Identification of a rat osteocalcin promoter 3′,5′-cyclic adenosine monophosphate response region containing two PuGGTCA steroid hormone receptor binding motifs

In MC3T3-E1 mouse osteoblastic cells, the adenylate cyclase activator forskolin increases osteocalcin (OC) mRNA levels. We have analyzed the effects of forskolin and 8-Br cAMP on the transcriptional activity of the rat OC promoter (with luciferase reporter) in MC3T3-E1 cells. Both forskolin and 8-Br cAMP activate the rat OC promoter 2- to 5-fold. By 5′ deletion analysis, we have mapped the cAMP response to the region -121 to -92. The 48-base pair rat OC promoter region -121 to -74 (hence denoted ROCRR) can confer cAMP responsiveness to an unresponsive heterologous minimal promoter. Crude nuclear extracts prepared from MC3T3-E1 cells form three complexes with the ROCRR by gel shift analysis. No specific change in nuclear factor binding in response to cellular forskolin treatment could be demonstrated. Intriguingly, two nuclear factor complexes bound to the ROCRR also recognized the thyroid hormone response element palindrome (AGGTCATGACCT) but did not bind the classic cAMP (TGACGTCA) or glucocorticoid (AGAACANNNTGTTCT) response elements. The rat OC promoter possesses two directly repeated PuGGTCA steroid hormone response element hexamer motifs (bottom strand) in the region -114 to-93 within the ROCRR, separated by a 10 nucleotide spacer. Oligos encoding the individual rat OC hexamer sites compete for the ROCRR DNA:protein complexes recognized by the thyroid hormone response element palindrome. Removal of the up-stream hexamer site by 5′ deletion (-121 to -100) in the context of the native OC promoter abrogates cAMP responsiveness. Taken together, these data suggest that this novel rat OC cAMP response region assembles a protein:DNA complex containing member(s) of the steroid hormone receptor superfamily. Transcriptional activity, but not DNA binding, is regulated by cAMP.