Osterix Regulation during Endochondral Ossification: Contribution to Chondrogenesis

INTRODUCTION: Insight into elucidating the transcriptional regulatory mechanisms underlying the successive events that mediate endochondral ossification is important in understanding possible mechanism of disease as well as cures to severe diseases ranging from skeletal dysplasia to fracture nonunion. The zinc finger-containing transcription factor Osterix (Osx) was demonstrated to be a key mediator of bone formation and mineralization. The role of Osx in mediating endochondral ossification is still unclear. MATERIALS AND METHODS: We performed real-time RT-PCR analyses using RNA extracted from E11.5 through E18.5 mouse embryo as previously described (Smith et al, 2005). Gene expression was normalized to the housekeeping gene βactin. Tissue representation of Osx was also determined by real time RTPCR using RNAs isolated from adult C57B6 mouse tissues (liver, heart, spleen, lung, kidney, calvaria, vertebrae, long bones, ribs, skin, fat, thymus and muscle). The distribution of Osx mRNA during embryonic endochondral ossification between E13.5 and E16.5 was assessed by in situ hybridization as previously reported (Smith et al, 2005) and was complemented with protein expression by immunohistochemical assay as described previously (Wang et al, 2005). We utilized the E11.5 limb bud-derived mesenchymal stem cells cultured in micromass model (Zhang et al, 2004) to recapitulate the early stages of endochondral ossification. We assessed Osx expression and regulation by real time RT-PCR after treatment with TGF-β (5 ng/ml) and BMP-2 (50 ng/ml) respectively known to delay and accelerate maturation. Gain and loss of function experiments, using adenoviral infections and RNA interference are underway and will reveal the function of Osx in mediating chondrogenesis and endochondral ossification. RESULTS: Our real time RT-PCR data show that Osx expression is dramatically induced between E11.5 and E13.5 (7 fold) in mouse embryos. Osx mRNA levels continue to be up-regulated through E16.5 (16 fold) concomitant with onset of skeletal mineralization (Fig. 1). This Osx regulation mimics that of Runx2 and precedes that of type X collagen. Osx transcripts are highly expressed in skeletal elements such as calvaria, vertebrae, femur, rib cage or xyphoid appendicular in comparison to fat, spleen or muscle. We further performed in situ hybridization to examine the spatial and temporal expression of Osx transcripts during mouse embryonic endochondral ossification between E13.5 and E16.5. We observe that Osx transcripts are expressed during early mouse endochondral ossification in the axial skeleton between E13.5 and E14.5 and remain expressed throughout embryogenesis to E16.5 (Fig. 2). We further performed immunohistochemical assays to determine the distribution of Osx proteins during embryonic development. Our immunohistochemical results show that between E14.5 and E15.5 Osx protein is expressed in the perichondrium and confined in the immature chondrocyte cell population while it is absent from cells that underwent hypertrophy. Between E16.5 and E17.5 when the axial skeleton becomes more ossified, Osx persists in osteoblastic cell populations as well as in the perichondrium and periosteum (Fig. 3). Real time PCR data show that Osx expression in limb bud micromass cultures between day 2 and day 16 is progressively induced. This upregulation follows that of Runx2 and is enhanced by BMP-2 while inhibited by TGF-β at all time points. Type X collagen, a marker of chondrocyte maturation, was also concomitantly expressed and regulated by TGF-β and BMP-2 with a similar way to Osx. Gain and loss of function studies are underway to determine the role of Osx in mediating chondrocyte terminal maturation. Osx