Loss of Dicer in Newborn Melanocytes Leads to Premature Hair Graying and Changes in Integrin Expression.

Premature hair graying occurs owing to the depletion of melanocyte stem cells in the hair follicle, which can be accelerated by stress caused by genetic or environmental factors. However, the connection between stress and melanocyte stem cell loss is not fully understood. MicroRNAs are molecules that control gene expression by regulating mRNA stability and translation and are produced by the enzyme Dicer, which is repressed under stress. In this study, using 2 mouse genetic models and human and mouse cell lines, we found that the inactivation of Dicer in melanocytes leads to misplacement of these cells within the hair follicle, resulting in a lack of melanin transfer to keratinocytes in the growing hair and the exhaustion of the melanocyte stem cell pool. We also show that miR-92b, which regulates ItgaV mRNA and protein levels, plays a role in altering melanocyte migration. Overall, our findings suggest that the Dicer-miR92b-ItgaV pathway serves as a major signaling pathway linking stress to premature hair greying.

UVB represses melanocyte cell migration and acts through ß-catenin.

The exposure of skin to ultraviolet (UV) radiation can have both beneficial and deleterious effects: it can lead, for instance, to increased pigmentation and vitamin D synthesis but also to inflammation and skin cancer. UVB may induce genetic and epigenetic alterations and have reversible effects associated with post-translational and gene regulation modifications. ß-catenin is a main driver in melanocyte development; although infrequently mutated in melanoma, its cellular localization and activity are frequently altered. Here, we evaluate the consequence of UVB on ß-catenin in the melanocyte lineage. We report that in vivo, UVB induces cytoplasmic/nuclear relocalization of ß-catenin in melanocytes of newborn mice and adult human skin. In mouse melanocyte and human melanoma cell lines in vitro, UVB increases ß-catenin stability, accumulation in the nucleus and cotranscriptional activity, leading to the repression of cell motility and velocity. The activation of the ß-catenin signalling pathway and its effect on migration by UVB are increased by an inhibitor of GSK3ß, and decreased by an inhibitor of ß-catenin. In conclusion, UVB represses melanocyte migration and does so by acting through the GSK3-ß-catenin axis.

The WNT-less wonder: WNT-independent ß-catenin signaling.

ß-catenin is known as an Armadillo protein that regulates gene expression following WNT pathway activation. However, WNT-independent pathways also activate ß-catenin. During the establishment of the melanocyte lineage, ß-catenin plays an important role. In the context of physiopathology, ß-catenin is activated genetically or transiently in various cancers, including melanoma, where it can be found in the nucleus of tumors. In this review, we discuss alternative pathways that activate ß-catenin independent of WNTs and highlight what is known regarding these pathways in melanoma. We also discuss the role of ß-catenin as a transcriptional regulator in various cell types, with emphasis on the different transcription factors it associates with independent of WNT induction. Finally, the role of WNT-independent ß-catenin in melanocyte development and melanomagenesis is also discussed.

Selective Targeting of Extracellular Insulin-Degrading Enzyme by Quasi-Irreversible Thiol-Modifying Inhibitors.

Many therapeutically important enzymes are present in multiple cellular compartments, where they can carry out markedly different functions; thus, there is a need for pharmacological strategies to selectively manipulate distinct pools of target enzymes. Insulin-degrading enzyme (IDE) is a thiol-sensitive zinc-metallopeptidase that hydrolyzes diverse peptide substrates in both the cytosol and the extracellular space, but current genetic and pharmacological approaches are incapable of selectively inhibiting the protease in specific subcellular compartments. Here, we describe the discovery, characterization, and kinetics-based optimization of potent benzoisothiazolone-based inhibitors that, by virtue of a unique quasi-irreversible mode of inhibition, exclusively inhibit extracellular IDE. The mechanism of inhibition involves nucleophilic attack by a specific active-site thiol of the enzyme on the inhibitors, which bear an isothiazolone ring that undergoes irreversible ring opening with the formation of a disulfide bond. Notably, binding of the inhibitors is reversible under reducing conditions, thus restricting inhibition to IDE present in the extracellular space. The identified inhibitors are highly potent (IC50(app) = 63 nM), nontoxic at concentrations up to 100 µM, and appear to preferentially target a specific cysteine residue within IDE. These novel inhibitors represent powerful new tools for clarifying the physiological and pathophysiological roles of this poorly understood protease, and their unusual mechanism of action should be applicable to other therapeutic targets.

Transcriptional control of late differentiation in human keratinocytes by TAp63 and Notch.

We previously showed that in cervical carcinoma cells, the TAp63ß isoform of the p63 transcription factor is negatively interfering with the carcinogenic pathways promoting anchorage-independent growth. In this study, we have defined the mechanisms underlying the effects of TAp63ß through a transcriptome analysis of human keratinocytes overexpressing this protein. TAp63ß modulated expression of 1203 genes (944 activated and 259 repressed; P-value <0.05), notably genes involved in epithelial development and keratinocyte differentiation. In comparison, while TAp63γ acts similarly to TAp63ß to transactivate a selected panel of target genes, other p63 isoforms, including ΔNp63α, which is highly expressed in keratinocytes, are inactive. Upon induction of differentiation of primary human keratinocytes, we observed endogenous expression of TAp63ß and γ isoforms, along with transcriptional activation of selected target genes. Intriguingly, our data also indicated that TAp63ß activates transcription of members of the Notch pathway, which is known to promote keratinocyte differentiation. By inhibiting and activating the Notch pathway, we revealed a subset of TAp63ß-activated genes that were co-dependent on Notch for their expression. Our work demonstrates that the shorter TAp63 isoforms (TAp63ß/γ) are specifically induced in human keratinocytes and cooperate with Notch signalling to activate transcription of late differentiation genes supporting their role as putative tumor suppressors in HPV-associated tumorigenesis.

Optimization of peptide hydroxamate inhibitors of insulin-degrading enzyme reveals marked substrate-selectivity.

Insulin-degrading enzyme (IDE) is an atypical zinc-metallopeptidase that degrades insulin and the amyloid ß-protein and is strongly implicated in the pathogenesis of diabetes and Alzheimer's disease. We recently developed the first effective inhibitors of IDE, peptide hydroxamates that, while highly potent and selective, are relatively large (MW > 740) and difficult to synthesize. We present here a facile synthetic route that yields enantiomerically pure derivatives comparable in potency to the parent compounds. Through the generation of truncated variants, we identified a compound with significantly reduced size (MW = 455.5) that nonetheless retains good potency (ki = 78 ± 11 nM) and selectivity for IDE. Notably, the potency of these inhibitors was found to vary as much as 60-fold in a substrate-specific manner, an unexpected finding for active site-directed inhibitors. Collectively, our findings demonstrate that potent, small-molecule IDE inhibitors can be developed that, in certain instances, can be highly substrate selective.