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Endocrinology, doi:10.1210/en.2006-1264
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Endocrinology Vol. 148, No. 7 3122-3130
Copyright © 2007 by The Endocrine Society

Insulin-Like Growth Factor-I and Fibroblast Growth Factor, But Not Growth Hormone, Affect Growth Plate Chondrocyte Proliferation

Michele R. Hutchison, Mary H. Bassett and Perrin C. White

Department of Pediatrics, The University of Texas Southwestern Medical Center, Dallas, Texas 75390

Address all correspondence and requests for reprints to: M. Hutchison, Division of Pediatric Endocrinology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9063. E-mail: michele.hutchison{at}utsouthwestern.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Of the many factors that regulate linear growth, IGF-I has a central role in epiphyseal chondrocyte development. Whether IGF-I is solely of systemic or also of local origin is uncertain, as is how other growth factors interact with IGF-I at the growth plate. We studied the proliferative effects of IGF-I on juvenile bovine epiphyseal chondrocytes fractionated by density gradient centrifugation. Cell density correlated with size, glycogen content, and gene expression patterns. There was a gradient of response to IGF-I, with the greatest proliferative response in high-density cells corresponding to the reserve zone, as measured by [3H]thymidine uptake. Low-density (hypertrophic zone) cells proliferated only when exposed to IGF-I and basic fibroblast growth factor (FGF). The gradient of IGF-I response correlated with [125I]IGF-I binding as determined by Scatchard analysis: IGF-I receptor number was 10-fold greater in reserve zone cells than in hypertrophic cells. When exposed to basic FGF for 24 hours, IGF-I binding in hypertrophic cells increased 3-fold. In contrast, no specific binding of GH was demonstrated in juvenile bovine chondrocytes. GH produced neither signal transducer and activator of transcription phosphorylation, increased proliferation, nor increased IGF-I mRNA levels in any chondrocyte fraction. IGF-I mRNA levels were extremely low at 800-1100 copies/µg 18S RNA in bovine chondrocytes. These results suggest that the major regulator of chondrocyte proliferation is systemic IGF-I; FGFs may influence the actions of IGF-I at the growth plate by altering its receptor number in chondrocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE LINEAR GROWTH of long bones is achieved at the growth plate, a layer of cartilage situated between the epiphysis and metaphysis. The chondrocytes of the growth plate exist within a cartilaginous matrix and are arranged in specific layers, or zones. In order from the epiphysis to the metaphysis, these are the reserve zone (RZ) (a layer of small, round cells irregularly arranged), the proliferative zone (wherein the cells divide along the long axis of the bone in regular columns), the prehypertrophic zone, and finally the hypertrophic zone (HZ) (large, glycogen-filled cells) (1, 2).

The cells of the proliferative zone have been regarded as crucial to the regulation of bone growth, because this zone is considered the site of active replication (3). The RZ may supply the proliferative zone from a subset of stem-like cells (4). Distal to the proliferative zone, the cells cease proliferating and begin to differentiate into postmitotic hypertrophic cells (5). Hypertrophic cells secrete the low-molecular-weight collagen, collagen X (COLX), synthesize large amounts of glycogen, and initiate the mineralization of the extracellular matrix. Once their glycogen stores have been depleted, HZ cells undergo apoptosis and leave behind lacunae into which osteoblasts and blood vessels migrate to complete the process of conversion of the cartilaginous matrix to trabecular bone (6).

Whereas the growth plate has been well characterized at the histomorphological level, much remains to be understood about the signals that regulate chondrocyte proliferation and differentiation. Some of the signals regulating proliferation are of systemic origin, and others are autocrine/paracrine in nature. The roles of GH and IGF-I in the regulation of bone growth have been extensively studied. IGF-I has been implicated as a major stimulator of growth plate chondrocyte proliferation by both in vitro studies (7, 8) and gene knockout experiments (9), but the source of IGF-I acting at the growth plate has been controversial. The "somatomedin hypothesis" states that the major role of GH in bone growth is through the regulation of levels of systemic IGF-I from the liver. IGF-I has more recently been proposed to have autocrine/paracrine roles in the growth plate in some (10, 11) but not other (12, 13) studies.

Fibroblast growth factors (FGFs) are also involved in the regulation of longitudinal bone growth. Mice overexpressing FGF2 have short limbs (14), and activating mutations in FGF receptor 3 impair bone growth as seen in achondroplasia and thanatophoric dysplasia (15, 16, 17). Some in vitro studies suggest that FGF2 inhibits growth plate chondrocyte proliferation by down-regulating growth-promoting proteins (18), whereas others have shown that FGF2 enhances proliferation (19, 20). As to the interaction of FGF and IGF-I at the growth plate, synergy was observed in the stimulation of proliferation in chick chondrocytes (21) but not in rat mandibular chondrocytes (22).

Thus far, most studies describing growth plate chondrocytes have used rodent models. Rodent growth plate physiology differs from that of larger mammals; mice and rats do not undergo a pubertal growth spurt, nor do their growth plates fuse completely with puberty as in humans (23). We studied chondrocytes isolated from juvenile cattle aged 6–8 months, believing that a bovine model would approximate the physiology of a growing child. Puberty in both bulls and heifers begins at 10 to 12 months; epiphyseal closure at the distal metacarpus begins at 18 months and is complete by 24 months (24). The relatively large numbers of chondrocytes thus obtained allowed us to use density centrifugation to effectively isolate cells from the various zones of the growth plate and characterize IGF-I action, as well as the interaction between IGF-I and FGF, in these growth plate zones. We were also able to use this system to demonstrate that autocrine actions of IGF-I are unlikely to be physiologically important at the bovine growth plate.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Growth factors and hormones
Recombinant human IGF-I, recombinant human basic FGF (bFGF), and porcine GH (pGH) were purchased from Sigma (St. Louis, MO); bovine GH (bGH) was a gift from Monsanto Corp. (Martinez, CA); calf serum was from Invitrogen (Carlsbad, CA).

Harvesting and separation of chondrocytes
Distal forelimbs of male dairy cattle aged 6–8 months were obtained from a local abattoir and placed immediately on ice for transport. The time from slaughter of the animals to completion of harvesting of the chondrocytes was 3 h or less. The hoof was detached, and the soft tissue was dissected from the metacarpus, which was then split lengthwise with a hammer and chisel. Placement of the split metacarpus in a vise allowed the bone to be broken open at the epiphysis. The chondrocytes thus exposed were mechanically curetted, with care taken to avoid the vascular perichondrium. Perichondrial tissue was obtained by additional curettage of the vascular tissue remaining after removal of growth plate cartilage. The cartilage fragments were placed in serum-free media consisting of DMEM/F12 (1:1) from Invitrogen with the addition of 1 mM cysteine, 1 mM pyruvate, 50 µg/ml ascorbate, and penicillin/streptomycin/amphotericin B (Invitrogen). At least six bones were used for each experiment, representing at least three animals. Any bones that did not break cleanly and readily across the epiphysis were considered potentially partly fused and were discarded. The curetted growth plate material was rinsed several times in PBS and placed in 50 ml serum-free media with 0.15% collagenase P (Roche, Indianapolis, IN) with gentle agitation for 4 h at 37 C. The chondrocytes were passed through a 50 µm mesh to remove occasional bony particulates and fragments of undigested cartilage.

The filtered chondrocytes were subjected to continuous density gradient centrifugation as described, with modifications (25). The cells were mixed with Iso-osmotic Percoll (Sigma) at a final concentration of 25% and transferred to 15 ml polypropylene tubes for centrifugation at 20,000 x g (for isopycnic gradient banding) in a fixed-angle rotor for 30 min at 18 C. In most experiments, each tube contained approximately 2.5 x 107 cells. Cells were removed from the gradient in four fractions of equal volume, with fraction 1 representing the lowest-density cells, and fraction 4 the highest density cells. The approximate density range for each fraction was 1.005–1.016 for fraction 1, 1.017–1.032 for fraction 2, 1.033–1.049 for fraction 3, and 1.050–1.065 for fraction 4. The four fractions were transferred to fresh tubes, the Percoll was diluted with PBS, and the cells were pelleted at 1000 x g for 5 min. At this point, cells were either plated in DMEM/F12 media or used immediately for RNA isolation.

Cell viability was determined using trypan blue staining. After Percoll gradient centrifugation and removal of Percoll with PBS, cell viability for all fractions was consistently between 90 and 95%.

Staining of fixed cells
For glycogen staining, cells from each fraction were removed directly from the Percoll gradient, rinsed once with PBS, and allowed to attach to plastic plates overnight in serum-free medium. Glycogen staining was performed using the Periodic Acid-Schiff Base kit from Sigma.

Microdissection of growth plate zones
Microdissection at x10 magnification was performed to manually separate the four anatomic growth plate zones. The anatomic zones were separated primarily based on morphology; previous in situ experiments for collagen I {alpha} 1 (COL1{alpha}1), COLX, and alkaline phosphatase (ALPL) had been performed to confirm that morphology (cell size and location) corresponded to known gene expression patterns across the bovine growth plate sections (data not shown). Each dissected zone was placed in a separate tube and digested with collagenase as described above, after which the four digests were subjected to Percoll gradient separation as detailed above. Each of the four gradients was removed into four equal fractions, and the cell number contained in these resulting fractions was counted with trypan blue staining.

Cell proliferation assays
Cells taken directly from the Percoll gradient were plated in serum-free DMEM/F12 medium plus 1% BSA on 60-mm plastic plates (Dow Corning, Midland, MI) at a density of 1 x 105 cells per plate and maintained in a humidified incubator at 37 C and 5% CO2. All cell samples were plated and assayed in triplicate. At 12 h, IGF-I (100 ng/ml), FGF (20 ng/ml), or bGH (100 ng/ml) were added as indicated. Eight hours after growth factor addition, [3H]thymidine (95 Ci/mmol; GE Healthcare, Little Chalfont, Buckinghamshire, UK) was added at 1 µCi/plate. Twenty-four hours after growth factor addition, the cells were rinsed once with PBS, scraped into 5% trichloroacetic acid, transferred to 24-mm Whatman (Clifton, NJ) glass filters, and rinsed three times with 5 ml of 5% trichloroacetic acid on a vacuum manifold. The filters were rinsed once with 70% ethanol, dried completely, and transferred to vials with scintillation fluid, and the incorporated radioactivity was measured in a scintillation counter.

RNA isolation, cDNA synthesis, and real-time RT-PCR
RNA was extracted from cells using RNA STAT-60 (Tel-Test, Friendswood, TX) following the instructions of the manufacturer. For each sample, 10 µg of total RNA was subjected to deoxyribonuclease I treatment to remove genomic DNA (DNA-free kit; Ambion, Austin, TX). Five micrograms of deoxyribonuclease I-treated RNA was then reverse transcribed using the High-Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA).

Real-time RT-PCR was performed using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems) following the protocol supplied by the manufacturer. COLX, ALPL, Indian hedgehog (IHH), COL1{alpha}1, collagen II {alpha} 1 (COL2{alpha}1), decorin (DCN), bovine IGF-I, rat IGF-I, bovine IGF-I receptor, and 18S detection was performed using the TaqMan Universal PCR Master Mix (Applied Biosystems) and 100 nM each primer and probe. All primers spanned an intron/exon boundary. All real-time RT-PCR reactions were confirmed to produce only a single PCR product. The primer and probe sets are displayed in Table 1Go.


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  		TABLE 1. Oligonucleotides used for real-time RT-PCR

 
Relative gene expression for each mRNA was calculated by the {Delta}{Delta}Ct method using fraction 1 as the calibrator. As an internal control, each individual sample was normalized to its 18S ribosomal RNA content. Results were expressed as the n-fold difference in gene expression and determined by the following formula: n-fold = 2–({Delta}Ct sample–{Delta}Ct calibrator), in which the parameter Ct (threshold cycle) is defined as the fractional cycle number at which the PCR reporter signal passes a fixed threshold. {Delta}Ct values of the sample and calibrator were determined by subtracting the average Ct value of the transcript under investigation from the average Ct value of the 18S rRNA gene for each sample. The {Delta}{Delta}Ct method is more fully described in User Bulletin 2 of the ABI Prism 7700 Sequence Detection system (Applied Biosystems).

Quantitation of IGF-I message
Cells from the four chondrocyte fractions were plated immediately after the Percoll gradient separation in serum-free medium at a density of 2 x 106 cells per 150-mm plate. Primary rat hepatocytes were generated as described previously (26) and plated in William’s medium (Invitrogen) plus 1% BSA at 2 x 106 cells per 150-mm plate. Cells were given either no treatment or 100 ng/ml bGH for either 6 or 24 h, after which the medium was aspirated and the cells were dissolved in RNA STAT-60 solution. RNA isolation and real-time RT-PCR was performed as described above. Primers used for the quantitation of bovine IGF-I message by TaqMan real-time RT-PCR were designed to span the boundary between exons 3 and 4; the four major human IGF-I transcripts include exons 3 and 4, and the same is presumed to be the case for cattle. For quantitation, a 418 bp fragment of bovine IGF-I was generated by PCR from cDNA made from whole bovine liver and inserted into pCRII-TOPO (Invitrogen). Similarly, an 187 bp fragment of 18S cDNA was inserted into pCR2.1-TOPO. Standard curves were generated with both constructs for use in real-time RT-PCR reactions. For quantitation of rat IGF-I message, primers were designed to span exons 2 and 3, and a 100 bp fragment of rat IGF-I was generated from whole rat liver and inserted into PCRII-TOPO.

Western immunoblotting for phospho-signal transducer and activator of transcription 5 (STAT5)
Cells were plated as described above for IGF-I quantitation and treated with 100 ng/ml bGH for 5, 10, or 30 min. Cells were then lysed in radioimmunoprecipitation assay buffer (1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and 1 mM orthovanadate in PBS) with protease cocktail (Sigma) added. Total protein concentrations were measured with the Bio-Rad (Hercules, CA) protein assay, equal amounts of total protein were separated on 10% polyacrylamide gels, and the protein was transferred electrophoretically to Hybond ECL membranes (GE Healthcare Biosciences). The membranes were blotted with rabbit anti-phospho-STAT5A/B (Sigma) according to the recommendations of the manufacturer. Chemiluminescent detection was performed with the ECL plus Western blotting detection system (GE Healthcare Biosciences).

Scatchard analyses
Each of the factors (IGF-I, pGH, and bGH) was labeled with 125I (GE Healthcare Biosciences) using Iodogen tubes from Pierce (Rockford, IL). Free 125I was separated from the labeled protein by passing over a Zeba desalt spin column (Pierce). Specific activities ranged from 100 to 400 µCi/µg. Cell fractions 1 and 4 from the Percoll gradient and primary rat hepatocytes were plated at 5 x 105 cells in 0.5 ml DMEM/F12 at 1:1 plus HEPES buffer (pH 7.4) and 1% BSA and were allowed to attach overnight, after which cells were incubated with labeled hormone at 4 C for 6 h. Labeled hormone concentrations ranged from 5 x 10–12 to 1 x 10–9 M. Nonspecific binding was determined by incubating cells under identical conditions with a 500-fold excess of unlabeled hormone. The cells were rinsed three times with cold PBS and solubilized in 0.3% SDS for measurement of bound radioactivity in a {gamma}-counter. The receptor number per cell and receptor dissociation constant (Kd) were estimated by Scatchard analysis of competition binding data (27), and statistical significance of differences was determined using Student’s t test (28). Curve fits for Scatchard analyses were linear regressions with R2 ≥ 0.95.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Verification of chondrocyte separation
Growth plate chondrocytes of the bovine distal metacarpus were excised, digested, and separated into four separate fractions based on their respective densities using Percoll gradients as described previously (25). In our hands, the cells were distributed continuously in these gradients rather than in distinct bands. Typically, six metacarpal growth plates yielded a total of 1–1.2 x 108 cells, distributed approximately 5–10% in fraction 1, 10–15% in fraction 2, 35–40% in fraction 3, and 35–40% in fraction 4.

Differentiation of growth plate chondrocytes results in glycogen accumulation and hypertrophy. Our cell fractions differed in glycogen content and overall size; fraction 1 cells were much larger those of fraction 4 (Fig. 1Go). Cells from all four fractions stained positively for glycogen but only faintly in fraction 4 cells.


Figure 1
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  		FIG. 1. Representative bright-field photomicrographs (x20 magnification, labeled with the fraction number) of chondrocytes from the four density fractions. Cells were removed from the Percoll gradient fractions, fixed to plastic, and stained for glycogen with periodic acid Schiff. Arrows indicate large, glycogen-stained hypertrophic cells.

 
To determine the extent to which Percoll gradient fractions corresponded to precise anatomic zones of the intact growth plate, we performed manual microdissection of fresh bovine growth plate sections and then subjected the dissected zones to Percoll gradient centrifugation as above. Figure 2Go shows a typical bovine growth plate section with the four anatomical zones as they were dissected. Cell counts revealed that, in fact, the gradient fractions do approximately correspond to the anatomical zones: 79% of the cells dissected from the RZ sedimented in the highest density fraction (fraction 4), and no cells sedimented in the low density fraction 1. Conversely, the dissected HZ produced a gradient wherein 83% of the cells sedimented in fraction 1, and no cells were present in either fraction 3 or 4. For the remainder of this paper, we will use the terms "fraction 1" and "HZ" interchangeably, as well as "fraction 4" and "RZ."


Figure 2
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  		FIG. 2. Representative hematoxylin-eosin stain of a bovine growth plate section, indicating the four zones. The zones were separated by manual microdissection, digested with collagenase, and subjected to density gradient centrifugation. Each gradient was divided into four equal fractions, and the cell number in each fraction was counted with trypan blue. Cell numbers shown are the average of two separate experiments, expressed as percentage for each gradient. Bold percentages represent cells appearing in their expected positions within each gradient. R, Reserve; P, proliferative; PH, prehypertrophic; H, hypertrophic.

 
The four chondrocyte fractions were analyzed by real-time RT-PCR for the expression of transcripts known to be enriched in hypertrophic cells. COLX, ALPL (29), and the paracrine hormone IHH (25) are specific to the HZ. Figure 3Go shows clear gradients of expression for COLX and IHH, with the highest expression in the least-dense fraction 1 cells. The relative abundance of COLX message of 10:1 in HZ compared with RZ cells agrees with that reported by others (30). ALPL, although clearly least abundant in the fraction 4 cells, has approximately equal levels in fractions 1 through 3. COL2A1, a marker of early-phase differentiation (31), was most highly expressed in fractions 2 and 3.


Figure 3
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  		FIG. 3. Relative levels of COLX, ALPL, IHH, and COL2A1 present in the four chondrocyte fractions. Real-time RT-PCR was used to quantitate mRNA levels of the marker proteins in the four density fractions of bovine growth plate chondrocytes. Data points were calculated using the {Delta}{Delta}Ct method and represent the mean ± SE of real-time data from at least five separate chondrocyte preparations, expressed as fold difference from fraction 4 (the calibrator). *, P < 0.016; **, P < 0.001 (compared with fraction 4).

 
To demonstrate that the chondrocyte preparations were not contaminated with osteoblasts, the cell fractions were analyzed by real-time RT-PCR for levels of the osteoblast-specific transcription factor runt-related transcription factor 2 (RUNX2). RUNX2 levels were low and did not differ significantly across the chondrocyte fractions (data not shown.) To ensure that the chondrocyte fractions were not contaminated with perichondrium, three separate experiments were performed in which perichondrial tissue was dissected from bones after the growth plate cartilage was removed. This was digested with collagenase and subjected to Percoll centrifugation. In all three experiments, the perichondrial cells sedimented in the portion of the gradient corresponding to fraction 4. Chondrocyte fractions and perichondrium were analyzed by real-time RT-PCR for levels of transcripts known to be specific to the perichondrium. Both COL1{alpha}1 and the proteoglycan DCN were enriched in perichondrial tissue (Fig. 4Go) but not in fraction 4, confirming that fraction 4 is not significantly contaminated with perichondrial cells. In addition, perichondrial cells did not proliferate in response to IGF-I (data not shown).


Figure 4
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  		FIG. 4. COL1A1 and DCN mRNA levels present in the four chondrocyte fractions and perichondrium (P) as determined by real-time RT-PCR. Data points were calculated using the {Delta}{Delta}Ct method and represent the mean ± SE of real-time data from three separate chondrocyte preparations, expressed as fold difference from fraction 4 (the calibrator). *, P < 0.01 (compared with fraction 4).

 
Chondrocyte proliferative responses to GH, IGF-I, and bFGF
Cell proliferation studies were performed with chondrocytes in low-density monolayer culture on plastic. Others have demonstrated that chondrocytes grown on naked plastic eventually dedifferentiate (32, 33). In our system, COLX expression is diminished by culture d 7 and is almost undetectable by d 14 (data not shown). For this reason, all proliferation studies were completed by d 2–3.

Because GH and IGF-I are thought to be the predominant systemic factors stimulating proliferation at the prepubertal growth plate, we initially examined the response of the four cell fractions to either 100 ng/ml bGH or 100 ng/ml IGF-I, with the expectation that the stimuli would have a greater proliferative effect in the high-density, less differentiated cells. GH did not increase proliferation in any fraction, and the combination of bGH and IGF-I did not increase proliferation beyond that seen with IGF-I alone (data not shown). There was a marked difference in response to IGF-I across the cell fractions. IGF-I alone did not stimulate proliferation in fraction 1 or 2 cells, whereas the greatest IGF-I-stimulated proliferation was consistently seen in fraction 4 cells, with an increase over baseline approximately 3-fold. Meanwhile, bFGF alone did not stimulate proliferation in any cell fraction (Fig. 5Go). IGF-I alone had little effect on proliferation in the more differentiated fraction 1 and 2 cells, but the combination of IGF-I and bFGF increased proliferation in these cells. In contrast, bFGF did not increase IGF-I-stimulated [3H]thymidine uptake in fraction 4 cells.


Figure 5
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  		FIG. 5. Cell proliferation of the four chondrocyte fractions (Fx) in response to IGF-I (100 ng/ml), FGF (20 ng/ml), or the two in combination. Proliferation assessed as counts per minute of [3H]thymidine uptake. Error bars represent 95% confidence intervals. n = 9 per bar. *, P < 0.0001; **, P < 0.001 (when compared with no addition).

 
Testing the somatomedin hypothesis in fractionated bovine growth plate chondrocytes
GH does not activate STAT5 in bovine chondrocytes.
Chondrocytes from all four gradient fractions were plated and exposed to bGH at 100 ng/ml for 5, 10, or 30 min. Anti-phospho-STAT5 raised to the tyrosine-phosphorylated sequence corresponding to amino acids 687–698 of human STAT5 (which is identical to the rat and bovine sequences) was used to assess STAT5 phosphorylation in response to bGH addition. We were unable to demonstrate phosphorylation of STAT5 in response to bGH in any chondrocyte fraction at any time point. As a positive control, rat primary hepatocytes were similarly treated; STAT5 activation was apparent at 10 min (Fig. 6Go), although the signal was somewhat weak, suggesting that the conditions used for hepatocyte culture may not have been ideal. However, hepatocytes under identical conditions responded to GH with a robust increase in IGF-I message levels (see below).


Figure 6
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  		FIG. 6. Western immunoblotting for phospho-STAT5 in response to bGH. Cells from each of the four fractions were exposed to bGH for 5, 10, or 30 min, and cells lysates were assessed for the presence of tyrosine-phosphorylated STAT5 protein. A representative blot is shown of fraction 1 (Fx1) cells exposed to bGH for 10 min. Rat primary hepatocytes (Hep) were used as a positive control. The arrow indicates the predicted location of phospo-STAT5.

 
GH does not increase IGF-I message in bovine chondrocytes.
There was no significant difference in IGF-I message content between the four fractions using cells taken directly from the gradient; moreover, IGF-I message was extremely low, at 0.8–1.1 x 103 copies of IGF-I message per microgram of 18S RNA (Table 2Go). For comparison, IGF-I message in bovine whole liver was more than 6000-fold higher, at 6.7 x 106 copies/µg 18S.


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  		TABLE 2. IGF-I mRNA quantitation in bovine chondrocytes and liver

 
To assess whether bGH could increase IGF-I message in the growth plate cells, real-time RT-PCR performed to assess the change in IGF-I mRNA after incubation with 100 ng/ml bGH at 6 and 24 h. There was no significant change in the level of IGF-I message with bGH in any of the four fractions at either time point (data shown for 24 h incubation), whereas bGH produced a 50-fold increase in IGF-I message in primary rat hepatocytes at 24 h (Table 2Go).

Scatchard analysis of hormone binding sites
Because the response to IGF-I varied across the chondrocyte fractions and we detected no measurable response to GH, we used classic ligand binding studies to assess the number of hormone binding sites present on fraction 1 and 4 cells. Neither pGH nor bGH showed any specific binding to either chondrocyte fraction. Figure 7Go shows the Scatchard plot of pGH binding to primary rat hepatocytes, with a dissociation constant of 1.05 nM and approximately 10,800 GH binding sites per hepatocyte, but no specific binding to fraction 1 chondrocytes. Similar results were obtained using chondrocytes from fraction 4 (data not shown). Specific binding of IGF-I to bovine chondrocytes was readily demonstrable. Scatchard analysis revealed a single class of high-affinity IGF-I binding sites in both cell fractions with a Kd of 0.31 ± 0.2 nM. The number of IGF-I receptors present in fraction 1 was 3100 ± 150 receptors per cell and in fraction 4 was 30,000 ± 1200 receptors per cell. Identical results were obtained in two separate experiments.


Figure 7
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  		FIG. 7. Representative Scatchard plot of [125I]pGH binding to hypertrophic chondrocytes ({square}). Similar results were obtained with [125I]bGH in both fraction 1 and 4 chondrocytes. Rat primary hepatocytes (bullet) were used as a positive control.

 
Because IGF-I did not produce a proliferative response in fraction 1 cells in the absence of bFGF, we questioned whether bFGF might potentiate IGF-I-stimulated proliferation by increasing IGF-I receptor number in these cells. Scatchard analysis of IGF-I binding to fraction 1 cells incubated with bFGF for 24 h showed an increase in IGF-I binding sites from 3000 ± 130 in the absence of bFGF to 9800 ± 300 in the presence of bFGF (Fig. 8Go). Nearly identical results were obtained in two separate experiments. The addition of bFGF to fraction 1 cells did not significantly affect IGF-I receptor affinity. Real-time RT-PCR revealed no significant difference in IGF-I receptor mRNA levels between fraction 1 and 4 cells. Moreover, fraction 1 cells incubated with bFGF under conditions identical to that which increased IGF-I receptor number did not show a significant increase in IGF-I receptor mRNA levels (data not shown).


Figure 8
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  		FIG. 8. Representative Scatchard plot of [125I]IGF-I binding to hypertrophic chondrocytes after 24 h incubation with ({blacksquare}) or without (bullet) 20 ng/ml bFGF.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Density fractionation of bovine growth plate chondrocytes
The zonal architecture of the growth plate was originally defined histomorphologically (34), and subsequent in vivo studies distinguished the cells in each zone using such characteristics as their proliferative responses to IGF-I or GH (35). Most in vitro studies used chondrocytes from rodents (7, 36, 37), chick sterna (21, 38), newborn (39) or fetal calves (30), or chondrocyte cell lines (40, 41), and most cell preparations were used without fractionation. We sought to establish a model of the growth plate that would approximate that of the growing, prepubertal child using bones from juvenile cattle that had not entered puberty.

We found that chondrocyte cell density correlates very well with size, glycogen content, gene expression patterns, and mitogenic response to IGF-1. Microdissection followed by gradient centrifugation demonstrated that the gradient fractions approximately correspond to the anatomic zones of the growth plate, with HZ cells primarily in fraction 1 and RZ cells primarily in fraction 4. Fractions 2 and 3 are likely to contain cells from the prehypertrophic zone and proliferative zone, respectively, because a marker for proliferative chondrocytes, COL2A1, had the highest expression in these cells. Additional work is needed to better characterize these cell fractions.

Differential response to IGF-I and bFGF across the chondrocyte fractions
The gradient of IGF-I responsiveness across the cell fractions had the opposite trend to that of bFGF. bFGF alone did not increase proliferation in any of the fractions but did facilitate IGF-I-stimulated proliferation in hypertrophic cells. The differential responses to IGF-I in HZ and RZ cells were correlated with numbers of IGF-I binding sites. Because the number of IGF-I binding sites increased in fraction 1 in the presence of bFGF, bFGF may facilitate IGF-I action in hypertrophic chondrocytes by increasing IGF-I receptor number. FGF2 is synthesized by fetal ovine chondrocytes (42), FGF18 is present in perichondrium (43), and we found that hypertrophic cells of the bovine growth plate synthesize FGF1 (our unpublished observations), suggesting that locally produced FGFs may regulate IGF-I receptor levels in the HZ. Epidermal growth factor modestly increases IGF-I receptor number in fetal bovine chondrocytes (39), making it likely that multiple factors influence IGF-I activity in the growth plate by regulating IGF-I receptor levels.

Several FGFs have been shown to regulate both chondrocyte proliferation and differentiation, and most of these actions are to be transduced by FGF receptor 3. In late mouse embryological development, FGF receptor 3 activation inhibits both chondrocyte proliferation and differentiation (44, 45). Mice made to overexpress FGF9 at the growth plate have reduced numbers of hypertrophic cells (46). In vitro, FGF2 appears to inhibit entry into the hypertrophic phenotype, whereas FGF9 inhibits terminal differentiation (19). Our results suggest that FGFs regulate IGF-I activity in terminally differentiated chondrocytes, because HZ cells were made to proliferate in the presence of IGF-I and bFGF. It is important to note that the proliferation seen in the HZ cells is nonphysiological, because in vivo studies suggest that no proliferation occurs in the HZ (35). The proliferation seen in vitro may reflect a loss of tonic inhibition that is present in situ from exposure to the extracellular matrix or secreted factors. bFGF might increase IGF-I receptor number in committed hypertrophic cells to encourage IGF-I-mediated hypertrophy rather than proliferation, because IGF-I is thought to promote hypertrophy via insulin-like actions (47). Thus, the model of FGF action that involves modulation of chondrocyte proliferation and hypertrophic differentiation may be extended to include the facilitation of IGF-I-dependent cellular hypertrophy.

Previous in vivo studies suggest that the greatest proliferation occurs in the high-density small cells of the so-named proliferative zone. Fraction 4, which corresponds to the RZ, consistently demonstrated the greatest proliferative response to IGF-I in vitro. Because fraction 4 contains approximately 35–40% of the total number of growth plate cells, it is likely to include cells from the proliferative zone. However, COL2A1 expression suggests that many PZ cells are found in fraction 3. Perhaps the in vitro proliferation of RZ cells, as well as of HZ cells, results from the loss, under the culture conditions used in our studies, of factors tonically inhibiting cell division in the intact growth plate.

GH action at the growth plate
The somatomedin hypothesis proposes that the role of GH is to directly stimulate synthesis of IGF-I in the liver. The GH receptor signals through the JAKs (Janus kinases) and STATs (48). The molecules responsible for transducing the GH-dependent signal for IGF-I synthesis in hepatocytes are JAK2 and STAT5 (49).

Although IGF-I clearly has a role in regulating chondrocyte proliferation, there has been controversy as to whether significant amounts of IGF-I are produced at the growth plate to act in an autocrine or paracrine manner. In rodents, IGF-I mRNA was detected in situ (50, 51) and in vitro (52) and was GH responsive (10), but these findings were not confirmed in other studies (12, 13). IGF-I message is very low in fetal ovine (53), fetal bovine (30), and fetal human (54) growth plate chondrocytes. We confirmed these latter observations in juvenile cattle. GH has no appreciable direct effect on mitogenesis, STAT5 activation, or IGF-I synthesis in bovine chondrocytes. Moreover, specific GH binding was not demonstrable in these cells, suggesting that GH does not act at the growth plate to increase IGF-I synthesis as it does in the liver. Together, these results support the somatomedin hypothesis of GH/IGF-I action and suggest that the major stimulus for growth in large mammals is IGF-I of systemic origin.


   Acknowledgments
 
We recognize the generosity of David Meyers, Alan Rubin, and their employees at Dallas City Packing Company for cheerfully supplying calf forelegs each week. We also thank Daniella Rogoff and Kelli Black for their assistance with the primary hepatocyte cultures.


   Footnotes
 
This work was supported by Grant DK073447 (to M.R.H.) from the National Institutes of Health.

Disclosure Statement: The authors have nothing to disclose.

First Published Online March 29, 2007

Abbreviations: ALPL, Alkaline phosphatase; bFGF, basic fibroblast growth factor; bGH, bovine GH; COL1{alpha}1, collagen I {alpha} 1; COL2{alpha}1, collagen II {alpha} 1; COLX, collagen X; DCN, decorin; FGF, fibroblast growth factor; HZ, hypertrophic zone; IHH, Indian hedgehog; pGH, porcine GH; RUNX2, runt-related transcription factor 2; RZ, reserve zone; STAT5, signal transducer and activator of transcription 5.

Received September 14, 2006.

Accepted for publication March 21, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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