A gene expression profile for endochondral bone formation: oligonucleotide microarrays establish novel connections between known genes and BMP-2-induced bone formation in mouse quadriceps

Endochondral bone formation has been fairly well characterized from a morphological perspective and yet this process remains largely undefined at molecular and biochemical levels. In vitro and in vivo studies have shown that human bone morphogenetic protein-2 (hBMP-2) is an important developmental growth and differentiation factor, capable of inducing ectopic bone formation in vivo. This study evaluated several aspects of the osteogenic effect of hBMP-2 protein injected into quadriceps of female C57B1/6J SCID mice. Mice were euthanized 1, 2, 3, 4, 7, and 14 days postinjection and muscles were collected for several methods of analysis. Hematoxylin and eosin-stained sections of muscles injected with formulation buffer showed no evidence of osteogenesis. In contrast, sections of muscles injected with hBMP-2 showed evidence of endochondral bone formation that progressed to mineralized bone by day 14. In addition, radiographs of mice injected with hBMP-2 showed that much of the quadriceps muscle had undergone mineralization by day 14. Labeled mRNA solutions were prepared and hybridized to oligonucleotide arrays designed to monitor ∼1300 murine, full-length genes. Changes in gene expression associated with hBMP-2 were determined from time-matched comparisons between buffer and hBMP-2 samples. A gene expression profile was created for 215 genes that showed greater than 4-fold changes at one or more of the indicated time points. One hundred twenty-two of these genes have previously been associated with bone or cartilage metabolism and showed significant increases in expression, e.g., aggrecan ( Agc1), runt related transcription factor 2 ( Runx2), bone Gla protein 1 ( Bglap1), and procollagens type II ( Col2a1) and X ( Col10a1). In addition, there were 93 genes that have not been explicitly associated with bone or cartilage metabolism. Two of these genes, cytokine receptor-like factor-1 ( Crlf1) and matrix metalloproteinase 23 ( Mmp23), showed peak changes in gene expression of 15- and 40-fold on days 4 and 7, respectively. In situ hybridizations of muscle sections showed that Mmp23 and Crlf1 mRNAs were expressed in chondrocytes and osteoblasts, suggesting a role for both proteins in some aspect of cartilage or bone formation. In conclusion, oligonucleotide arrays enabled a broader view of endochondral bone formation than has been reported to date. An increased understanding of the roles played by these gene products will improve our understanding of skeletogenesis