Immunologic Regulation of Bone Development

A regulatory network comprised of transcription factors PU.1, Ikaros, E2A, EBF, and Pax5 control B cell fate specification and differentiation. Early B Cell Factor-1 (EBF-1) is essential for B cell fate specification while Pax5 is required for B cell development. Mice deficient in Pax5 or EBF-1 have a developmental arrest of B cell differentiation at the pro-B cell stage, which results in the absence of mature B cells. We analyzed the bone phenotype of Pax5 and EBF-1 wild-type (+/+) and homozygous mutant (–/–) mice to determine if the loss of these transcription factors regulated bone cell development.

Bones from Pax5–/– mice were strikingly osteopenic 15 days after birth, with increased numbers of osteoclasts, and decreased trabecular number. The number of osteoblasts in Pax5–/– bones and their function in vitro were not different from controls. In addition, Pax5 was not expressed by wild-type osteoblasts. To investigate the origin of the in vivo increase in osteoclasts, Pax5–/– or +/+ spleen cells were cultured with M-CSF and RANKL and multinucleated, TRAP⁺ cells counted. Cells from Pax5–/– spleen produced 5-10 times more osteoclasts than did controls.

Tibia from EBF-1–/– mice had a striking increase in osteoblasts lining bone surfaces. Consistent with this was an increase in osteoid thickness and in the bone formation rate. This correlated with a 2-fold increase in serum osteocalcin. However, in vitro proliferation and ALP of mutant osteoblasts did not differ from control. In contrast, osteoclast number was similar in 4 week-old +/+ and –/– mice; however, at 12 weeks the number of osteoclasts was more than twice that of controls. These data correlated with an increase in bone volume at 12 weeks of age. The most striking aspect of the EBF-1–/– bones was the presence of adipocytes, which filled the marrow space. The adipocytes in the marrow were present at both 4 and 12 weeks of age. Increased fat was also seen in the liver of mutant mice. However, subcutaneous fat was almost absent in EBF-1–/– mice. Importantly, EBF-1 mRNA was expressed in wild-type osteoblasts and in adipocytes. Loss of EBF-1 and Pax5 causes distinct, non-overlapping bone phenotypes. It is important to understand why this network of transcription factors, which are so important for B cell development, have such striking effects on bone cell growth and development.