The cartilaginous growth plate determines the size and shape of a long bone by maintaining sequential differentiation of chondrocytes from proliferation to hypertrophy, and by regulating the production rate of hypertrophic chondrocytes. Misregulation of this process may lead to chondrodysplasia. The signaling pathways regulating growth plate chondrocyte hypertrophy are not completely understood. There are two major transition points regulating chondrocyte hypertrophy. The first is the entry of the hypertrophic stage from proliferation, which occurs mainly in the pre-hypertrophic zone that expresses the Ihh and PTHrP receptors. Previous studies have suggested a negative feedback loop in which Ihh induces PTHrP at the proximal end of the growth plate, whose interaction with its receptor in the prehypertrophic zone potently inhibits chondrocyte entry into hypertrophy (Vortkamp et al., 1996
). The second transition point is the exit of hypertrophic cartilage to bone, which occurs at the chondro-osseous junction. Previous studies have shown that this transition requires degradation of hypertrophic cartilage, which is not only mediated by metalloproteinases including MMP 9 and 13 in hypertrophic chondrocytes (Vu et al., 1998
), but also is induced by the presence of cells from the bone marrow area in the chondro-osseous junction (Cole et al., 1992
). However, the molecular nature of this induction remains elusive.
Our study suggests that the chemokine stromal cell-derived factor 1 (SDF-1) may be involved in this process. We found that SDF-1 and its receptor CXCR4 are expressed in a complementary pattern at the chondro-osseous junction in the growth plate, with hypertrophic cartilage positive for the receptor CXCR4, and its adjacent bone marrow expressing the ligand SDF-1. Interestingly, such a complementary expression pattern of SDF-1/CXCR4 exists in a wide variety of opposed tissue pairs during development, including gastrular mesoderm/ectoderm, vascular endothelium/mesoderm, thyroid endodermal epithelium/mesenchyme, and nasal ectodermal epithelium/mesenchyme (McGrath et al., 1999
). Such a complementary gene expression pattern results in a paracrine regulatory mechanism in which an SDF-1 producing tissue induces the development of the opposing tissue that expresses CXCR4. Based on this complementary gene expression pattern in the growth plate, we hypothesized that SDF-1 from bone marrow induces and/or enhances differentiation and degradation of hypertrophic cartilage, which expresses CXCR4.
In this study, we tested this hypothesis directly by examining the role of the chemokine SDF-1 and its receptor CXCR4 in this regulatory process in cells, organ cultures, and in vivo
. We found that transfection of CXCR4 into proliferative chondrocytes resulted in a dose-dependent increase of MMP-13 and type X collagen in response to SDF-1 treatment. Both MMP-13 and type X collagen are well-known markers for hypertrophic chondrocytes. In organ culture, SDF-1 is capable of diffusing into sternal cartilage rapidly and accumulates around chondrocytes. This suggests that SDF-1 synthesized by cells in bone marrow may be capable of diffusing into the adjacent cartilage in the chondro-osseous junction. Previous data have shown that SDF-1 binds to glycosaminoglycans in the extracellular matrix or on the cell surface (Fermas et al., 2008
; Uchimura et al., 2006
; Mbemba et al., 1999
). Such binding may stabilize SDF-1 and result in its accumulation around chondrocytes.
We have shown that, when a tibia growth plate was incubated with SDF-1, SDF-1 stimulated elongation of the hypertrophic zone at the distal end of the growth plate towards the proximal end. However, it did not result in a neo hypertrophic zone at the top of the growth plate. How is this polarity achieved? Our data suggest that this zone specific induction may be due to the specific expression of the SDF-1 receptor CXCR4 in the hypertrophic cartilage. We have shown that induction of hypertrophic chondrocyte markers, including Runx2, Col X, and MMP-13 in chondrocytes, requires the presence and interaction of both SDF-1 and CXCR4. In organ culture, although SDF-1 infiltrates the growth plate tissue in all directions, only the distal region of a growth plate contains CXCR4, and this is the area where hypertrophy is enhanced by SDF-1. Since a major source of SDF-1 in vivo
is from cells in the bone marrow area at the chondro-osseous junction adjacent to the hypertrophic cartilage, these cells may be involved in inducing the hypertrophic cartilage phenotype including synthesis of type X collagen and MMP-13 (Inada et al., 2004
The in vivo effects of SDF-1 on gene expression in the growth plate were consistent with our in vitro
findings. Elevation of SDF-1 concentration in rabbit growth plates in vivo
leads to increased type X collagen gene expression, degradation of the cartilage matrix, potentially from MMP-13, and premature closure of the growth plate (Lee et al., 2007
). Thus, the closure of growth plate in the SDF-1-treated physis is associated with stimulation of chondrocyte hypertrophy.
Interaction of SDF-1 and CXCR4 in articular chondrocytes results in up-regulation and release of MMP-3, −9, and −13 (Kanbe et al., 2002
; Kanbe et al., 2004
). Interestingly, high concentration of SDF-1, which occurs in the synovium of rheumatoid arthritis and osteoarthritis patients, results in death of articular chondrocytes (Lei Wei, 2006
). Since chondrocytes from the hypertrophic zone of a growth plate and those from OA articular cartilage share some common features including up-regulation of Col X and MMP-13, one may hypothesize that a high concentration of SDF-1 may also contribute to cell death occurring in the chondro-osseous junction in the growth plate, similar to that in OA cartilage. This hypothesis remains to be tested.
Our study reveals a role for Runx2, a transcription factor critical for bone formation (Dong et al., 2006
; Ducy et al., 1997
; Kamekura et al., 2006
; Komori et al., 1997
; Yoshida et al., 2004
; Zou et al., 2006
) in regulating chondrocyte hypertrophy by SDF-1/CXCR4 signaling. This role is twofold. First, Runx2 mediates SDF-1 induction of chondrocyte hypertrophy. We have shown that treating the CXCR4 cDNA transfected proliferating chondrocytes with SDF-1 resulted in a significant increase of Runx2 mRNA and protein levels. Runx2, in turn, was involved in activating Col X expression. Recent data have shown that SDF-1 also regulates osteoblast properties through Runx2 (Wei Zhu, 2007
). Second, Runx2 up-regulates CXCR4 in hypertrophic chondrocytes. Through Runx2 over-expression and knock-down experiments, we demonstrated that Runx2 plays an important role for CXCR4 expression in hypertrophic chondrocytes. Thus, our findings suggest a positive feedback loop in which Runx2 regulates chondrocyte differentiation through activation of SDF-1/CXCR4 signaling; and activation of SDF-1/CXCR4 signaling further enhances Runx2 expression, thereby inducing chondrocyte hypertrophy.