br Acknowledgements br The oxygenation level has an

Acknowledgements
The oxygenation level has an impact on various aspects of skin physiology, but mechanisms of these effects, including the role of hypoxia in epidermal differentiation, are still poorly understood. The main components of the hypoxia pathway are short-lived transcription factor HIF1a (stabilized by hypoxia) and its dimerization partner ARNT. To elucidate the role of hypoxia and HIF pathway in control of epidermal differentiation we used primary and immortalized human keratinocyte monolayer cultures along with skin equivalents developed on alginate matrices using these cells. It appeared that hypoxia positively regulates the TBB of early differentiation markers, such as CK1, acting synergistically with Ca-dependent activation of these genes. However, in keratinocytes with attenuated HIF1 activity (Arnt-KD) hypoxia had no effect on CK1 expression. The effect of Ca (w/o hypoxia conditioning) on CK1 was the same in both control and Arnt-KD cells. Microarray analysis of keratinocytes under 1% O and low/high Ca revealed a few genes regulated by hypoxia and Ca independently. But the majority of differentiation-associated genes were mutually regulated by both, while the mode of this co-regulation was variable. Furthermore, hypoxia also affects expression of genes implicated in Ca turnover. Our results revealed a complex and context-dependent interplay between Ca and hypoxia/HIF1 pathways during epidermal differentiation suggesting that concomitant modulation of their activity may serve as a tool to control differentiation in both monolayer cultures and in 3D skin equivalents. The relevance of these findings to epidermal homeostasis and disease remains to be elucidated.
Introduction Guanine-rich single-stranded DNA can form four-stranded DNA secondary structures called G-quadruplexes (G4) [1]. The formation of G-quadruplex has been initially proposed to occur at telomeric region where G-rich repeats are present within an extended 3′ single-strand protruding portion involving the interaction of four guanine bases in a square planar arrangement stabilized by central cations [2], [3]. G-quadruplexes can sequester the 3′ end of the telomeric DNA and prevent it from being extended by telomerase, making this structure promising anti-cancer drug target [3]. Some studies on telomeric DNA G-quadruplex binding ligands show that these ligands can inhibit telomerase activity and induce telomere shortening and replicative senescence by blocking the binding of telomerase with G-overhang [4], [5], [6]. It is clear how the telomeric DNA G-quadruplex binding ligands work in telomerase positive cancer cells. Significantly, tumor cells stabilize their telomeres, either rely on upregulating telomerase or activating an alternative lengthening of telomeres (ALT) mechanism [7]. Although recent data report that G-quadruplex binding ligands induce an important telomere degradation of ALT cell lines, it has been suggested that additional mechanisms may explain the biological activity of the G-quadruplex binding ligands in these tumor cell lines [8], [9], [10]. However, how these G-quadruplex binding ligands induce replicative mortality in telomerase negative cancer cells remains unclear. To address this question, it is necessary to mention telomeric repeat–containing RNA (TERRA) and telomeric repeat factor 2 (known as TRF2 or TERF2). Telomeric repeat-containing RNA (TERRA) is a large non-coding RNA in mammalian cells, which forms an integral component of telomeric heterochromatin [11], [12]. Individual TERRA molecules start with a subtelomeric RNA tract followed by a variable number of telomeric G-rich repeats (5′-UUAGGG-3′ in vertebrates) [13]. Phan and co-workers found that long telomeric RNAs form G-quadruplexes comprising four UUAGGG repeats, adopting a parallel conformation containing three G-tetrad layers [14]. TERRA G-quadruplexes are ideal therapeutic targets, because they are required for telomere heterochromatin formation in all cancer cells, even in those that do not require telomerase (ALT-positive tumors) [15]. Several types of TERRA G-quadruplex binding ligands have been reported, which may inspire new strategies for targeting telomere-related diseases [16], [17], [18], [19].