Cloning and in vitro characterization of alpha 1(I)-collagen 11 beta-hydroxysteroid dehydrogenase type 2 transgenes as models for osteoblast-selective inactivation of natural glucocorticoids

The NAD-dependent enzyme, 11 beta -hydroxysteroid dehydrogenase type II (11 beta HSD2), catalyzes the unidirectional conversion of biologically active glucocorticoids to inactive metabolites. In vivo, 11 beta HSD2 protects th e mineralocorticoid receptor from activation by glucocorticoids in mineralo corticoid target tissues such as kidney. The goal of the present study was to use targeted overexpression of 11 beta HSD2 as a novel means of disrupti ng glucocorticoid signaling in osteoblastic cells. Rat 11 beta HSD2 complem entary DNA was cloned downstream of a 2.3- and 3.6-kb alpha1(I)-collagen (C ol1a1) promoter fragment to produce the expression plasmids Co12.3-HSD2 and Co13.6-HSD2, respectively, which were transiently and/or stably transfecte d in osteoblastic ROS 17/2.8 and MC3T3-E1 cells. Transgene messenger RNA an d protein were detected in transfected cells by Northern blot analysis and immunostaining, respectively. Transfection of 11 beta HSD2 led to higher ra tes of conversion of [H-3]corticosterone to [H-3]dehydrocorticosterone and reduced glucocorticoid-dependent regulation of a mouse mammary tumor virus promoter-reporter construct, cell growth, and messenger RNA markers compare d with transfection of a control vector. Expression of 11 beta HSD2 under t he control of Col1a1 promoter fragments may provide a novel model to study the role of glucocorticoid signaling in osteoblastic cells.