Lysosomes Support the Degradation, Signaling, and Mitochondrial Metabolism Necessary for Human Epidermal Differentiation

Abstract

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.

Figure 1.. Lysosomes are necessary for epidermal differentiation.

(a) 2 μM Lys05, for 48-hours, blocked lysosome-mediated degradation of autophagosomes, resulting in the retention of p62, LC3AII, and LC3BII. (b) Over the 9-day time course of this experiment Lys05-treated keratinocytes proliferated at nearly the same rate as control keratinocytes (Log₂ of means ± s.d.). (c) Control versus Lys05 or bafilomycin-A1 treated organotypic cultures. Lysosomal inhibition results in undifferentiated tissue lacking normal epidermal architecture (H+E), including the expression of keratin 10 (red), filaggrin (green), involucrin (red), and loricrin (green), nuclei (blue). (d) Nonsilencing control (NS) and vATPase shRNA knockdown (vATPasei) organotypic cultures highlighting keratin-10 (red), filaggrin (green), and nuclei (blue). vATPase gene knockdown was confirmed by qPCR. All scale bars = 100μM.

Figure 2:. Epidermal differentiation requires autophagy.

(a) Control, SBI, and Spautin-1 organotypic cultures examined by H & E and immunofluorescence. Inhibition of autophagy blocked epidermal differentiation, including the expression of Keratin 10 (red) and filaggrin (green). Nuclei are (blue). (b) ATG7 depletion inhibited differentiation as demonstrated by loss of keratin-10 (red) and filaggrin (green). ATG7 knockdown was confirmed by qPCR. (c) Cryosections of NS and beclin-1i (shRNA knockdown) organotypic cultures highlighting keratin-10 (red), filaggrin (green), and nuclei (blue). Knockdowns of beclin-1 were confirmed by qPCR. All scale bars = 100μM.

Figure 3:. Lysosomes support the mTOR-signaling that promotes protein translation and mitochondrial metabolism.

(a-b) Lysosomal inhibition lead to loss of both pS6 and TFAM. (a) pS6 (red), ColVII (green), and nuclei (blue) or (b) for TFAM (red) and nuclei (blue). All scale bars = 100μM. (c) Seahorse analysis shows reduced baseline and maximal oxygen consumption in keratinocytes treated with Lys05 or bafilomycin-A1.

Figure 4:. Reactive Oxygen Species are critical for epidermal architecture.

(a) The ROS-scavenger N-acetylcysteine (NAC) or mitochondrial inhibitor dideoxycytidine (DDC) inhibit epidermal differentiation indicated by keratin-10 (red), filaggrin (green) and nuclei (blue). (b) Lysosomal inhibition diminished ROS, as demonstrated by the expression of NRF2 (red), with ColVII (green), and nuclei (blue). (c) Diffuse localization of cytochrome-c (red) in suprabasal layers of OTC reflects mitochondrial depolarization. All scale bars = 100μM.

Figure 5:. Exogenous ROS restores differentiation in epidermis with inhibited lysosomes.

(a) Exogenous ROS restored differentiation to Lys05 or bafilomycin-A1 OTCs as demonstrated by immunofluorescent probing for keratin-10 (red) and filaggrin (green), with nuclei (blue). (b) Exogenous ROS rescued LC3A (red) localization in Lys05 and bafilomycin-A1 OTCs. All scale bars = 100μM.