1,25-DIHYDROXYVITAMIN D-3 INHIBITION OF COL1A1 PROMOTER EXPRESSION INCALVARIAE FROM NEONATAL TRANSGENIC MICE

We studied the effect of 1,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3) on organ cultures of transgenic mouse calvariae containing segments of t he Collal promoter extending to -3518, -2297, -1997, -1794, -1763, and -1719 bp upstream of the transcription start site fused to the chlora mphenicol acetyltransferase (CAT) reporter gene. 1,25(OH)(2)D-3 had a dose-dependent inhibitory effect on the expression of the -3518 bp pro moter construct (ColCAT3.6), with maximal inhibition of about 50% at 1 0 nM. This level of inhibition was consistent with the previously obse rved effect on the endogenous Coital gene in bone cell models. All of the shorter constructs were also inhibited by 10 nM 1,25(OH)(2)D-3, su ggesting that the sequences required for 1,25(OH)(2)D-3 inhibition are downstream of -1719 bp. The inhibitory effect of 1,25(OH)(2)D-3 on tr ansgene mRNA was maintained in the presence of the protein synthesis i nhibitor cycloheximide, suggesting that the inhibitory effect on Colla l gene transcription does not require de novo protein synthesis. We al so examined the in vivo effect of 1,25(OH)(2)D-3 treatment of transgen ic mice on ColCAT activity, and found that 48 h treatment caused a dos e-dependent inhibition of CAT activity in calvariae comparable to that observed in organ cultures. In conclusion, we demonstrated that 1,25( OH)(2)D-3 inhibits Col1A1 promoter activity in transgenic mouse calvar iae, both in vivo and in vitro. The results indicate that there is a 1 ,25(OH)(2)D-3 responsive element downstream of -1719 bp, The inhibitor y effect does not require new protein synthesis. (C) 1998 Elsevier Sci ence B.V, All rights reserved.