The antioxidant stress effect of granulin precursor in vitiligo

Abstract

The imbalance of the skin redox system is regarded as a crucial factor contributing to the loss of melanocytes in vitiligo. However, it remains unclear whether alterations in signal transmission between melanocytes and other cells impact the homeostasis of the skin microenvironment. Hence, leveraging single-cell sequencing and microarray data, we investigated the role of cell-cell and ligand receptor interactions in the pathogenesis of vitiligo. We discovered that the granulin-sortilin 1 ligand-receptor serves as an essential bridge for communication between melanocytes and other skin cells in normal skin, yet it is significantly downregulated in vitiligo lesions. Enrichment analysis indicates that the activation of granulin-sortilin 1 ligand-receptor is closely associated with the regulation of oxidative stress. In vitro experiments have verified that progranulin, the protein encoded by the granulin gene, enhances the ability of melanocytes to resist cell death induced by reactive oxygen species and markedly upregulates the expression of nuclear factor erythroid 2-related factor 2 and Heme Oxygenase-1. Notably, this process can be impeded by the interaction inhibitor. Moreover, the expression of nuclear factor erythroid 2-related factor 2 might be linked to the transcription of transcription factor EB activated by progranulin. In conclusion, the granulin-sortilin 1 ligand-receptor can activate the intracellular antioxidant system to counteract melanocyte death. The impairment of the granulin-sortilin 1 ligand-receptor may be implicated in melanocyte loss in vitiligo.

Keywords: GRN; Melanocyte; NRF2; Oxidative stress; Vitiligo.

Fig. 1

Melanocytes receive signals from various cells in normal skin. (A) This study’s analysis flowchart is presented. (B) The bubble map depicts the top five marker genes within different cell clusters in normal skin. (C) Uniform Manifold Approximation and Projection (UMAP) analysis shows the distribution, quantity of cells, and cell types across all cell clusters. (D) The network diagram illustrates the cellular communication and the interaction intensity among 10 types of cells. (E) Melanocytes receive signals from keratinocytes, basal keratinocytes, fibroblasts, antigen presenting cells, hair follicle cells, and stromal cells. (F) The granulin (GRN) signaling pathway represents a functional signal transmitted by various cells to melanocytes.

Fig. 2

The signals received by melanocytes were disrupted in vitiligo. (A) Uniform Manifold Approximation and Projection (UMAP) analysis shows the distribution, number of cells, and cell types within all cell clusters in both lesional and non—lesional areas of vitiligo. (B) This demonstrates the cellular communication received by melanocytes in both lesional and non—lesional skin. (C) The signals received by melanocytes experience substantial alterations in terms of both quantity and significance (weight). (D) The Macrophage Migration Inhibitory Factor—Cluster of Differentiation 74/Cluster of Differentiation 44 (MIF—CD74/CD44) ligand—receptor is significantly upregulated in the cellular communication received by melanocytes in skin lesions (p < 0.001), whereas the granulin—sortilin 1 (GRN—SORT1) ligand—receptor is significantly downregulated (p < 0.05). (E) The GRN gene significantly downregulated in various cells of vitiligo lesions.

Fig. 3

GRN—SORT1 might be linked to the oxidative stress imbalance in vitiligo lesions. (A-B) Gene Ontology analyses were conducted between the high and low—scoring groups based on the granulin—sortilin 1 (GRN—SORT1) ligand—receptor from vitiligo lesions. Signaling pathways associated with reactive oxygen species, such as the respiratory electron transport chain, electron transfer activity, cellular detoxification, cellular oxidant detoxification, and antioxidant activity, were significantly enriched (p < 0.001). In addition to the enrichment of oxidative stress—related pathways, lysosomal membranes (p < 0.001) and proton transporting V—type ATPase complexes (p < 0.001) were also significantly enriched.

Fig. 4

Progranulin may inhibit H₂O₂—induced cell death in melanocytes via the NRF2/HO-1 signaling pathway. (A) Treat the PIG1 and MNT1 cells with different concentrations of hydrogen peroxide (H₂O₂) respectively. H₂O₂ at a concentration higher than 0.08 mM can induce the death of PIG1 cells, while a concentration higher than 0.5 mM can induce the death of MNT1 cells. (B) PIG1 cells were treated with 0.1 mM H₂O₂, while MNT1 cells were treated with 0.7 mM H₂O₂ to induce the oxidative stress model. The CCK8 assay confirms that progranulin (PGRN) can reverse H₂O₂—induced cell death, while the inhibitor can suppress this reversal process. (C) Under H₂O₂ stimulation, PGRN upregulates the expression of nuclear factor erythroid 2—related factor 2 (NRF2) and Heme Oxygenase—1 (HO—1), and this upregulation can be counteracted by the inhibitor. (D) A reactive oxygen species (ROS) probe was used to detect the production of ROS in PIG1 and MNT1 cells. H₂O₂ significantly elevated the intracellular ROS level. However, the addition of PGRN effectively reduced ROS production. This beneficial effect of PGRN was, nonetheless, hindered by the inhibitor (The scale bar in the figure is 100 µm). A p—value of less than 0.05 was considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001).