Involvement of hypoxia-inducible factor activity in inevitable air-exposure treatment upon differentiation in a three-dimensional keratinocyte culture

Formation of the human skin epidermis can be reproduced by a three-dimensional (3D) keratinocyte culture system, in which air-exposure is inevitable upon initiation of differentiation. In the continuous submerged culture without air-exposure, even with a differentiation-compatible medium, several keratinocyte-specific proteins were not induced resulting in the formation of aberrant epidermal layers. To clarify the mechanism by which air-exposure promotes keratinocyte differentiation, we performed a comparative analysis on biological properties between submerged and air-liquid interphase culture systems. By transcriptomic analysis, hypoxia-inducible factor (HIF)-related genes appeared to significantly change in these cultured cells. In submerged culture, the transcriptional activity of HIF on its canonical response element was enhanced, while air-exposure treatment drastically reduced the transcriptional activity despite the high HIF protein level. Regulating HIF activity through reagents and genetic manipulation revealed that the reduced but retained HIF-transcriptional activity was essentially involved in differentiation. Furthermore, we showed, for the first time, that artificial supplementation of oxygen in the submerged culture system could restore keratinocyte differentiation as observed in the air-exposed culture. Thus, we mechanistically evaluated how HIF regulates the air-exposure-dependent differentiation of keratinocytes in a 3D culture system.

Automated summary

The formation of human skin epidermis can be replicated in a 3D keratinocyte culture system with air-exposure being crucial for differentiation. An analysis of biological properties in submerged and air-liquid interphase culture systems revealed significant changes in hypoxia-inducible factor (HIF)-related genes. Despite high HIF protein levels, air-exposure treatment drastically reduced the transcriptional activity of HIF on its canonical response element, which was found to be essential for differentiation. Oxygen supplementation in submerged culture restored keratinocyte differentiation, highlighting the role of HIF in air-exposure-dependent differentiation in a 3D culture system.