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. 2011 Nov;26(11):2622-33.
doi: 10.1002/jbmr.502.

The critical role of the epidermal growth factor receptor in endochondral ossification

Xianrong Zhang  1 , Valerie A Siclari, Shenghui Lan, Ji Zhu, Eiki Koyama, Holly L Dupuis, Motomi Enomoto-Iwamoto, Frank Beier, Ling Qin
Affiliations

The critical role of the epidermal growth factor receptor in endochondral ossification

Xianrong Zhang et al. J Bone Miner Res. 2011 Nov.

Abstract

Loss of epidermal growth factor receptor (EGFR) activity in mice alters growth plate development, impairs endochondral ossification, and retards growth. However, the detailed mechanism by which EGFR regulates endochondral bone formation is unknown. Here, we show that administration of an EGFR-specific small-molecule inhibitor, gefitinib, into 1-month-old rats for 7 days produced profound defects in long bone growth plate cartilage characterized by epiphyseal growth plate thickening and massive accumulation of hypertrophic chondrocytes. Immunostaining demonstrated that growth plate chondrocytes express EGFR, but endothelial cells and osteoclasts show little to no expression. Gefitinib did not alter chondrocyte proliferation or differentiation and vascular invasion into the hypertrophic cartilage. However, osteoclast recruitment and differentiation at the chondro-osseous junction were attenuated owing to decreased RANKL expression in the growth plate. Moreover, gefitinib treatment inhibited the expression of matrix metalloproteinases (MMP-9, -13, and -14), increased the amount of collagen fibrils, and decreased degraded extracellular matrix products in the growth plate. In vitro, the EGFR ligand transforming growth factor α (TGF-α) strongly stimulated RANKL and MMPs expression and suppressed osteoprotegerin (OPG) expression in primary chondrocytes. In addition, a mouse model of cartilage-specific EGFR inactivation exhibited a similar phenotype of hypertrophic cartilage enlargement. Together our data demonstrate that EGFR signaling supports osteoclastogenesis at the chondro-osseous junction and promotes chondrogenic expression of MMPs in the growth plate. Therefore, we conclude that EGFR signaling plays an essential role in the remodeling of growth plate cartilage extracellular matrix into bone during endochondral ossification.

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Figures

Figure 1
Figure 1. In vivo inhibition of EGFR activity expands the long bone growth plates
One-month-old rats were treated with either vehicle (A,C,E) or gefitinib (B,D,F) for 7 days and their tibiae were processed for MMA sections (A-D) or paraffin sections (E-F) for histological examination. Staining of the bone with Goldner’s trichrome staining showed a striking enlargement of the growth plate, particularly the hypertrophic zone, in the gefitinib-treated animals (the whole bone at low magnification (A-B) and the growth plate region at high magnification (C-D)). PZ: proliferative zone; HZ: hypertrophic zone. (E-F) Safranin O staining confirms the increase in the length of hypertrophic zone. (G) Quantification of the lengths of whole growth plate (GP) and each zones. n=5/group. a: p<0.001 vs. vehicle. (H) Quantification of chondrocyte number along the longitudinal length of cartilage.
Figure 2
Figure 2. Gefitinib treatment expands the growth plate and decreases bone volume in the primary spongiosa (PS) and secondary spongiosa (SS) of rat long bones
(A)Magnified image of COJ and primary spongiosa in Goldner’s trichrome-stained rat tibiae treated with vehicle or gefitinib. Arrows indicate cartilage remnants (light blue) in the bone spicules. (B) MicroCT images of longitudinal sections of distal femurs of vehicle- and gefitinib-treated rats. (C) Structural parameters of trabecular bone in the primary spongiosa. (D) Structural parameters of trabecular bone in the secondary spongiosa. BV/TV: trabecular bone volume/tissue volume; TbTh: trabecular thickness; TbSp: trabecular separation; TbN: trabecular number. n=6/group. b: p<0.01; c: p<0.05 vs. vehicle.
Figure 3
Figure 3. Gefitinib treatment does not alter chondrocyte proliferation, differentiation and vascular invasion
(A) BrdU incorporation (brown staining) in the proliferative zone of growth plates from vehicle- and gefitinib-treated rats. (B) qRT-PCR analysis of gene expression of stage-specific chondrocyte markers (aggrecan, col2a1, Ihh, and Col10a1) and transcription factors (Sox9 and Runx2). b: p<0.01 vs. vehicle. (C) H&E staining of rat tibiae show that there is no overt difference in localization, size or morphology of blood vessels in the gefitinib-treated rats compared to vehicle-treated rats. Red blood cells are stained red with no blue nuclei.
Figure. 4
Figure. 4. Osteoclast differentiation at the COJ iss delayed in the gefitinib-treated rats
(A) TRAP staining (red) of rat tibiae reveals that gefitinib-treated rats have fewer osteoclasts at the COJ but comparable osteoclast number in the primary spongiosa underneath the junction. (B) qRT-PCR measurement of mRNA expression of RANKL and OPG in the growth plate dissected from vehicle- and gefitinib-treated rats. (C) qRT-PCR measurement of mRNA expression of RANKL and OPG in primary chondrocytes treated by TGFα for indicated time points. The mRNA level of the control group at 1 h was set to 1. (D) qRT-PCR demonstrates that 5 μM gefitinib abolishes the effects of 24 h of TGFα treatment on RANKL and OPG expression in primary chondrocytes. a: p<0.001; b: p<0.01; c: p<0.05 vs. control.
Figure 5
Figure 5. Blocking EGFR activity suppresses the expression of MMPs and ECM degradation in growth plate
(A) qRT-PCR analyses of MMP mRNA expression in the growth plate tissues from vehicle- and gefitinib-treated rats. b: p<0.01; c; p<0.05 vs. vehicle. (B) Western blotting shows that the protein levels of MMP9, 13, and 14 in the growth plates from gefitinib-treated rats were decreased to 7.9±2.1%, 14.0±12.6%, and 39.2±10.5%, respectively, of those from vehicle group as measured by densitometric analyses. (C) In situ hybridization reveals a decrease of MMP13 expression in the gefitinib-treated growth plate. (D) Gelatin zymography analysis of protein lysates from growth plate further confirms the decrease of MMP9 activity in gefitinib-treated animals. The activity of MMP2 was shown here as an internal control. The amounts of pro (a) and mature (b) MMP9 in gefitinib group decreased to 30% and 47% of that in vehicle group as measured by densitometric analysis. (E) Polarized light microscopy of unstained tibial paraffin sections from vehicle and gefitinib-treated rats. The sections were oriented with maximum birefringency (white blue). COJ is depicted as a dashed line separating growth plate (GP) and primary spongiosa (PS) areas. The intensity of birefringency was then analyzed and quantified by ImageJ. n=6/group. b: p<0.01 vs. vehicle. (F) Immunostaining of type II collagen in the growth plate. (G) Immunostaining of collagenase-produced 3/4 cleavage fragment of type II collagen. (H) Western blot of protein lysates from growth plate tissues using an antibody to the MMP-cleaved aggrecan product.
Figure 6
Figure 6. EGFR signaling regulates MMP9, 13 and 14 expression in primary chondrocytes
(A) Time course of TGFα regulation of MMP expression. The mRNA levels were analyzed by qRT-PCR and adjusted to the mRNA level in the control harvested at 1 h. a: p<0.001; c: p<0.05 vs. control. (B) Western blots of MMP proteins in the primary chondrocytes after 4 days of TGFα treatment. Medium was changed after 2 days along with the addition of fresh TGFα. In the gefitinib lanes, 5 μM gefitinib was added to the cells 1 h prior to TGFα treatment. The blots were quantified by densitometric measurement. The data were normalized against β-actin and the corresponding values were shown underneath each blot.
Figure 7
Figure 7. Mice deficient in chondrogenic EGFR activity exhibit enlarged hypertrophic zone
(A) H&E staining of tibial growth plate from Col2-Cre EgfrWa5/f mice and controls. Double arrow lines indicate the hypertrophic zone. (B) Quantification of the lengths of proliferative (PZ) and hypertrophic (HZ) zones. n=4-5/group. a: p<0.001 vs Egfr+/f; b: p<0.005 vs EgfrWa5/f; c: p<0.05 vs Egfr+/f. (C) A model of how EGFR signaling stimulates cartilage degradation. Two mechanisms contribute to this function of EGFR. First (1), EGFR signaling up-regulates the expression of MMPs (, , and 14) in the growth plate and thus is responsible for cartilage ECM degradation. Second (2), EGFR signaling is important for RANKL expression in the growth plate and thus is responsible for osteoclastogenesis at the COJ.
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