Alteration of gene expression in human keratinocytes by aryl hydrocarbon receptor and retinoic acid receptors
Description
Investigation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) response in HuE-1 and SCC-12F keratinocyte cell lines revealed differential responses. Exposure of SCC-12F keratinocytes to dioxin produced a cytochrome P450 1A1 (CYP1A1) response, assayed by ethoxycoumarin-o-deethylase activity (15-35 pmol/mg protein/minute at 1-100 nM TCDD). Neither HuE-1 keratinocytes or HEK 293 cells (human embryonic kidney) produced this response (5 pmol or 1 pmol/mg protein/min for HuE-1 and HEK, respectively at 100 nM TCDD). HuE-1 cells possess truncated ARNT protein, which could cause lack of dioxin signaling in these cells. Transfection of HEK 293 cells with a CYP1A1 promoter construct, followed by dioxin exposure, induced reporter gene activity 2-3 fold, indicating these cells responded to dioxin but the endogenous CYP1A1 gene was defective Transforming growth factor beta (TGF-$\beta$), upregulated in keratinocyte culture medium by trans-RA exposure (683.2 pmol vs. 157 pmol in control), was suppressed by TCDD (41 pmol). The dominant TCDD effect was due, at least in part, to TCDD-mediated loss of trans-RA binding to its receptor (RARs) (Kd = 0.13 nM, 1.8 fmol binding sites vs. Kd = 0.13 nM, 4.3 fmol binding sites in control). Loss of binding affected trans-RA-mediated transcription, determined by transfection with a retinoic acid responsive reporter plasmid (trans-RA reporter activity = 48.1% of control, TCDD activity = 164% of control, TCDD + RA activity = 171.3% of control) Investigation of epidermal growth factor receptor (EGFR) production in HuE-1 keratinocytes revealed RAR-$\alpha$ suppressed EGFR transcription, determined by a luciferase reporter assay in absence of added ligand in every case (5.4% control). When cells were confluent and differentiating, a second RAR isoform, RAR-$\gamma ,$ was less effective at suppressing EGFR production (93% of control). When cells were actively proliferating, both RAR-$\alpha$ and RAR-$\gamma$ appeared equally effective in this action (23% vs 28% of control, for RAR-$\alpha$ or RAR-$\gamma ).$ Examination of RAR protein in HuE-1 cells revealed RAR-$\gamma$ protein was always present. RAR-$\alpha$ was only detected when added exogenously via an expression plasmid. This revealed the required up-regulation of RAR-$\alpha$ to aid cells through differentiation, while RAR-$\gamma ,$ present in high abundance, appears ineffective in mediating differentiation