Mechanisms of Cortisol Action in Bone

St. Francis Hospital And Medical Center, Hartford CT

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Abstract

DESCRIPTION (provided by applicant): Investigations from our laboratory revealed that glucocorticoids induce Notch1 mRNA in cells of the osteoblast lineage. Consequently, we explored the function of Notch1 in osteoblastic cells and found that Notch1 determines the cellular fate of undifferentiated stromal cells. Overexpression of the active Notch1 intracellular domain (NotchlC) induced adipogenesis and prevented osteoblast differentiation in cultures of undifferentiated stromal cells and in cultures of osteoblasts. The objective of this project is to understand the function of Notch1 in bone in vivo and in vitro and define mechanisms involved in its inhibitory effects on osteoblastogenesis. For this purpose, we are creating transgenic mouse lines overexpressing Notch1 under the control of either the type I collagen or the osteocalcin promoter and obtained Notch1ox/1ox mice for the creation of conditional knockouts. Our Specific Aims are: 1) to determine the function of Notch1 in vivo by transgenic overexpression of NotchlC under the control of the type I collagen or the osteocalcin promoter so that expression occurs in undifferentiated cells and in osteoblasts in the bone microenvironment The skeletal phenotype of transgenic mice will be compared to that of wild-type mice and determined by histomorphometry, contact radiography, densitometry and micro CT scanning. 2) To determine the function of Notch1 in vivo by targeted gene deletion in osteoblasts, achieved by intercrosses between transgenic mice expressing the Cre recombinase under the control of the osteocalcin or type I collagen promoter and mice in which the Notch1 gene is flanked by IoxP sequences. The skeletal phenotype will be assessed with histomorphometric and radiological techniques, and if indicated, by examining structural and crystal properties of the skeleton. 3) To determine the mechanism of action of Notch in vitro in stromal and osteoblastic cells overexpressing Notch1 and examining the behavior of Notch1 null cells. The impact of Notch1 on the differentiation of cells of the osteoblastic lineage and pathways involved will be determined. In addition, we will explore whether Notch1 mediates effects of glucocorticoids on osteoblastic cell differentiation in vivo and in vitro. These investigations should clarify the role of Notch1 in bone cell function.