Characterisation of the Up-stream components in the stretch-induced ERK-1/2 MAP kinase cascade in Human Myometrial Cells

Abstract

The mechanisms underlying stretch-induced signalling in the human myometrium are poorly understood. Previous work in the group has demonstrated that in vitro stretch of human myometrial cells leads to increased expression of the pro-labour factors COX-2 and IL-8 via activation of the ERK-1/2 MAPK cascade, however the identities of components upstream in this pathway have yet to be elucidated. I over expressed constitutively active Ras in human myometrial cells, leading to increased ERK-1/2 phosphorylation and elevated COX-2 and IL-8 mRNA expression. I then inhibited Ras using both the chemical inhibitor manumycin A and transfection with dominant-negative mutant Ras. Each resulted in decreased ERK-1/2 phosphorylation and COX-2 expression in response to stretch. I performed immunoprecipitations to investigate which factors associated with Ras and found that stretch increased its association with SOS-1 and Grb-2. I then confirmed that Grb-2 was essential for the propagation of the stretch signal to ERK-1/2 by using siRNA to silence Grb- 2 and found that this resulted in decreased phosphorylation of ERK-1/2 and COX-2 and IL-8 protein synthesis. Silencing of SOS-1 decreased COX-2 protein expression but had little effect on ERK-1/2 phosphorylation. I then investigated the role of FAK, a constituent of stretch sensing focal adhesions, in the transduction of the stretch signal. First I used the inhibitors Y-27632 and PF-573,228, however neither inhibited the stretch-induced increase in ERK-1/2 phosphorylation or COX-2 and IL-8 mRNA expression In contrast, using siRNA to silence FAK expression reduced both the stretch-induced increase in ERK-1/2 phosphorylation and COX-2 and IL-8 protein synthesis. Moreover, transfection with dominant-negative FRNK reduced ERK-1 phosphorylation and COX-2 protein synthesis. Cells were also treated with the Src family kinase inhibitor PP-2 resulting in a decrease in stretch-induced ERK-1/2 phosphorylation and COX-2 protein synthesis. These data suggest that Ras, Grb-2 and FAK all act upstream of ERK-1/2 to mediate stretchinduced up-regulation of COX-2 and IL-8 in human myometrial cells, the role of SOS-1 is less clear, but also appears to be involved in stretch-induced expression of COX-2.