Bone sialoprotein (BSP) is a major non-collagenous protein in bone and other mineralized tissues. Using two transgenic mouse lines in which 4.8 kb (mBSP4.8Luc) or 9.0 kb (mBSP9.0Luc) of the BSP promoter was ligated to a luciferase reporter gene, we found that the 9.0 kb mouse BSP promoter is necessary for tissue-specific expression of BSP. mBSP9.0Luc or mBSP4.8Luc transgenic mice were also crossbred with Runx2+/- mice. We found that the direct effect of Runx2 on mouse BSP promoter is to enhance its activity, while the suppression effect of Runx2 on BSP promoter is in an indirect way.
BSP-Luc/ACTB-EGFP transgenic mice were created with two genetic markers: an enhanced green fluorescent protein which can be used to track the migration of all transplanted cells, and a luciferase reporter gene serving as a marker for osteogenic differentiation of these cells. Using bone marrow stromal cells (BMSCs) isolated from these mice, we demonstrated that transplanted BMSCs can access to the wound sites from peripheral circulation, and participate in wound healing processes.
BSP-TVA transgenic mice showed a specific expression of an avian retroviral receptor gene (TVA) in BSP-expressing cells. After systemic infection with an avian retrovirus expressing a mutant Cbfa1/Runx2 (Cbfa1mu), which acts as a dominant-negative regulator of Cbfa1/Runx2, the BSP-TVA mice showed successful Cbfa1mu expression and suppressed BSP expression in bone tissues. Therefore, by targeting genes to sites of osteogenesis, BSP-TVA transgenic mouse model is a unique system for studying molecular events associated with bone formation in vivo.
Although BSP expression has been shown to be coincident with de novo bone formation, substantial evidences suggested that BSP also plays a role in bone resorption. We have provided in vitro evidence showing that BSP acts synergistically with RANKL/RANK signaling to induce osteoclastogenesis in RAW264.7 cells. CMV-BSP transgenic mice were created in which constitutively over-expressed BSP is driven by the cytomegalovirus (CMV) promoter. These transgenic mice exhibit phenotypes including thinner bone tissues, increased osteoclast numbers, and lower bone mineral density, all of which indicated an increase in bone resorption.
We also applied transgenic mouse models in the study of breast cancer metastasis. We found that CMV-BSP mice receiving intracardiac cancer cell inoculation developed more severe metastases in multiple vital organs, although originally these cancer cells showed a characteristic of bone-seeking metastases. Using CtpsK/BSP transgenic mice in which targeted BSP overexpression in osteoclasts was driven by a Cathepsin K (CtpsK) promoter, we found that after intracardiac and intratibial inoculation of cancer cells, more CtpsK/BSP mice developed bone metastatic lesions, and the osteolytic lesion area was significantly larger in CtpsK/BSP mice than in the wild-type mice.
In conclusion, these transgenic mouse lines, serving as invaluable tools in bone and mineralized tissue studies, could be further utilized in future studies aimed at investigating molecular events in bone remodeling, developing new strategies in tissue engineering, and developing new therapies to osteolytic bone diseases. (Supported by NIH grants DE14537 and DE16710)