Parathyroid hormone represses alpha 1(I) collagen promoter activity in cultured calvariae from neonatal transgenic mice.
We examined the effect of PTH on the activity of alpha 1(I) collagen promoter fusion genes in cultured calvariae from transgenic mice. The parent construct, ColCAT 3.6, contains 3520 basepairs of 5' rat alpha 1(I) collagen DNA, 115 basepairs of untranslated alpha 1(I) collagen-coding DNA, and the bacterial chloramphenicol acetyltransferase reporter gene, while the 5'-deletion ColCAT 2.3 contains 2296 kilobases of rat alpha 1(I) collagen promoter sequence. Transgenic mouse lines harboring these collagen promoter fusion genes were developed using the oocyte microinjection technique, and for each construct, three different lines of mice were tested. Calvariae from 6- to 8-day-old transgenic mice were cultured for 48 h with or without bovine PTH-(1-34). ColCAT 3.6 and ColCAT 2.3 were expressed at comparable levels in calvariae and were inhibited by PTH. There were parallel decreases in the incorporation of [3H]proline into collagen and levels of the endogenous alpha 1(I) collagen mRNA and transgene mRNA. Forskolin at 10 microM mimicked the inhibitory effect of PTH on promoter activity in ColCAT 3.6 and ColCAT 2.3 calvariae. A RNase protection assay showed that the transgene was initiated correctly from the transgene promoter. These data show that PTH and cAMP can repress collagen promoter activity in calvariae from transgenic mice, suggesting that the alpha 1(I) collagen promoter may contain cis elements down-stream of -2.3 kilobases that mediate PTH and cAMP repression of collagen gene expression in bone. Cultured bone explants from transgenic mice can be used as a model to study hormonal regulation of alpha 1(I) collagen promoter constructs.
Kream
B E
BE
Department of Medicine, University of Connecticut Health Center, Farmington 06030.
LaFrancis
D
D
Petersen
D N
DN
Woody
C
C
Clark
S
S
Rowe
D W
DW
Lichtler
A
A
D000818:Animals; D000831:Animals, Newborn; D001842:Bone and Bones / Q000378:metabolism*; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D003001:Cloning, Molecular; D005576:Colforsin / Q000494:pharmacology; D003094:Collagen / Q000096:biosynthesis* / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D015151:Immunoblotting; D051379:Mice; D008822:Mice, Transgenic; D009924:Organ Culture Techniques; D010281:Parathyroid Hormone / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D012886:Skull / Q000378:metabolism; D014158:Transcription, Genetic
D010281:Parathyroid Hormone; D012333:RNA, Messenger; D005576:Colforsin; D003094:Collagen; D015500:Chloramphenicol O-Acetyltransferase