Abstract
Abnormal keratinocyte differentiation is fundamental to pathologies such as skin cancer and mucosal inflammatory diseases. The ability to grow keratinocytes in vitro allows the study of differentiation however any translational value is limited if keratinocytes get altered by the culture method. Although serum lipids (SLPs) and phenol red (PR) are ubiquitous components of culture media their effect on differentiation is largely unknown. We show for the first time that PR and SLP themselves suppress expression of differentiation-specific keratins K1, K10 and K2 in normal human epidermal keratinocytes (NHEK) and two important cell lines, HaCaT and N/TERT-1. Removal of SLP increased expression of K1, K10 and K2 in 2D and 3D cultures, which was further enhanced in the absence of PR. The effect was reversed for K1 and K10 by adding all-trans retinoic acid (ATRA) but increased for K2 in the absence of PR. Furthermore, retinoid regulation of differentiation-specific keratins involves post-transcriptional mechanisms as we show KRT2 mRNA is stabilised whilst KRT1 and KRT10 mRNAs are destabilised in the presence of ATRA. Taken together, our results indicate that the presence of PR and SLP in cell culture media may significantly impact in vitro studies of keratinocyte differentiation.
Highlights
Skin is the main body barrier against environmental stresses including UV, chemicals and microbes as well as providing protection against water loss[1,2]
The protein expression truly mimicked the mRNA expression for KRT1 and KRT10 with much higher protein expression in serum lipids (SLPs)−/phenol red (PR)+ compared with the control SLP+/PR+ but did not change significantly in SLP+/PR−
FCS and PR are routinely found in medium used to culture epidermal keratinocytes, the effect of SLP, present in FCS, and PR themselves on keratinocyte differentiation have never been fully investigated
Summary
Skin is the main body barrier against environmental stresses including UV, chemicals and microbes as well as providing protection against water loss[1,2]. This differentiation programme produces several cell layers containing keratinocytes at different stages of differentiation until the cells are terminally differentiated before shedding off from the skin surface In hyperproliferative situations such as during wound healing, psoriasis, pathological scarring and in some cancers, keratins K6, K16 and K17 are induced[20,21,22,23]. We demonstrate that the increase in KRT2 transcript was because of stabilisation of its mRNA by ATRA whereas KRT1 and KRT10 mRNAs were destabilised This is the first report where differential effect of retinoids on the expression of differentiation-specific keratins has been ascribed to the stability of their transcripts
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