Egr-1 mRNA induction by medium flow involves mRNA stabilization and is enhanced by the p38 inhibitor SB203580 in osteoblast-like cells


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Abstract

AimMechanical stimuli are important for maintaining organ structure and tissue function. To elucidate signalling pathways activated by mechanical stimuli, the contribution of mRNA stabilization to induction of egr-1 mRNA by medium flow was examined and the mechanisms responsible for stabilization were analysed. An early-response gene that encodes a transcription factor, egr-1, activates transcription of several genes in response to mechanical stimuli, and was therefore selected to resolve how early-induced signals are integrated and connected to subsequent response.MethodsMouse osteoblast-like MC3T3E1 cells were stably transfected with the chloramphenicol acetyltransferase (CAT) gene linked to the egr-1 promoter, and inductions of endogenous egr-1 and transfected CAT mRNA following medium flow were compared using real-time reverse transcriptase PCR. The mechanism of induction was examined using a transcription inhibitor and mitogen-activated protein (MAP) kinase inhibitors. Activation of MAP kinases by medium flow was investigated using western blotting.ResultsInduction of egr-1 mRNA by medium flow was twofold higher than CAT mRNA induction. Induction of egr-1 mRNA was also observed in cells pre-treated with transcription inhibitor. The p38 inhibitor SB203580 enhanced induction of egr-1 mRNA by medium flow. Extracellular signal regulated kinase (ERK), p38 and c-Jun N-terminal kinase (JNK) were activated by medium flow.ConclusionA considerable part of egr-1 mRNA induction by medium flow may be due to mRNA stabilization. The p38 inhibitor SB203580 enhances induction.

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