Krüppel-like factor 9 is a circadian transcription factor in human epidermis that controls proliferation of keratinocytes.

Circadian clocks govern a wide range of cellular and physiological functions in various organisms. Recent evidence suggests distinct functions of local clocks in peripheral mammalian tissues such as immune responses and cell cycle control. However, studying circadian action in peripheral tissues has been limited so far to mouse models, leaving the implication for human systems widely elusive. In particular, circadian rhythms in human skin, which is naturally exposed to strong daytime-dependent changes in the environment, have not been investigated to date on a molecular level. Here, we present a comprehensive analysis of circadian gene expression in human epidermis. Whole-genome microarray analysis of suction-blister epidermis obtained throughout the day revealed a functional circadian clock in epidermal keratinocytes with hundreds of transcripts regulated in a daytime-dependent manner. Among those, we identified a circadian transcription factor, Krüppel-like factor 9 (Klf9), that is substantially up-regulated in a cortisol and differentiation-state-dependent manner. Gain- and loss-of-function experiments showed strong antiproliferative effects of Klf9. Putative Klf9 target genes include proliferation/differentiation markers that also show circadian expression in vivo, suggesting that Klf9 affects keratinocyte proliferation/differentiation by controlling the expression of target genes in a daytime-dependent manner.

Real-time monitoring of membrane cholesterol reveals new insights into epidermal differentiation.

Cholesterol is organized in distinctive liquid-ordered micro-domains within biological membranes called lipid rafts. These micro-domains direct multiple physiological functions in mammalian cells by modulating signaling processes. Recent findings suggest a role for lipid rafts in cellular processes in human keratinocytes such as early differentiation and apoptosis. However, research of lipid rafts is hindered by technological limitations in visualizing dynamic cholesterol organization in plasma membranes. This study addresses a real-time, non-invasive method for the long-term observation of cholesterol reorganization in plasma membranes. In addition, this study also addresses the dynamic process of cholesterol depletion and repletion in primary human keratinocytes. Cholesterol reorganization was measured by observed changes in cellular impedance. Disruption of lipid rafts with low concentrations of methyl-beta-cyclodextrin (MbetaCD) resulted in an increase in the proliferative capacity of keratinocytes, which was assessed using real-time proliferation curves and adenosine triphosphate (ATP)-based proliferation assays. Quantitative PCR showed a concomitant decrease in messenger RNA (mRNA) expression of the early differentiation markers keratins 1 and 10. Conversely, specific cholesterol reintegration led to a 4.5-fold increase in keratin 2 mRNA expression, a marker for late keratinocyte differentiation, whereas depletion resulted in a significant downregulation. These findings imply a strictly controlled mechanism for the regulation of membrane cholesterol composition in both early and terminal keratinocyte differentiation. The impedance-based method that this study addresses further enhances our understanding of how physiological processes in keratinocytes are controlled by membrane cholesterol.