ARNT controls the expression of epidermal differentiation genes through HDAC- and EGFR-dependent pathways
Authors
E. Douglas Robertson, Lynda Weir, Malgorzata Romanowska, Irene M. Leigh and Andrey A. Panteleyev
Institution
Ninewells Hospital, University of Dundee
Country
United Kingdom
Year
2012
Journal
Journal of Cell Science
Abstract
Previously we showed that spatial and developmental modulation of ARNT (HIF1β) expression in mouse epidermis is essential for maintenance of keratinocyte differentiation, proper formation of the barrier and normal desquamation. Here, using lentiviral suppression or induction of ARNT in TERT-immortalized (N-TERT) and HaCaT cells we assessed the nature and mechanisms of ARNT involvement in control of differentiation in human epidermal keratinocytes. ARNT depletion did not affect the levels of basal keratins K5/14 but significantly induced expression of several key differentiation markers (an effect abolished by EGF supplementation). Furthermore, ARNT deficiency resulted in the downregulation of amphiregulin (AREG) - the most highly expressed EGFR ligand in human keratinocytes, whilst upregulation of ARNT showed the opposite. In ARNT-deficient monolayer cultures and 3D epidermal equivalents the downregulation of AREG was concurrent with a decline of EGFR and ERK1/2 phosphorylation. TSA, a potent suppressor of HDAC activity, abolished the effects of ARNT deficiency implying a role for HDACs in ARNT-dependent modulation of AREG/EGFR pathway and downstream epidermal genes. Total HDAC activity was significantly increased in ARNT-depleted cells and decreased with ARNT overexpression. ARNT-dependent shifts in HDAC activity were specifically attributed to significant changes in the levels of HDAC 1, 2 and 3 proteins (but not mRNA) in both monolayer and 3D cultures. Collectively our results suggest that ARNT controls AREG expression and the downstream EGFR/ERK pathway in keratinocytes at least in part by modulating HDAC activity. This novel regulatory pathway targeting advanced stages of epidermal differentiation may have important implications for skin pathology such as psoriasis, atopic dermatitis and cancer.
Product use
3D culture of immortalised keratinocytes using 3D Prime culture medium