TY - JOUR
T1 - 1,25-Dihydroxyvitamin D3 inhibition of Col1a1 promoter expression in calvariae from neonatal transgenic mice
AU - Bedalov, Antonio
AU - Salvatori, Roberto
AU - Dodig, Milan
AU - Kapural, Belinda
AU - Pavlin, Dubravko
AU - Kream, Barbara E.
AU - Clark, Stephen H.
AU - Woody, Charles O.
AU - Rowe, David W.
AU - Lichtler, Alexander C.
N1 - Funding Information:
This work was supported by the following grants from the National Institutes of Health: AR29983 (A.C.L.), AR38933 (B.E.K., S.H.S., and A.C.L.) and AR29850 (B.E.K.), and grants from NASA and the American Heart Association, AHA 92015860 (D.W.R.). Dr. Clark was supported in part by the Medical Research Service, Department of Veterans Affairs.
PY - 1998/7/9
Y1 - 1998/7/9
N2 - We studied the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on organ cultures of transgenic mouse calvariae containing segments of the Col1a1 promoter extending to -3518, -2297, -1997, -1794, -1763, and -1719 bp upstream of the transcription start site fused to the chloramphenicol acetyltransferase (CAT) reporter gene. 1,25(OH)2D3 had a dose-dependent inhibitory effect on the expression of the -3518 bp promoter construct (ColCAT3.6), with maximal inhibition of about 50% at 10 nM. This level of inhibition was consistent with the previously observed effect on the endogenous Col1a1 gene in bone cell models. All of the shorter constructs were also inhibited by 10 nM 1,25(OH)2D3, suggesting that the sequences required for 1,25(OH)2D3 inhibition are downstream of -1719 bp. The inhibitory effect of 1,25(OH)2D3 on transgene mRNA was maintained in the presence of the protein synthesis inhibitor cycloheximide, suggesting that the inhibitory effect on Col1a1 gene transcription does not require de novo protein synthesis. We also examined the in vivo effect of 1,25(OH)2D3 treatment of transgenic mice on ColCAT activity, and found that 48 h treatment caused a dose-dependent inhibition of CAT activity in calvariae comparable to that observed in organ cultures. In conclusion, we demonstrated that 1,25(OH)2D3 inhibits Col1A1 promoter activity in transgenic mouse calvariae, both in vivo and in vitro. The results indicate that there is a 1,25(OH)2D3 responsive element downstream of -1719 bp. The inhibitory effect does not require new protein synthesis.
AB - We studied the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on organ cultures of transgenic mouse calvariae containing segments of the Col1a1 promoter extending to -3518, -2297, -1997, -1794, -1763, and -1719 bp upstream of the transcription start site fused to the chloramphenicol acetyltransferase (CAT) reporter gene. 1,25(OH)2D3 had a dose-dependent inhibitory effect on the expression of the -3518 bp promoter construct (ColCAT3.6), with maximal inhibition of about 50% at 10 nM. This level of inhibition was consistent with the previously observed effect on the endogenous Col1a1 gene in bone cell models. All of the shorter constructs were also inhibited by 10 nM 1,25(OH)2D3, suggesting that the sequences required for 1,25(OH)2D3 inhibition are downstream of -1719 bp. The inhibitory effect of 1,25(OH)2D3 on transgene mRNA was maintained in the presence of the protein synthesis inhibitor cycloheximide, suggesting that the inhibitory effect on Col1a1 gene transcription does not require de novo protein synthesis. We also examined the in vivo effect of 1,25(OH)2D3 treatment of transgenic mice on ColCAT activity, and found that 48 h treatment caused a dose-dependent inhibition of CAT activity in calvariae comparable to that observed in organ cultures. In conclusion, we demonstrated that 1,25(OH)2D3 inhibits Col1A1 promoter activity in transgenic mouse calvariae, both in vivo and in vitro. The results indicate that there is a 1,25(OH)2D3 responsive element downstream of -1719 bp. The inhibitory effect does not require new protein synthesis.
KW - Bone
KW - Calcium regulating hormone
KW - Collagen
KW - Osteoblast
KW - Transcription
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U2 - 10.1016/S0167-4781(98)00079-7
DO - 10.1016/S0167-4781(98)00079-7
M3 - Article
C2 - 9655920
AN - SCOPUS:0032500077
SN - 0167-4781
VL - 1398
SP - 285
EP - 293
JO - Biochimica et Biophysica Acta - Gene Structure and Expression
JF - Biochimica et Biophysica Acta - Gene Structure and Expression
IS - 3
ER -