Doctor of Philosophy
Judith A. Cole
Charles A Lessman
Andrew C Liu
Carrie Hayes Sutter
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly lipophilic polyaromatic hydrocarbon and a persistent environmental contaminant. In the human epidermis, TCDD causes enhanced proliferation and altered, accelerated differentiation. However, epidermal differentiation, mediated by calcium (Ca), opposes and attenuates proliferative signaling, driven by the epidermal growth factor receptor (EGFR). Using a monolayer cell culture model, confluent NHEKs were treated with high Ca (1.8 mM) or TCDD (10 nM). EGFR down-regulation, signaling, and proliferation were then examined to assess the proliferative capacity of differentiating skin versus pathophysiological, TCDD-mediated differentiation. We found that while Ca and TCDD caused marked (~50% and ~30%) loss of [125I]-EGF binding, the effects of this down-regulation differed. ERK activity decreased 45% in Ca-treated cells and increased 300% in TCDD-treated cells. Utilizing ligand-specific ELISAs to interrogate cell-secreted EGFR ligands which might drive ERK activity, we found that Ca increased transforming growth factor-alpha (TGF-α) secretion, while TCDD increased TGF-α and EREG secretion relative to basal cells. Amphiregulin (AREG) increased with time in all treatments. Inhibiting ligand secretion with the MMP inhibitor, batimastat, or action with ligand-specific neutralizing antibodies in TCDD-treated cells reduced the increased ERK activity to basal levels but could not rescue ERK activity in Ca-treated cells. Similarly, in TCDD-treated NHEKs, neutralizing AREG or EREG reversed EGFR down-regulation, and removal of TGF-α further reduced biotin-EGF binding. In Ca-treated cells, neutralizing EGFR ligands had no effect on EGFR down-regulation. We then investigated how these treatments influenced proliferative capacity of cell populations through dsDNA quantitation and EdU labelling. We found that while Ca-treatment led to ~15% fewer cells than basal they still retained an equal (~5%) amount of EGFR-dependent proliferating cells. Conversely, TCDD-treatment led to equal cell numbers relative to basal cells, both of which were reduced following ligand neutralization. However, TCDD-treatment led to a significant increase in the population of proliferating cells (~8%) relative to basal. Taken together, our data suggests that in Ca-treated cells, EGFR down-regulation is ligand-independent and correlated with a loss of proliferative signaling capacity while TCDD-treated cells down-regulate the EGFR in a ligand-dependent manner, and that TGF-α retains a population of surface-associated receptors which may drive increased proliferation.
Dissertation or thesis originally submitted to the local University of Memphis Electronic Theses & dissertation (ETD) Repository.
Campion, Christina M., "The Role of the EGF Receptor and Its Ligands in Differentiation and 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Toxicity" (2016). Electronic Theses and Dissertations. 1538.