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Molecular Endocrinology, doi:10.1210/me.2002-0441
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Molecular Endocrinology 18 (10): 2440-2450
Copyright © 2004 by The Endocrine Society

Down-Regulation by Nuclear Factor {kappa}B of Human 25-Hydroxyvitamin D3 1{alpha}-Hydroxylase Promoter

Regina Ebert, Marlena Jovanovic, Matthias Ulmer, Doris Schneider, Jutta Meissner-Weigl, Jerzy Adamski and Franz Jakob

Orthopedic Department (R.E., M.J., M.U., D.S., J.M.-W., F.J.), University of Wuerzburg, Wuerzburg, Germany; and Institute of Experimental Genetics (J.A.), GSF-National Research Center for Environment and Health, Neuherberg, Germany

Address all correspondence and requests for reprints to: Franz Jakob, M.D., Experimental and Clinical Osteology, Orthopedic Department, University of Wuerzburg, Brettreichstrasse 11, D-97074 Wuerzburg, Germany. E-mail: f-jakob. klh{at}mail.uni-wuerzburg.de.

1,25-(OH)2 vitamin D3 is important for calcium homeostasis and cell differentiation. The key enzyme for the activation of liver-derived 25(OH) vitamin D3 is 25-hydroxyvitamin D3 1{alpha}-hydroxylase. It is expressed mainly in the kidney but also in peripheral tissues. A 1413-bp fragment of the 1{alpha}-hydroxylase promoter was cloned into luciferase vectors pGL2basic and pGL3basic. Sequence analyses revealed four base exchanges and three base deletions compared with the published sequence which were identically found in five control persons. In silico promoter analyses revealed 17 putative nuclear factor (NF){kappa}B sites, 10 of which were found to bind NF{kappa}B in EMSA experiments. Cotransfection of NF{kappa}B p50 and p65 subunits resulted in dramatic reduction of the promoter activity of the full-length construct as well as a series of 5'-deletion constructs. Deletion of the farmost 3'-situated NF{kappa}B-responsive element almost abolished NF{kappa}B responsiveness. Treatment of human embryonic kidney 293 cells with sulfasalazine, a NF{kappa}B inhibitor, resulted in enhanced 1{alpha}-hydroxylase mRNA production. Down-regulation of 1{alpha}-hydroxylase promoter through NF{kappa}B signaling may contribute to the pathogenesis of inflammation-associated osteopenia/osteoporosis.




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