Investigation of the effects of injury upon intracellular signalling pathways and expression of inflammatory response genes in articular cartilage
Author(s)
Watt, Fiona Elizabeth
Type
Thesis
Abstract
Damage to joints predisposes to osteoarthritis. The mechanism by which injury to
cartilage might lead to net matrix loss and cartilage degeneration remains unknown.
Following experimental sharp injury to porcine articular cartilage (dissection from, or
scoring of the articular surface), our group has previously shown rapid activation of
the 3 mitogen activated protein kinase (MAPK) pathways in cartilage. Activation of
ERK, and probably also p38 MAPK, is due to release of fibroblast growth factor
(FGF) from the matrix after injury. However, despite a long search, the cause of JNK
activation following sharp injury remains unknown. I investigated the extent and
regulation of inflammatory signalling after cartilage injury and whether it was
sufficient to cause expression of inflammatory response genes.
In this work, I show that a number of intracellular signalling pathways including PI3
kinase and IκB kinase (IKK) (which leads to activation of NFκB) are activated by
sharp injury to cartilage. The signalling following injury was sufficient to induce a
wide range of inflammatory response mRNAs, including pro-inflammatory cytokines
such as IL-6, COX-2 and proteinases such as MMP-1 and ADAMTS-4 in a pattern
which was not entirely IL-1-like. Pharmacological inhibition experiments suggested
that the production by injured cartilage of an inflammatory response gene which could
be assayed at the protein level, activin A, was regulated by FGF-mediated pathways
(ERK) as well as by NFκB and tyrosine kinases (src family kinases).
Given these findings, the role of tyrosine kinases in the early response of cartilage to
injury was explored. By phosphotyrosine immunoprecipitation and purification from
injured cartilage lysates, FAK and its substrate paxillin were identified from silverstained
gels by mass spectrometry. The phosphorylation of these src substrates accounted for rapidly inducible bands seen on phosphotyrosine western blotting of
injured cartilage lysates. However, no evidence of a role for src kinases in the
regulation of MAPK/IKK signalling upon injury was found. In contrast, blockade of
another tyrosine kinase, the FGF receptor, led to partial inhibition not only of the
ERK pathway following sharp injury, but also the other MAPKs and IKK. Whilst
activation of the same pathways was also seen following injury to synovium, FGF
receptor inhibition had no effect on this signalling. This suggested that FGF may have
a pro-inflammatory action following injury in vivo which is a particular feature of
cartilage.
cartilage might lead to net matrix loss and cartilage degeneration remains unknown.
Following experimental sharp injury to porcine articular cartilage (dissection from, or
scoring of the articular surface), our group has previously shown rapid activation of
the 3 mitogen activated protein kinase (MAPK) pathways in cartilage. Activation of
ERK, and probably also p38 MAPK, is due to release of fibroblast growth factor
(FGF) from the matrix after injury. However, despite a long search, the cause of JNK
activation following sharp injury remains unknown. I investigated the extent and
regulation of inflammatory signalling after cartilage injury and whether it was
sufficient to cause expression of inflammatory response genes.
In this work, I show that a number of intracellular signalling pathways including PI3
kinase and IκB kinase (IKK) (which leads to activation of NFκB) are activated by
sharp injury to cartilage. The signalling following injury was sufficient to induce a
wide range of inflammatory response mRNAs, including pro-inflammatory cytokines
such as IL-6, COX-2 and proteinases such as MMP-1 and ADAMTS-4 in a pattern
which was not entirely IL-1-like. Pharmacological inhibition experiments suggested
that the production by injured cartilage of an inflammatory response gene which could
be assayed at the protein level, activin A, was regulated by FGF-mediated pathways
(ERK) as well as by NFκB and tyrosine kinases (src family kinases).
Given these findings, the role of tyrosine kinases in the early response of cartilage to
injury was explored. By phosphotyrosine immunoprecipitation and purification from
injured cartilage lysates, FAK and its substrate paxillin were identified from silverstained
gels by mass spectrometry. The phosphorylation of these src substrates accounted for rapidly inducible bands seen on phosphotyrosine western blotting of
injured cartilage lysates. However, no evidence of a role for src kinases in the
regulation of MAPK/IKK signalling upon injury was found. In contrast, blockade of
another tyrosine kinase, the FGF receptor, led to partial inhibition not only of the
ERK pathway following sharp injury, but also the other MAPKs and IKK. Whilst
activation of the same pathways was also seen following injury to synovium, FGF
receptor inhibition had no effect on this signalling. This suggested that FGF may have
a pro-inflammatory action following injury in vivo which is a particular feature of
cartilage.
Date Issued
2009-04
Date Awarded
2009-10
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Saklatvala, Jeremy
Creator
Watt, Fiona Elizabeth
Publisher Department
The Kennedy Institute of Rheumatology
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)