Interleukin-6 (IL-6) has a complex role in bone that may involve effects on both resorption and formation. However, its exact actions on bone cells are unclear. This proposal will examine the effects that IL-6 has in bone through the construction of a transgenic mouse that expresses human IL-6 (hIL-6) protein to high levels in a limited number of tissues. to do this, I will employ a construct that uses the rat alpha1(I) collagen promoter to drive transcription of a hIL-6 cDNA. The advantage of this construct is that the pattern of expression by this promoter is well established. In previous studies it was demonstrated that the highest levels of expression of protein by the rat alpha1(I) collagen promoter was in only a few organs (tendon, teeth and bone) and that in teeth and bone, expression was highest in mature odontoblasts and osteoblasts. Hence, this construct should permit me to target the production of hIL-6 in transgenic mice and examine the effects that the overexpression of IL-6 in osteoblasts has on bone formation and resorption. Specific experiments will be: 1. Construct a vector that can express hIL-6 under the influence of the 3.6 kb rat alpha1(I) collagen promoter (ColhIL-6). Demonstrate that this construct expresses hIL-6 in experiments that employ transient in vitro transfections of ROS 17/2.8 cells. Construct transgenic mice that overexpress hIL-6 protein under the influence of the 3.6 kb rat alpha1(I) collagen promoter (ColhIL-6 mice). 2. Perform histomorphometric comparison of the tissues in ColhIL-6 mice with two control mice lines: 1) wild type mice and 2) transgenic mice that express the chloramphenicol acetyltransferase (CAT) marker gene under the influence of the 3.6 kb rat alpha1(I) collagen promoter (ColCAT mice). 3. Measure osteoclast formation rates in bone marrow cultures from control and ColhIL-6 mice. Determine the effect that coculture of osteoblast-enriched cell populations and marrow cells has on the formation of osteoclast-like cells in these cultures. 4. Compare in vitro bone resorption rates (45 Ca release and the rate osteoclasts form) and in vitro bone formation rates (the rate that [3H]proline is incorporated into collagen and steady state levels of alpha1(I) collagen mRNA) in calvariae cultures from control and ColhIL-6 mice. 5. Measure the effects that ovariectomy and estrogen replacement has on the bones of sexually mature control and ColhIL-6 mice.
