Keratinocytes maintain epidermal integrity through cellular differentiation. This process enhances intraorganelle digestion in keratinocytes to sustain nutritional and calcium-ionic stresses observed in upper skin layers. However, the molecular mechanisms governing keratinocyte differentiation and concomitant increase in lysosomal function is poorly understood. Here, by using primary neonatal human epidermal keratinocytes, we identified the molecular link between signaling pathways and cellular differentiation/lysosome biogenesis. Incubation of keratinocytes with CaCl2 induces differentiation with increased cell size and early differentiation markers. Further, differentiated keratinocytes display enhanced lysosome biogenesis generated through ATF6-dependent ER stress signaling, but independent of mTOR-MiT/TFE pathway. In contrast, chemical inhibition of mTORC1 accelerates calcium-induced keratinocyte differentiation, suggesting that activation of autophagy promotes the differentiation process. Moreover, differentiation of keratinocytes results in lysosome dispersion and Golgi fragmentation, and the peripheral lysosomes showed colocalization with Golgi-tethering proteins, suggesting that these organelles possibly derived from Golgi. In line, inhibition of Golgi function, but not the depletion of Golgi-tethers or altered lysosomal acidity, abolishes keratinocyte differentiation and lysosome biogenesis. Thus, ER stress regulates lysosome biogenesis and keratinocyte differentiation to maintain epidermal homeostasis.
Objectives To demonstrate the synergistic effect of 4‐hexylresorcinol (4‐HR) with niacinamide in boosting anti‐melanogenic efficacy in vitro and establish the in vivo efficacy and safety of the combination in a human trial. Methods Primary human epidermal melanocytes and 3D pigmented skin equivalents were treated with 4‐HR, niacinamide, and their combinations for their effect on pigmentation. This was followed by a randomized, double‐blind, split‐face clinical study in Chinese subjects, and effects on skin tone, hyperpigmentation, fine lines and wrinkles, hydration, and skin firmness were measured for a 12‐week study period. Results In vitro tyrosinase enzyme activity studies showed that 4‐HR is one of the most potent tyrosinase inhibitors. The combination of 4‐HR and niacinamide showed a synergistic reduction in melanin production in cultured melanocytes and lightened the 3D skin equivalent model. In vitro as well as in the human trial, the combination of 4‐HR and niacinamide showed significantly improved efficacy over niacinamide alone on hyperpigmentation spots as measured by L*, the visual appearance of fine lines and wrinkles in crow's feet and perioral area and skin firmness, with no product‐related adverse events. Conclusions A formulation containing a combination of 4‐HR and niacinamide delivered superior skin tone and anti‐ageing benefits significantly better than niacinamide alone with no adverse events. This study demonstrates that a product designed to affect multiple pathways of melanogenesis, inflammation, and ageing may provide an additional treatment option, beyond hydroquinone and retinoids, for hyperpigmentation and ageing.
Keratinocytes maintain epidermis integrity and function including physical and antimicrobial barrier through cellular differentiation. This process is predicted to be controlled by calcium ion gradient and nutritional stress. Keratinocytes undergo proteome changes during differentiation, which enhances the intracellular organelle digestion to sustain the stress conditions. However, the molecular mechanism between epidermal differentiation and organelle homeostasis is poorly understood. Here, we used primary neonatal human epidermal keratinocytes to study the link between cellular differentiation, signaling pathways and organelle turnover. Upon addition of calcium chloride (2 mM) to the culture medium, keratinocytes increased their cell size and the expression of differentiation markers. Moreover, differentiated keratinocytes showed enhanced lysosome biogenesis that was dependent on ATF6-arm of UPR signaling but independent of mTOR-MiT/TFE transcription factors. Furthermore, chemical inhibition of mTOR has increased keratinocyte differentiation and relocalized the MiT/TFE TFs to the lysosome membranes, indicating that autophagy activation promotes the epidermal differentiation. Interestingly, differentiation of keratinocytes resulted in dispersal of fragmented Golgi and lysosomes, and the later organelles showed colocalization with Golgi-tethering proteins, suggesting that these lysosomes possibly originated from Golgi, hence named as Golgi-associated lysosomes (GALs). Consistent to this prediction, inhibition of Golgi function using brefeldin A completely abolished the formation of GALs and the keratinocyte differentiation. Thus, ER stress regulates the biogenesis of GALs, which maintains keratinocyte differentiation and epidermal homeostasis.
Following publication of this article, the authors realized there was an error in Fig. 2b that needed correction. The TFEB panel of Fig. 2b (total lysate) appears to be the same as the TFEB panel of Fig. 2e (cytosolic fraction); the TFE3 panels of Fig. 2b (total lysate) appear to be the same as the TFE3 panels of Fig. 2e (cytosolic fraction) which happened during image assembly. The corrected figure is provided below. This error did not impact the scientific conclusions of the article. We apologize for any inconvenience to the readers. This has been corrected in both the PDF and HTML versions of the Article.
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