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, 11beta-hydroxysteroid dehydrogenase type II (11 beta HSD2), catalyzes the unidirectional conversion of biologically active glucocorticoids to inactive metabolites. In vivo, 11 beta HSD2 protects the mineralocorticoid receptor from activation by glucocorticoids in mineralocorticoid 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 disrupting glucocorticoid signaling in osteoblastic cells. Rat 11 beta HSD2 complementary DNA was cloned downstream of a 2.3- and 3.6-kb alpha 1(I)-collagen (Col1a1) promoter fragment to produce the expression plasmids Col2.3-HSD2 and Col3.6-HSD2, respectively, which were transiently and/or stably transfected in osteoblastic ROS 17/2.8 and MC3T3-E1 cells. Transgene messenger RNA and protein were detected in transfected cells by Northern blot analysis and immunostaining, respectively. Transfection of 11 beta HSD2 led to higher rates of conversion of [(3)H]corticosterone to [(3)H]dehydrocorticosterone and reduced glucocorticoid-dependent regulation of a mouse mammary tumor virus promoter-reporter construct, cell growth, and messenger RNA markers compared with transfection of a control vector. Expression of 11 beta HSD2 under the control of Col1a1 promoter fragments may provide a novel model to study the role of glucocorticoid signaling in osteoblastic cells.
Woitge
H
H
Department of Medicine, School of Medicine, University of Connecticut Health Center, Farmington, Connecticut 06030, USA.
Harrison
J
J
Ivkosic
A
A
Krozowski
Z
Z
Kream
B
B
D042842:11-beta-Hydroxysteroid Dehydrogenases; D000818:Animals; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003001:Cloning, Molecular; D003094:Collagen / Q000235:genetics*; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D003345:Corticosterone / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000502:physiology; D005938:Glucocorticoids / Q000494:pharmacology / Q000502:physiology*; D006854:Hydrocortisone / Q000494:pharmacology; D006913:Hydroxysteroid Dehydrogenases / Q000235:genetics* / Q000378:metabolism; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D014162:Transfection; D019076:Transgenes
D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D005938:Glucocorticoids; D012333:RNA, Messenger; D003907:Dexamethasone; D003094:Collagen; D006913:Hydroxysteroid Dehydrogenases; D042842:11-beta-Hydroxysteroid Dehydrogenases; D015500:Chloramphenicol O-Acetyltransferase; C003552:11-dehydrocorticosterone; D003345:Corticosterone; D006854:Hydrocortisone