Abstract
Osteoblastic cells response to mechanical forces by activating signal transduction cascades and altering gene expression patterns. We examined the responses of MC3T3E1 mouse osteoblasts to short term, low level (1000 microstrain, 1Hz) loads applied by cyclic deformation of the growth surface. At these load levels, daily short-term loading significantly retards the ascorbate induced differentiation of the cells as measured by alkaline phosphatase and osteopontin expression. This effect peaked at 5 minutes of loading per day; loads of 1 or more hours per day accelerated the differentiation process slightly as measured by the same criteria. C-fos is known to respond to mechanical loading of bones in vivo, we therefore examined the effect of brief loading bouts on c-fos promoter activity.
Stable lines of MC3T3E1 cells carrying the fos promoter driving a luciferase reporter gene were loaded for 0, 5 or 60 minutes. For these experiments cells were grown in MEM without ascorbate and were then either supplemented or not with 37.5mM ascorbate-2-phosphate at confluence. In cells which had not been pre-treated with ascorbate the c-fos promoter was essentially unresponsive to loads. Following 24 hours of ascorbate treatment (placing these cells at the earliest stages differentiation) a 5 minute loading bout resulted in a marked (~ 50%) decrease in luciferase activity with a trough at 6–8 hours. Loading for 60 minutes caused a similar, but accelerated inhibition of luciferase activity with a trough at 2–4 hours after loading. 24 hours after loading, fos promoter activity had returned to baseline in cells loaded for 60 minutes but remained depressed at 75% of baseline in cells loaded for 5 minutes.
Ets family transcription factors have been implicated in gene regulation in response to mechanical stimulation in several systems. The c-fos promoter contains a Serum Response Element which contains both a CarG motif responsible for binding the Serum Response Factor and an ets core motif CAGGT which can bind ets factors. We therefore repeated these experiments using a mutant c-fos promoter in which the ets binding site is destroyed. The response of this mutant to loading for 60 minutes was indistinguishable from that of the wt-promoter. However, in contrast to the wt-promoter, the ets-mutated promoter responded to a 5 minute loading with a rapid increase in activity (~150%) which peaked at 10 hours before returning to baseline at 24 hours.
These results suggest that although similar in magnitude, the inhibition of the c-fos promoter by 5 and 60 minute loading bouts are regulated by different mechanisms, and implicate the ets family of transcription factors in the response to the briefer loading events.
The abstracts were prepared by Orah Naor. Correspondence should be addressed to him at the Israel Orthopaedic Association, PO Box 7845, Haifa 31074, Israel.
