Hypoxia-mediated control of HIF/ARNT machinery in epidermal keratinocytes

Abstract

Transcriptional activity of hypoxia-induced factor 1 (HIF1) - a heterodimer of HIF1α and ARNT (HIF1β) - is essential for cellular adaptation to environmental stress and plays an important role in skin development, wound healing, tumorigenesis and barrier function. Using primary mouse and human epidermal keratinocytes at ambient or hypoxic (1% O(2)) conditions we studied effects of hypoxia upon HIF protein expression. Significant nuclear levels of ARNT and HIF1α along with high HIF1 activity in normoxic keratinocytes suggest an as yet uncharacterised oxygen-independent role for HIF pathway in the epidermis. Acute hypoxia results in an instant but transient increase of HIF1α protein accompanied by a gradual decrease in its mRNA, while ARNT expression remains unchanged. In prolonged (chronic) hypoxia both HIF1α and Arnt are downregulated along with decline of HIF1 activity. However, expression of classical HIF1 targets such as Selenbp1 and Vegfa remains high. Thus, keratinocytes respond to acute hypoxia with immediate block of HIF1α protein degradation and concomitant increase of HIF activity, while under chronic hypoxia pro-angiogenic signalling is maintained through HIF1-independent pathway(s). Decline of HIF1α during chronic exposure is controlled at both mRNA and protein levels, while Arnt is downregulated post-translationally. Distinct transcription levels of Hif1α and Hif3α splice variants under normoxia and their differential response to hypoxia suggest functional diversity of Hif-α isoforms and highlight the complexity of HIF machinery control in epidermal keratinocytes.

ARNT controls the expression of epidermal differentiation genes through HDAC- and EGFR-dependent pathways

“…Protein lysates were obtained from 70-80% confluent keratinocytes as reported previously (Geng et al, 2006) and processed for WB (Weir et al, 2011a) using appropriate antibodies (supplementary material Table S3). …”

“…RNA was diluted 1:10 and used in qPCR reactions with Invitrogen Superscript III Platinum SYBR Green One-Step qRT-PCR. Reactions were run on a Bio-Rad mini-opticon realtime PCR machine as previously described (Weir et al, 2011a). Primers used are shown in supplementary material Table S2.…”

Hypoxic Upregulation of ARNT (HIF-1β): A Cell-Specific Attribute with Clinical Implications

“…Exposure of primary mouse keratinocytes to acute hypoxia (1% O 2 ) resulted in an upregulation of ARNT after 4 and 5 h, respectively [16]. However, this effect was not statistically significant.…”

“…Putative alterations on ARNT mRNA expression were also evaluated in this cell model. In contrast, time-course experiments revealed no apparent changes on mRNA level in murine keratinocytes cultured in hypoxia up to 48 h [16]. The selection of an inappropriate internal control in qPCR analysis can also lead to different expression levels in normoxia and hypoxia [17].…”

Vascular endothelial growth factor production is induced by histone deacetylase 1 and suppressed by von Hippel-Lindau protein in HaCaT cells

“…In keratinocytes, factors such as transforming growth factor-alpha (TGF-alpha), epidermal growth factor, and insulin-like growth factor I and II(IGF-I/II) are involved in the induction of VEGF production. [5] Although HIF-1alpha is constitutively expressed in normal keratinocytes, [6][7] it is not known if HDAC1 and VHL can regulate the levels of HIF-1alpha in keratinocytes to alter VEGF production.…”