Tumor Necrosis Factor-alpha mediated transactivation of the Epidermal Growth Factor Receptor and Keratinocytes Open Access

Maffei, Vincent J (2011)

Permanent URL: https://etd.library.emory.edu/concern/etds/j098zb527?locale=en
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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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