The underlying hypothesis for this application is that metabolites of the enzyme 5-lipoxygenase (5LO) play an important role in bone by stimulating osteoclastic bone resorption and inhibiting osteoblastic bone formation. 5LO metabolites of arachidonic acid may also be partially responsible for the bone loss which occurs due to estrogen deficiency.
The specific aims of the proposal are: 1. To determine if the osteoclast is a producer of 5LO metabolites. 2. To determine the effects of 5LO metabolites on osteoblast function. 3. To characterize the dynamic and static features of bone in the 5LO knockout mice, and 4. To determine if 5LO plays a role in the increased bone loss associated with estrogen withdrawal.
For Specific Aim 1, in situ hybridization for 5LO mRNA expression in bone and bone cells and quantitation of 5LO metabolite production by isolated osteoclasts will be performed. To determine if bone resorbing agents regulate mRNA and protein for 5LO, ribonuclease protection assays, immunoprecipitation, and immunoblotting will be performed.
For Specific Aim 2, the effects of 5LOand bone-like nodule formation will be tested, as well as effects one bone formation in calvarial organ cultures. Osteoblast function in 5LO knockout animals will be compared to osteoblast function in wild- type animals. As the 5LO knockout mice have increased cortical bone, Specific Aim 3 will be performed to determine if this increase in bone is due to a decrease in osteoblast numbers or activity, an increase in osteoblast function, or a combination. The biomechanical properties of these bones will also be determined.
In Specific Aim 4, it will be determined if 5LO metabolites mediate the effects of estrogen withdrawal by comparing bone loss in ovariectomized 5LO knockout mice with their wild-type controls. The proposed studies will provide important new insights into the role of 5LO metabolites in bone-a research are which is so far relatively unexplored. These studies may have clinical relevance to bone diseases such as osteoporosis and bone loss due to inflammatory conditions. Hence, these studies may also have profound implications for the increasing number of asthmatic patients who are currently being treated with steroids and 5LO inhibitors.
