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. 2010 May 1;341(1):236-45.
doi: 10.1016/j.ydbio.2010.02.033. Epub 2010 Mar 4.

Stimulation of chondrocyte hypertrophy by chemokine stromal cell-derived factor 1 in the chondro-osseous junction during endochondral bone formation

Lei Wei  1 , Katsuaki Kanbe, Mark Lee, Xiaochun Wei, Ming Pei, Xiaojuan Sun, Richard Terek, Qian Chen
Affiliations

Stimulation of chondrocyte hypertrophy by chemokine stromal cell-derived factor 1 in the chondro-osseous junction during endochondral bone formation

Lei Wei et al. Dev Biol. .

Abstract

During endochondral bone formation, chondrocytes undergo differentiation toward hypertrophy before they are replaced by bone and bone marrow. In this study, we found that a G-protein coupled receptor CXCR4 is predominantly expressed in hypertrophic chondrocytes, while its ligand, chemokine stromal cell-derived factor 1 (SDF-1) is expressed in the bone marrow adjacent to hypertrophic chondrocytes. Thus, they are expressed in a complementary pattern in the chondro-osseous junction of the growth plate. Transfection of a CXCR4 cDNA into pre-hypertrophic chondrocytes results in a dose-dependent increase of hypertrophic markers including Runx2, Col X, and MMP-13 in response to SDF-1 treatment. In organ culture SDF-1 infiltrates cartilage and accelerates growth plate hypertrophy. Furthermore, a continuous infusion of SDF-1 into the rabbit proximal tibial physis results in early physeal closure, which is accompanied by a transient elevation of type X collagen expression. Blocking SDF-1/CXCR4 interaction suppresses the expression of Runx2. Thus, interaction of SDF-1 and CXCR4 is required for Runx2 expression. Interestingly, knocking down Runx2 gene expression results in a decrease of CXCR4 mRNA levels in hypertrophic chondrocytes. This suggests a positive feedback loop of stimulation of chondrocyte hypertrophy by SDF-1/CXCR4, which is mediated by Runx2.

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Figures

Fig. 1
Fig. 1. Distribution of SDF-1 and CXCR4 in the growth plate
Immunohistochemical staining for CXCR4 and SDF1 was performed on one day old mouse proximal tibial growth plates. CXCR4 expression was strongest in the hypertrophic zone (red color) (A), while SDF-1 was most strongly expressed in bone marrow (red color) adjacent to the hypertrophic chondrocytes(B). Scale bar = 20 µm.
Fig. 2
Fig. 2. Expressions of CXCR4, Type X, and Runx2 in proliferative, pre-hypertrophic, and hypertrophic chondrocytes
Total RNA was isolated from three different zones of 17-day-old chicken embryonic sterna and qRT-PCR was carried out to quantitate CXCR4 mRNA. CXCR4 mRNA was much higher in hypertrophic chondrocytes than in proliferative and pre-hypertrophic chondrocytes (A), similar to the up-regulation of hypertrophic markers type X collagen and Runx2 (B). Bar graphs show the averages of quantified data from three independent experiments using Real Time PCR. *: p<0.05 compared to proliferative chondrocytes.
Fig. 3
Fig. 3. SDF-1/CXCR4 induction of chondrocyte hypertrophy
To determine whether SDF-1/CXCR4 signaling induces chondrocytes hypertrophy, 50% confluent of proliferative chondrocytes were infected with chicken fibroblast conditional medium containing RCAS-CXCR4 or RCAS alone for 4 days and then treated with 100 ng SDF-1/mL for one day. CXCR4 expression in proliferative chondrocytes transfected with RACS-CXCR4 was detected by immuno-fluorescent staining with myc-tag antibody but not in RACS transfected group (A, red color for CXCR4). qRT-PCR was performed for MMP-13 (B) and Type X collagen (C). MMP-13 and Type X collagen mRNA expression increased after treatment of proliferative chondrocytes transfected with RCAS-CXCR4 with SDF-1 in a dose dependent manner. Similar results were observed in three experiments for mmp-13. Bar graphs show the averages of quantified data of type X collagen using Real time PCR from three independent experiments. *: Significant difference in comparison to the non- SDF-1 treated group, p<0.05. Scale bar = 20 µm. CECs: Chicken Embryonic Proliferative Chondrocytes
Fig. 4
Fig. 4. SDF-1 can diffuse into cartilage
To determine whether SDF-1 can diffuse into cartilage, 17-day-chicken embryonic sternal cartilage was incubated with SDF-1 (100ng/mL) or without SDF-1 for 1h, 3h, and 24h. 10 µm frozen sections were used to detect SDF-1 by immuno-fluorescent staining with mAb against SDF-1. Fluorescence microscopy showed a progressive increase in SDF-1 staining (red color) surrounding chondrocytes during the 24 h time course (A, B,C) compared to control at 24 h (D). Scale bar = 20 µm.
Fig. 5
Fig. 5. SDF-1 accelerates growth plate chondrocyte hypertrophy in organ culture
To confirm that SDF-1 induces chondrocyte hypertrophy in growth plates, 12-day-old chicken tibia growth plates were cultured in the presence of SDF-1 (100ng/mL) or in the absence of SDF-1 for 2, 4, and 6 days. 10 µm frozen sections were used to detect Type X collagen expression by immuno-fluorescent staining with mAb against type X collagen. A progressive increase in the size of the hypertrohic growth plate based on Type X collagen staining was seen. The ratio of the length of the hypertrophic zone to that of the total growth plate was calculated at the different time points (B). (* p<0.05). Scale bar = 100 µm.
Fig. 6
Fig. 6. SDF-1/CXCR4 signaling regulates Runx2 expression
50% confluent proliferative chondrocytes were infected with RACS-CXCR4 or RCAS for 4 days and then treated with 100 ng/ml SDF-1 for one day. Real-Time PCR was used to quantify Runx2 gene expression and western blot was used to detect Runx2 protein levels. (A) Runx2 mRNA expression increased after RACS-CXCR4 transfection and SDF-1 exposure, however, the increase of Runx2 expression was inhibited by CXCR4 inhibitor AMD3100. * p<0.05 compared to RACS; #: compared to RACS-CXCR4 and SDF-1 treatment. (B) Western blot showed similar changes in Runx2 protein.
Fig. 7
Fig. 7. Runx2 regulates CXCR4, MMP-13, and Type X collagen expression
2.5×105 hypertrophic chondrocytes from 17-day-chicken sterna embryo were seeded in F12 culture medium overnight and transfected with 1 µg human full length Runx2 DNA or co-transfected with 1 µg human full length Runx2 DNA combined with Runx 2 Si RNA (5nM /mL) two days in 6-well plates. mRNA levels of CXCR4, MMP-13, and Type X collagen were quantified by Real time PCR. Over-expression of Runx2 increased CXCR4 (A), MMP-13 (B), and Type X collagen expression while down-regulation of Runx2 using SiRNA inhibited their endogenous and Runx2 induction CXCR4, MMP-13, and Type X collagen expression. *: Significant difference in comparison to mock control, p<0.05. #: Statistically significant in comparison to Runx2 treatment alone.
Figure 8
Figure 8. SDF-1 infusion results in the tibial physeal closure and stimulates type X collagen expression
Tissue sections of rabbit proximal tibiae treated with SDF-1 (SDF-1 treatment) or PBS (No-SDF-1 treatment) for eight weeks were stained with safranin O for gross morphology (A–a and B–a). Unstained sections were used for chondrocyte microdissection by laser capture with captured area indicated by blue shade (A–b, A–c and B–c). The PBS-treated control rabbit physis (A–a) demonstrates normal growth plate morphology with a distal hypertrophic zone. The SDF-1-treated rabbit physis (B–a) exhibits marked thinning without distinction between proliferative and hypertrophic zone remnants. There was disorganization of cell columns; scattered hypertrophic chondrocytes; and vascular, as well as osseous, invasion into the growth plate remnant (B–a and B–b). These features are consistent with physeal closure. Brackets on two photomicrographs (A–c and B–c) indicate the height of the physis, defined from the resting zone to the last intact hypertrophic lacuna. Cells were microdissected from the proliferative and hypertrophic zone respectively in the control physis (A–b and A–c), and from the remnant of the growth plate in the SDF-1-treated physis (B–c). Real time PCR results indicate that the expression of type X mRNA was not detectable in the proliferative chondrocytes. Type X collagen expression was significantly increased in the hypertrophic zone of the SDF-1-treated physis at four-week time point in comparison to those from the PBS-treated physis, and remained at the same level in the remnant cartilage when the growth plate was closed after the physis was treated with SDF-1 for eight weeks (C).
Figure 8
Figure 8. SDF-1 infusion results in the tibial physeal closure and stimulates type X collagen expression
Tissue sections of rabbit proximal tibiae treated with SDF-1 (SDF-1 treatment) or PBS (No-SDF-1 treatment) for eight weeks were stained with safranin O for gross morphology (A–a and B–a). Unstained sections were used for chondrocyte microdissection by laser capture with captured area indicated by blue shade (A–b, A–c and B–c). The PBS-treated control rabbit physis (A–a) demonstrates normal growth plate morphology with a distal hypertrophic zone. The SDF-1-treated rabbit physis (B–a) exhibits marked thinning without distinction between proliferative and hypertrophic zone remnants. There was disorganization of cell columns; scattered hypertrophic chondrocytes; and vascular, as well as osseous, invasion into the growth plate remnant (B–a and B–b). These features are consistent with physeal closure. Brackets on two photomicrographs (A–c and B–c) indicate the height of the physis, defined from the resting zone to the last intact hypertrophic lacuna. Cells were microdissected from the proliferative and hypertrophic zone respectively in the control physis (A–b and A–c), and from the remnant of the growth plate in the SDF-1-treated physis (B–c). Real time PCR results indicate that the expression of type X mRNA was not detectable in the proliferative chondrocytes. Type X collagen expression was significantly increased in the hypertrophic zone of the SDF-1-treated physis at four-week time point in comparison to those from the PBS-treated physis, and remained at the same level in the remnant cartilage when the growth plate was closed after the physis was treated with SDF-1 for eight weeks (C).
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