Transgenic expression of COL1A1-chloramphenicol acetyltransferase fusion genes in bone: differential utilization of promoter elements in vivo and in cultured cells.
To directly compare the patterns of collagen promoter expression in cells and tissues, the activity of COL1A1 fusion genes in calvariae of neonatal transgenic mice and in primary bone cell cultures derived by sequential digestion of transgenic calvariae was measured. ColCAT3.6 contains 3.6 kb (positions -3521 to +115) of the rat COL1A1 gene ligated to the chloramphenicol acetyltransferase (CAT) reporter gene. ColCAT2.3 and ColCAT1.7 are 5' deletion mutants which contain 2,296 and 1,672 bp, respectively, of COL1A1 DNA upstream from the transcription start site. ColCAT3.6 activity was 4- to 6-fold lower in primary bone cell cultures than in intact calvariae, while ColCAT2.3 activity was at least 100-fold lower in primary bone cells than in calvariae. These changes were accompanied by a threefold decrease in collagen synthesis and COL1A1 mRNA levels in primary bone cells compared with collagen synthesis and COL1A1 mRNA levels in freshly isolated calvariae. ColCAT3.6 and ColCAT2.3 activity was maintained in calvariae cultured in the presence or absence of serum for 4 to 7 days. Thus, when bone cells are removed from their normal microenvironment, there is parallel downregulation of collagen synthesis, collagen mRNA levels, and ColCAT3.6 activity, with a much greater decrease in ColCAT2.3. These data suggest that a 624-bp region of the COL1A1 promoter between positions -2296 and -1672 is active in intact and cultured bone but inactive in cultured cells derived from the bone. We suggest that the downregulation of COL1A1 activity in primary bone cells may be due to the loss of cell shape or to alterations in cell-cell and/or cell-matrix interactions that normally occur in intact bone.
Krebsbach
P H
PH
Department of Periodontology, University of Connecticut Health Center, Farmington 06030.
Harrison
J R
JR
Lichtler
A C
AC
Woody
C O
CO
Rowe
D W
DW
Kream
B E
BE
D000818:Animals; D001842:Bone and Bones / Q000166:cytology / Q000378:metabolism*; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003094:Collagen / Q000096:biosynthesis / Q000235:genetics*; D005786:Gene Expression Regulation; D051379:Mice; D008822:Mice, Transgenic; D009924:Organ Culture Techniques; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics
D012333:RNA, Messenger; D003094:Collagen; D015500:Chloramphenicol O-Acetyltransferase