INVOLVEMENT OF NUCLEAR ORPHAN RECEPTOR NGFI-B IN TRANSCRIPTIONAL ACTIVATION OF SALIVARY-SPECIFIC R15 GENE BY CAMP

Proline rich proteins (PRPs) are selectively expressed in the acinar c ells of the salivary glands and are inducible by beta-agonist isoprote renol and dietary tannins, In the previous studies of rat PRP gene, R1 5, the 5'-flanking region up to -1.7 kilobase pairs (kb), which was th ought to contain the necessary proximal regulatory elements, failed to confer the catecholamine isoproterenol and dietary tannin inducibilit y to the transgene expression in the salivary glands of transgenic mic e, Here we analyzed distal 5'-flanking region of R15 in order to under stand 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 sa livary-specific and inducible reporter gene expression in vivo, by tra nsgenic approach, Element(s) within the 0.7-kb (-2.4 to -1.7) region t hat 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 se quences are identified within a 184-base pair (bp) minimal control reg ion extended from -1995 to -1812 nucleotides relative to the transcrip tion start site. When reporter gene containing this 184-bp control reg ion and heterologous promoter was cotransfected with the NGFI-B expres sion construct, a transactivation that mimics the effect of cAMP is ob served in the parotid cells, Finally, mutations on all three identifie d NGFI-B binding sites and coexpression of a dominant negative mutant construct, pCMV-NGFI-B(Delta 25-195), abolish this transactivation med iated by NGFI-B. In summary, these data suggest that the inducible nuc lear orphan receptor NGFI-B may participate in the regulation of saliv ary acinar cell-specific and inducible expression of the rat R15 gene via three distinct distal NGFI-B sites.