduced circulating IGF-I serum levels, the animals did not show any obvious impairment of postnatal body growth.
The possibility that the in situ IGF-I hybridization signals in our study are due to cross-reactivity with IGF-II mRNA appears unlikely, because the IGF-I cDNA probe used (spanning a region from the middle of the B domain to the middle of the E domain) did not hybridize under the applied experimental conditions with rat IGF-II mRNA or with IGF-I mRNA of a variety of other animal species (not shown), and because the IGF-I mRNA signal corresponded very closely with IGF-I immunoreactivity. It is also unlikely that the IGF-I immunoreactivity resulted from cross-reaction with IGF-II. This has been excluded by the outcome of our absorption controls and in earlier studies on different organs of different species (12, 20), including fish chondrocytes (13). Therefore, the data reported by Shinar et al. (8) and Wang et al. (9) remain unexplained.
The absence of increased IGF-I immunoreactivity within the growth plate during IGF-I infusion argues against the possibility that immunoreactivity in chondrocytes results from circulating IGF-I taken up by the chondrocytes. As infused IGF-I stimulates chondrocyte maturation and thus longitudinal growth in hypox rats (21), it must reach chondrocyte type 1 IGF receptors by diffusion through the extracellular matrix. Apparently, concentrations of diffusing IGF-I in the matrix are much lower than those produced within the chondrocytes and can therefore not be traced by our immunohistochemical method. This may also explain why we did not detect IGF-I secreted by the chondrocytes into the extracellular space. Consequently, one would expect that diffusion of GH-induced circulating IGF-I into the matrix compartment is not detectable either, so that its potential relative contribution to GH-stimulated bone growth cannot be assessed.
In contrast to the growth plate, articular cartilage height, IGF-I mRNA expression, or IGF-I immunoreactivity did not respond to hypophysectomy or GH or IGF-I treatment. Comparable results have been obtained with cultured articular chondrocytes; GH had no stimulatory effect on the proliferation of rabbit (22, 23), human (24), and bovine (25) articular chondrocytes. Furthermore, IGF-I production by rabbit articular chondrocytes was not stimulated by GH (26).
One peculiarity of this study concerns the lower hypertrophic zone in the growth plate of normal rats. Here the number of chondrocytes exhibiting IGF-I mRNA signals by far exceeded the number of IGF-I-immunoreactive chondrocytes. A possible explanation for this discrepancy is that the cells of the lower hypertrophic zone release IGF-I rapidly after its synthesis. The chondrocytes of the lower hypertrophic zone are entering the phase of degeneration, and the zone is invaded by blood vessels, giving rise to mineralization and bone maturation. Therefore, IGF-I released from lower hypertrophic chondrocytes may contribute to the building of new bone. In bone, osteoblasts appear to be the major target of IGF-I. IGF-I stimulates osteoblast replication and enhances osteoblast recruitment (27).
The present study also shows that hypophysectomy does not equally affect all layers of growth plate chondrocytes. Whereas the reduction of IGF-I mRNA and immunoreactivity was most pronounced in the stem cell and proliferative phase (6- to 7-fold), expression remained unchanged in the upper hypertrophic zone. In the lower hypertrophic zone expression was reduced 2- to 3-fold. GH restored expression to normal in all chondrocyte layers and stimulated expression to higher than normal levels in the upper hypertrophic zone. Thus, in contrast to the finding reported by Nilsson et al. (6, 16), stem cells also responded to GH with increased IGF-I expression within 24 h. Although this finding does not exclude an additional priming effect of GH on stem cells to promote their differentiation independently of IGF-I, as postulated by the dual effector theory (28), the production of IGF-I by stem cells is compatible with IGF-I-mediated GH-controlled stem cell maturation. This reasoning is in line with the finding that IGF-I infused into hypox rats significantly enhances stem cell maturation (21), but is in contrast to the findings of Ohlsson et al. (29). This group injected GH (1 µg/day) or IGF-I (10 µg/day) locally above the tibial epiphysial plate of hypox rats for 12 days. Thereafter, they administered GH (20 µg/day) together with [3H]thymidine systemically for 14 days by sc implanted miniosmotic pumps to chase and dilute labeled cells with a high turnover. They found that only GH, not IGF-I, pretreatment increased the number of labeled cells in the germinal layer (which they considered stem cells). It was concluded that in contrast to GH, IGF-I acts only on the proliferation of the resulting chondrocytes. The reasons(s) for the discrepancy between these (29) and our previous (21) findings might lie in the different experimental approaches and evaluation methodologies and possibly also in the different bioactivities of the recombinant IGF-I preparations used by the two groups. Nevertheless, the dramatic reduction of the stem cell cycling time from 50 to 15 days by infused IGF-I as previously reported (21) together with the conspicuous increase in IGF-I mRNA and peptide in the stem cells during GH treatment demonstrated in the present study suggest that stem cell activation by GH is at least in part mediated via IGF-I produced and secreted by these cells.
Finally, the question of whether GH exerts direct (IGF-I-independent) effects in addition to IGF-I-mediated effects on stem cells of the growth plate cartilage in vivo cannot be unambiguously answered at present. Although there is little doubt that IGF-I is required for normal skeletal growth, as exemplified by the homozygous IGF-I knockout mouse (30, 31), it appears difficult, if not impossible, to dissect the local in vivo effects of GH and IGF-I with respect to their action on the growth plate, especially on the stem cell population.
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Acknowledgments |
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Footnotes |
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Received February 18, 2000.
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