PMID- 27117977 OWN - NLM STAT- MEDLINE DCOM- 20170130 LR - 20181202 IS - 1872-7786 (Electronic) IS - 0009-2797 (Print) IS - 0009-2797 (Linking) VI - 253 DP - 2016 Jun 25 TI - Role of EGF receptor ligands in TCDD-induced EGFR down-regulation and cellular proliferation. PG - 38-47 LID - S0009-2797(16)30154-5 [pii] LID - 10.1016/j.cbi.2016.04.031 [doi] AB - In cultures of normal human epidermal keratinocytes (NHEKs), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces the expression of the epidermal growth factor receptor ligands transforming growth factor-alpha (TGF-alpha) and epiregulin (EREG). TCDD also down-regulates EGF receptors (EGFR), suggesting that decreases in signaling contribute to the effects of TCDD. In this study, we treated post-confluent NHEKs with 10 nM TCDD and assessed its effects on EGFR binding, EGFR ligand secretion, basal ERK activity, and proliferation. TCDD caused time-dependent deceases in [(125)I]-EGF binding to levels 78% of basal cell values at 72 h. Amphiregulin (AREG) levels increased with time in culture in basal and TCDD-treated cells, while TGF-alpha and epiregulin (EREG) secretion were stimulated by TCDD. Inhibiting EGFR ligand release with the metalloproteinase inhibitor batimastat prevented EGFR down-regulation and neutralizing antibodies for AREG and EREG relieved receptor down-regulation. In contrast, neutralizing TGF-alpha intensified EGFR down-regulation. Treating NHEKs with AREG or TGF-alpha caused rapid internalization of receptors with TGF-alpha promoting recycling within 90 min. EREG had limited effects on rapid internalization or recycling. TCDD treatment increased ERK activity, a response reduced by batimastat and the neutralization of all three ligands indicating that the EGFR and its ligands maintain ERK activity. All three EGFR ligands were required for the maintenance of total cell number in basal and TCDD-treated cultures. The EGFR inhibitor PD1530305 blocked basal and TCDD-induced increases in the number of cells labeled by 5-ethynyl-2'-deoxyuridine, identifying an EGFR-dependent pool of proliferating cells that is larger in TCDD-treated cultures. Overall, these data indicate that TCDD-induced EGFR down-regulation in NHEKs is caused by AREG, TGF-alpha, and EREG, while TGF-alpha enhances receptor recycling to maintain a pool of EGFR at the cell surface. These receptors are required for ERK activity, maintenance of total cell number, and stimulating the proliferation of a small subset cells. CI - Copyright (c) 2016 Elsevier Ireland Ltd. All rights reserved. FAU - Campion, Christina M AU - Campion CM AD - Department of Biological Sciences, University of Memphis, Memphis, TN 38152, USA. FAU - Leon Carrion, Sandra AU - Leon Carrion S AD - Department of Biological Sciences, University of Memphis, Memphis, TN 38152, USA. FAU - Mamidanna, Gayatri AU - Mamidanna G AD - Department of Biological Sciences, University of Memphis, Memphis, TN 38152, USA. FAU - Sutter, Carrie Hayes AU - Sutter CH AD - Department of Biological Sciences, University of Memphis, Memphis, TN 38152, USA. FAU - Sutter, Thomas R AU - Sutter TR AD - Department of Biological Sciences, University of Memphis, Memphis, TN 38152, USA. FAU - Cole, Judith A AU - Cole JA AD - Department of Biological Sciences, University of Memphis, Memphis, TN 38152, USA. Electronic address: jcole2@memphis.edu. LA - eng GR - R01 ES017014/ES/NIEHS NIH HHS/United States PT - Journal Article DEP - 20160423 PL - Ireland TA - Chem Biol Interact JT - Chemico-biological interactions JID - 0227276 RN - 0 (Amphiregulin) RN - 0 (Iodine Radioisotopes) RN - 0 (Ligands) RN - 0 (Polychlorinated Dibenzodioxins) RN - 0 (Quinazolines) RN - 0 (Transforming Growth Factor alpha) RN - 62229-50-9 (Epidermal Growth Factor) RN - EC 2.7.10.1 (ErbB Receptors) RN - TC62B68RSL (4-((3-bromophenyl)amino)-6,7-dimethoxyquinazoline) SB - IM MH - Amphiregulin/analysis/metabolism MH - Cell Proliferation/*drug effects MH - Cells, Cultured MH - Down-Regulation/*drug effects MH - Enzyme-Linked Immunosorbent Assay MH - Epidermal Growth Factor/chemistry/metabolism MH - ErbB Receptors/chemistry/*metabolism MH - Humans MH - Iodine Radioisotopes/chemistry MH - Ligands MH - Polychlorinated Dibenzodioxins/*toxicity MH - Protein Binding MH - Quinazolines/pharmacology MH - Transforming Growth Factor alpha/analysis/metabolism PMC - PMC4892999 MID - NIHMS784321 OTO - NOTNLM OT - 2,3,7,8-Tetrachlorodibenzo-p-dioxin OT - EGF receptors OT - Extracellular signal-regulated kinase OT - Human keratinocytes COIS- Conflict of interest The authors declare that there are no conflicts of interest. EDAT- 2016/04/28 06:00 MHDA- 2017/01/31 06:00 PMCR- 2017/06/25 CRDT- 2016/04/28 06:00 PHST- 2016/01/25 00:00 [received] PHST- 2016/03/30 00:00 [revised] PHST- 2016/04/22 00:00 [accepted] PHST- 2016/04/28 06:00 [entrez] PHST- 2016/04/28 06:00 [pubmed] PHST- 2017/01/31 06:00 [medline] PHST- 2017/06/25 00:00 [pmc-release] AID - S0009-2797(16)30154-5 [pii] AID - 10.1016/j.cbi.2016.04.031 [doi] PST - ppublish SO - Chem Biol Interact. 2016 Jun 25;253:38-47. doi: 10.1016/j.cbi.2016.04.031. Epub 2016 Apr 23.