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Keratinocytes undergo significant structural remodeling during epidermal differentiation, including a broad transformation of the proteome coupled with a reduction in total cellular biomass. This suggests that intracellular digestion of proteins and organelles is necessary for keratinocyte differentiation. Here, we use both genetic and pharmacologic approaches to demonstrate that autophagy and lysosomal functions are required for keratinocyte differentiation in organotypic human skin. Lysosomal activity was required for mechanistic target of rapamycin signaling and mitochondrial oxidative metabolism. In turn, mitochondrial reactive oxygen species, produced as a natural byproduct of oxidative phosphorylation, were necessary for keratinocyte differentiation. Finally, treatment with exogenous reactive oxygen species rescued the differentiation defect in lysosome-inhibited keratinocytes. These findings highlight a reciprocal relationship between lysosomes and mitochondria, in which lysosomes support mitochondrial metabolism and the associated production of mitochondrial reactive oxygen species. The mitochondrial reactive oxygen species released to the cytoplasm in suprabasal keratinocytes triggers autophagy and lysosome-mediated degradation necessary for epidermal differentiation. As defective lysosome-dependent autophagy is associated with common skin diseases including psoriasis and atopic dermatitis, a better understanding of the role of lysosomes in epidermal homeostasis may guide future therapeutic strategies.

This study addresses the role of glycogen synthase kinase (GSK)-3β signaling in the tumorigenic behavior of melanoma. Immunohistochemical staining revealed GSK3β to be focally expressed in the invasive portions of 12 and 33% of primary and metastatic melanomas, respectively. GSK3 inhibitors and small interfering RNA (siRNA) knockdown of GSK3β were found to inhibit the motile behavior of melanoma cells in scratch wound, three-dimensional collagen–implanted spheroid, and modified Boyden chamber assays. Functionally, inhibition of GSK3β signaling was found to suppress N-cadherin expression at the messenger RNA and protein levels, and was associated with decreased expression of the transcription factor Slug. Pharmacological and genetic ablation of GSK3β signaling inhibited the adhesion of melanoma cells to both endothelial cells and fibroblasts and prevented transendothelial migration, an effect rescued by the forced overexpression of N-cadherin. A further role for GSK3β signaling in invasion was suggested by the ability of GSK3β inhibitors and siRNA knockdown to block phosphorylation of focal adhesion kinase (FAK) and increase the size of focal adhesions. In summary, we have, to our knowledge, demonstrated a previously unreported role for GSK3β in modulating the motile and invasive behavior of melanoma cells through N-cadherin and FAK. These studies suggest the potential therapeutic utility of inhibiting GSK3β in defined subsets of melanoma.