Tumor Necrosis Factor-alpha mediated transactivation of the Epidermal Growth Factor Receptor and Keratinocytes Público
Maffei, Vincent J (2011)
Published
Abstract
Tumor Necrosis Factor-alpha mediated transactivation of the Epidermal Growth Factor Receptor and Keratinocytes
Vincent J. Maffei
Tumor Necrosis Factor-alpha (TNF) has been implicated in a
variety of signaling networks
especially in the immune system. As such, TNF dysregulation has
been linked to numerous
autoimmune diseases particularly ones of chronic inflammation. TNF
has been characterized
historically as an inducer of nuclear factor kappa-B (NFkB) as well
as an inducer of apoptosis.
However, TNF in chronic inflammatory diseases such as psoriasis
promotes hyper-proliferation
of keratinocytes. The epidermal growth factor receptor family
(EGFR) is a group of cell-surface
proteins also involved in keratinocyte proliferation. This work
reports that TNF induces EGFR
activation in HaCaT keratinocytes through a matrix-metalloprotease
(MMP) mechanism, which,
in turn, leads to mitogen activated protein kinase activity (MAPK).
MMP cleavage of EGFR-
ligands is the proposed method of TNF-mediated EGFR activity.
Moreover, TNF induction of
the EGFR and NFkB pathways are independent and separate, which is
contrary to current
perspectives on TNF signaling. While TNF-induced EGFR activation is
insufficient for cell
cycle entry, it does protect keratinocytes from TNF-mediated
apoptosis. Additionally,
interleukin-8 expression, a marker of inflammation, by TNF occurs
through mutually activated
EGFR and NFκB pathways. Finally, neutralizing antibodies for
the EGFR family were used to
dissect individual contributions of receptor pairs towards a common
signal output. The
observations found for TNF-induced, EGFR-mediated IL-8 expression
were plausible given
findings in other studies. This work reveals TNF's direct effects
on keratinocytes that may
contribute to inflammation pathophysiology.
Table of Contents
Table of Contents
ABSTRACT...................................................................................................................................iv
ACKNOWLEDGEMENTS.................................................................................................................vi
1. INTRODUCTION
1.1 Tumor Necrosis
Factor-alpha.......................................................................................................1
1.2 Epidermal Growth Factor
Receptor...............................................................................................2
1.3 Inflammation and the
EGFR........................................................................................................4
2. MATERIALS AND METHODS
2.1 Cell
Culture.............................................................................................................................6
2.2 RNA Extraction & Real-time
PCR..................................................................................................6
2.3 Western
Blotting......................................................................................................................7
2.4 Cytokines & Pharmacologic
Inhibitors...........................................................................................8
2.5 Annexin-V staining and Fluorescence Assisted Cell
Sorting...............................................................8
2.6 Inverse matrix evaluation of linear
systems..................................................................................9
2.7
Statistics...............................................................................................................................10
3. RESULTS
3.1 TNF induces expression of egr-1 though the
EGFR.........................................................................10
3.2 TNF induction of Erk MAPK phosphorylation is dependent on the
EGFR and
extracellular matrix
metalloproteases..............................................................................................13
3.3 TNF induction of the EGFR/Erk/egr-1 pathway and NFκB
activation occur
irrespective of each
other..............................................................................................................15
3.4.1 TNF-induced EGFR activity and inflammatory
signaling................................................................15
3.4.2 TNF-mediated, EGFR-dependent proliferation and
apoptosis.........................................................16
3.4.3 TNF activation of the EGFR and NFκB are required for
optimal IL-8
expression..................................................................................................................................19
3.5 TNF activation of the erbb receptor network: inverse matrix
analysis of receptor
contributions...............................................................................................................................21
4.
DISCUSSION..........................................................................................................................24
5.
CONCLUSIONS.......................................................................................................................31
6.
REFERENCES..........................................................................................................................34
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