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TGF-beta regulation of nuclear proto-oncogenes and TGF-beta gene expression in normal human osteoblast-like cells
M Subramaniam1, M J Oursler, K Rasmussen
1Department of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, Minnesota 55905.
Abstract:
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.
Insights
Transforming growth factor-beta (TGF-beta) rapidly alters proto-oncogene expression in human osteoblasts, influencing cell growth and differentiation. These rapid gene changes, including c-fos and jun-B, are transcription-dependent and independent of new protein synthesis.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Transforming growth factor-beta (TGF-beta) is crucial for bone biology, regulating osteoblast function.
- Understanding TGF-beta's molecular mechanisms in osteoblasts is key to bone health research.
Purpose of the Study:
- To investigate the effects of TGF-beta on proto-oncogene mRNA levels in human osteoblasts.
- To elucidate the molecular pathways mediating TGF-beta's action on osteoblast gene expression.
Main Methods:
- Treatment of normal human osteoblast-like cells (hOB) and MG-63 cells with TGF-beta 1.
- Measurement of steady-state mRNA levels for c-fos, c-jun, and jun-B proto-oncogenes.
- Use of actinomycin-D and cycloheximide to assess transcriptional and translational dependence.
- Reporter gene assays to study jun-B promoter activity.
Main Results:
- TGF-beta rapidly increased c-fos mRNA (max 10-fold at 30 min) and gradually increased jun-B mRNA (max at 2 h).
- TGF-beta decreased c-jun mRNA levels in a time-dependent manner.
- TGF-beta's induction of c-fos mRNA was abolished by actinomycin-D, indicating transcriptional regulation.
- c-fos expression was independent of new protein synthesis, as shown by cycloheximide treatment.
- TGF-beta treatment also increased its own mRNA and protein secretion.
- TGF-beta regulated jun-B promoter activity.
Conclusions:
- TGF-beta significantly impacts proto-oncogene expression in human osteoblasts.
- Proto-oncogenes act as rapid, early genes in a cascade model of TGF-beta's hormonal action.
- These findings provide insights into TGF-beta signaling in bone cells.