The ultimate goal of this laboratory is to understand the ability of bone to adapt to mechanical forces. This adaptability can cause a counterproductive loss of bone mass which can result in fractures, e.g. during prolonged immobilization, space travel, or stress shielding by implants. Under these conditions it would be desirable to prevent or reduce bone loss, or even to increase bone mass. To rationally design this kind of intervention requires a complete understanding of the link between mechanical loading of the bone and the resulting cellular responses. Remodeling in response to altered mechanical loads affects the macrostructure of the mineralized extracellular matrix which carries the mechanical forces, but the effectors of the remodeling are bone cells. These cells act in response to changes in their local mechanical environment. An altered program of protein synthesis is a central component of the cellular activities which occur during the remodeling process, and this pattern of protein synthesis depends to a very large part on the spectrum of active transcription factors in the cell. It is therefore our hypothesis that bone cells perceive mechanical stimulation as an appropriate signal to trigger a series of specific transcriptional events. Thus, one of the earliest and most important responses of bone cells to mechanical input must be the activation and/or inactivation of a specific set of transcription factors. The identification of this specific set of transcription factors is a critically important step in understanding the link between loading of the whole bone, and the ultimate responses of the individual cells. As the basis for the present proposal, we argue that studying the promoter responses of genes that react to alterations in mechanical loading will enable us to identify the transcription factors that mediate these responses. For this purpose, we have selected a set of gene promoters specifically because they are known to be regulated by mechanical stimulation of bone or bone cells, and because there is evidence that their gene product plays a role in the response of bone to mechanical input. These are the transcription factor c-fos, insulin- like growth factor-1 (IGF-1), the inducible nitric oxide synthase (iNOS), and beta1 integrin. Although these promoters have been partially characterized in other systems, the elements which control their response to loads remain unknown. We propose to address this critical issue by these specific aims: (1) Establish a tissue culture model to study gene expression in bone cells growing in a mechanically active environment. (2) Screen the promoters of several genes which are known to respond to mechanical input, to identify load responsive cis-acting regulatory regions. (3) Identify the trans-acting factors which interact with these elements. Future studies beyond the conclusion of the proposed work would make use of the information gathered from analysis of these candidate genes to identify common upstream regulatory events. To confirm the role of specific transcription factors in the cellular response to mechanical loading, and to begin to design therapeutic interventions, we will proceed to directly manipulate the levels of these factors, either pharmacologically, or by overexpression or inactivation, using antisense, gene knockout, or transdominant negative approaches.
