Krox20 and Bone Mass. We propose to delineate the role of the transcription factor Krox20 in the regulation of osteoblast function and bone mass control. During the previous funding cycle of this project, we discovered that Krox20 expression and transcriptional activity were strongly inhibited by glucocorticoids (GCs), contributing to the repression of osteocalcin transcription in cultured osteoblasts. We propose to expand the project beyond the problem of GC-induced osteoporosis (GIO) and study the role of Krox20 in basic osteoblast biology in vivo and in vitro. Such a role is suggested by: (i) developmental arrest of trabecular bone formation in Krox20-/- mice;(ii) low trabecular bone mass and decreased bone formation rate in mature Krox20 mice;(iii) impaired mineralization in Krox20 and Krox20-/- osteoblast cultures;(iv) stimulation of Krox20 expression and activity by the Wnt signaling pathway;and (v) expression pattern in vivo and in vitro consistent with a role for Krox20 in osteoblast function. We will analyze as a function of age the skeletal phenotype of mice with insufficient and/or osteoblast-specific excess of Krox20. In vivo approaches will include microcomputed tomography (mu CT) as well as histological and serological analyses. The cellular and molecular targets of Krox20 will be determined analyzing osteoblast cultures in which Krox20 expression has been altered by either genetic manipulations or viral infection. The in vitro analyses will be performed at short intervals during the development of the osteoblast phenotype to identify the Krox20-sensitive differentiation stage. The specific roles of Krox20 will be determined with respect to cell cycle progression, apoptosis, development of biochemical markers and expression of genes that promote the osteoblast phenotype, including genes potentially regulated directly by Krox20 and indirectly by the Wnt signaling pathway. Finally, we will return to the investigation of GIO. We will assess the effect of GCs on Krox20 expression in vivo in the context of a detailed description (including mu CT and apoptosis) of GIO development in the mouse;and, we will determine whether treatment with a Krox20 virus or recombinant Wnt3A antagonize adverse effects of GCs in osteoblasts. These studies will elucidate novel regulatory mechanisms that control osteoblast function and bone formation in health and disease, and open new avenues in the pursuit of bone anabolics.
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