Characterisation of the Up-stream components in the stretch-induced ERK-1/2 MAP kinase cascade in Human Myometrial Cells
Author(s)
Cryar, Benjamin Joseph
Type
Thesis
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.
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.
Date Issued
2011-09
Date Awarded
2012-04
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Johnson, Mark
Griffiths, Mark
Publisher Department
Surgery and Cancer
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)