TGF-beta regulation of nuclear proto-oncogenes and TGF-beta gene expression in normal human osteoblast-like cells.
Transforming growth factor-beta (TGF-beta) is present in high levels in bone and plays an important role in osteoblast growth and differentiation. In order to dissect the molecular mechanisms of action of TGF-beta on osteoblasts, the effects of TGF-beta on the steady state mRNA levels of c-fos, c-jun, and jun-B proto-oncogenes on normal human osteoblast-like cells (hOB) and a transformed human osteoblast cell line (MG-63) were measured. Treatment of hOBs with 2 ng/ml of TGF-beta 1 resulted in a rapid increase in c-fos mRNA levels as early as 15 min post-treatment. A maximum (10-fold) increase was observed at 30 min after TGF-beta treatment followed by a decrease to control values. Similar responses were measured whether the cells were rapidly proliferating or quiescent. TGF-beta 1 induced jun-B mRNA levels more gradually with steady increase initially observed at 30 min and a maximum induction measured at 2 h post-TGF-beta treatment. In contrast, TGF-beta treatment caused a time dependent decrease in the c-jun mRNA levels, an opposite pattern to that of jun-B mRNA. Treatment of hOBs with TGF-beta 1 in the presence of actinomycin-D abolished TGF-beta 1 induction of c-fos mRNA, suggesting that TGF-beta action is mediated via transcription. In the presence of cycloheximide, TGF-beta causes super-induction of c-fos mRNA at 30 min, indicating that the c-fos expression by TGF-beta is independent of new protein synthesis. Further, transfection of 3 kb upstream region of jun-B promoter linked to a CAT reporter gene into ROS 17/2.8 cells was sufficient to be regulated by TGF-beta 1. Interestingly, TGF-beta treatment also increased the mRNA levels of TGF-beta 1 itself at 4 h post TGF-beta treatment, with a maximum increase observed at 14 h of treatment. TGF-beta 1 treatment for 30 min were sufficient to cause a delayed increase in TGF-beta protein secretion within 24 h. These data support that TGF-beta has major effects on hOB cell proto-oncogene expression and that the nuclear proto-oncogenes respond as rapid, early genes in a cascade model of hormone action.
Subramaniam
M
M
Department of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, Minnesota 55905.
Oursler
M J
MJ
Rasmussen
K
K
Riggs
B L
BL
Spelsberg
T C
TC
D000818:Animals; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D012516:Osteosarcoma / Q000473:pathology; D000090063:Proto-Oncogene Mas; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics; D011519:Proto-Oncogenes / Q000235:genetics* / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta / Q000235:genetics* / Q000494:pharmacology / Q000502:physiology
C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta