The dermis contains two distinct layers: the papillary and the reticular layers. In vitro cultures of the fibroblasts from these layers show that they are different. However, no molecular markers to differentiate between the two subtypes of fibroblasts are known. We performed gene expression analysis on cultured fibroblasts isolated from the papillary and reticular dermis. In all, 116 genes were found to be expressed differentially. Of these, 13 were validated by quantitative reverse transcriptase-PCR analysis and two markers could be validated at the protein level in monolayer cultures. Three markers showed differential expression in in vivo skin sections. The identified, characteristic markers of the two fibroblast subpopulations provide useful tools to perform functional studies on reticular and papillary fibroblasts.

We investigated the expression of microRNAs (miRNAs) associated with replicative senescence in human primary keratinocytes. A cohort of miRNAs up-regulated in senescence was identified by genome-wide miRNA profiling, and their change in expression was validated in proliferative versus senescent cells. Among these, miRNA (miR)-138, -181a, -181b, and -130b expression increased with serial passages. miR-138, -181a, and -181b, but not miR130b, overexpression in proliferating cells was sufficient per se to induce senescence, as evaluated by inhibition of BrdU incorporation and quantification of senescence-activated β-galactosidase staining. We identified Sirt1 as a direct target of miR-138, -181a, and -181b, whereas ΔNp63 expression was inhibited by miR-130b. We also found that ΔNp63α inhibits miR-138, -181a, -181b, and -130b expression by binding directly to p63-responsive elements located in close proximity to the genomic loci of these miRNAs in primary keratinocytes. These findings suggest that changes in miRNA expression, by modulating the levels of regulatory proteins such as p63 and Sirt1, strongly contribute to induction of senescence in primary human keratinocytes, thus linking these two proteins. Our data also indicate that suppression of miR-138, -181a, -181b, and -130b expression is part of a growth-promoting strategy of ΔNp63α in epidermal proliferating cells.

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Short p63 isoform, ΔNp63, is crucial for epidermis formation, and it plays a pivotal role in controlling the turnover of basal keratinocytes by regulating the expression of a subset of genes involved in cell cycle and cell adhesion programs. The glycolytic enzyme hexokinase 2 (HK2) represents the first step of glucose utilization in cells. The family of HKs has four isoforms that differ mainly in their tissue and subcellular distribution. The preferential mitochondrial localization of HK2 at voltage-dependent anion channels provides access to ATP generated by oxidative phosphorylation and generates an ADP/ATP recycling mechanism to maintain high respiration rates and low electron leak. Here, we report that ΔNp63 depletion in human keratinocytes impairs mitochondrial basal respiration and increases mitochondrial membrane polarization and intracellular reactive oxygen species. We show ΔNp63-dependent regulation of HK2 expression, and we use ChIP, validated by p63-Chip sequencing genomewide profiling analysis, and luciferase assays to demonstrate the presence of one p63-specific responsive element within the 15th intronic region of the HK2 gene, providing evidence of a direct interaction. Our data support the notion of ΔNp63 as a master regulator in epithelial cells of a combined subset of molecular mechanisms, including cellular energy metabolism and respiration. The ΔNp63-HK2 axis is also present in epithelial cancer cells, suggesting that ΔNp63 could participate in cancer metabolic reprogramming.p63 | HK2 | keratinocytes | oxidative metabolism | mitochondria