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Volume 271, Number 44, Issue of November 1, 1996 pp. 27637-27644
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Involvement of Nuclear Orphan Receptor NGFI-B in Transcriptional Activation of Salivary-specific R15 Gene by cAMP

(Received for publication, May 2, 1996, and in revised form, August 20, 1996)

From the Department of Molecular Pharmacology and Toxicology, University of Southern California, Los Angeles, California 90033

Proline-rich proteins (PRPs) are selectively expressed in the acinar cells of the salivary glands and are inducible by -agonist isoproterenol and dietary tannins. In the previous studies of rat PRP gene, R15, the 5-flanking region up to 1.7 kilobase pairs (kb), which was thought to contain the necessary proximal regulatory elements, failed to confer the catecholamine isoproterenol and dietary tannin inducibility to the transgene expression in the salivary glands of transgenic mice. Here we analyzed distal 5-flanking region of R15 in order to understand the mechanisms of tissue-specific and inducible gene regulation. An upstream regulatory region located between 2.4 and 1.7 kb of the R15 5-flanking region is demonstrated to be indispensable for the salivary-specific and inducible reporter gene expression in vivo, by transgenic approach. Element(s) within the 0.7-kb (2.4 to 1.7) region that is able to cis-activate the expression of a heterologous reporter gene expression is further elucidated by transient transfection assays in vitro. Three distinct nuclear orphan receptor NGFI-B regulatory sequences are identified within a 184-base pair (bp) minimal control region extended from 1995 to 1812 nucleotides relative to the transcription start site. When reporter gene containing this 184-bp control region and heterologous promoter was cotransfected with the NGFI-B expression construct, a transactivation that mimics the effect of cAMP is observed in the parotid cells. Finally, mutations on all three identified NGFI-B binding sites and coexpression of a dominant negative mutant construct, pCMV-NGFI-B(25-195), abolish this transactivation mediated by NGFI-B. In summary, these data suggest that the inducible nuclear orphan receptor NGFI-B may participate in the regulation of salivary acinar cell-specific and inducible expression of the rat R15 gene via three distinct distal NGFI-B sites.

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