The M3 Muscarinic Acetylcholine Receptor Promotes Epidermal Differentiation

Abstract

The M3 muscarinic acetylcholine receptor is predominantly expressed in the basal epidermal layer where it mediates the effects of the autocrine/paracrine cytotransmitter acetylcholine. Patients with the autoimmune blistering disease pemphigus develop autoantibodies to M3 muscarinic acetylcholine receptor and show alterations in keratinocyte adhesion, proliferation, and differentiation, suggesting that M3 muscarinic acetylcholine receptor controls these cellular functions. Chmr3^-/- mice display altered epidermal morphology resembling that seen in patients with pemphigus vulgaris. In this study, we characterized the cellular and molecular mechanisms through which M3 muscarinic acetylcholine receptor controls epidermal structure and function. We used single-cell RNA sequencing to evaluate keratinocyte heterogeneity and identify differentially expressed genes in specific subpopulations of epidermal cells in Chmr3^-/- neonatal mice. We found that Chmr3^-/- mice feature abnormal epidermal morphology characterized by accumulation of nucleated basal cells, shrinkage of basal keratinocytes, and enlargement of intercellular spaces. These morphologic changes were associated with upregulation of cell proliferation genes and downregulation of genes contributing to epidermal differentiation, extracellular matrix formation, intercellular adhesion, and cell arrangement. These findings provide, to our knowledge, previously unreported insights into how acetylcholine controls epidermal differentiation and lay a groundwork for future translational studies evaluating the therapeutic potential of cholinergic drugs in dermatology.

Figure 1.. Chrm3^−/− thickens mouse epidermis.

(a) The differences between epidermal morphology between WT versus Chrm3^(−/−) mice were manifested by an increased thickness of the basal layer comprised of crowded and slightly shrunk keratinocytes in KO mice. Bar = 10μm. (b) Quantitative analysis of the differences in epidermal morphology between WT and Chrm3^(−/−) mice showed an increase in both the thickness of epidermis and the number of nucleated basal cells within IFE in KO mice. P - value was calculated from two-tailed t-test. (c) UMAP showing the clusters of seven major epidermal cell types, including IFE, HF, melanocytes, Langerhans cells, Merkel cells, sebaceous gland, and T cells. The marker gene list is shown in Supplementary Table S1. (d) All the seven epidermal cell types are present in both WT and Chrm3^(−/−) epidermis. (e) WT and Chrm3^(−/−) Cellular composition of epidermis. The black box highlights the basal populations, which are increased in the Chrm3^(−/−) epidermis. HF, hair follicle; IFE, interfollicular epidermis; KO, knockout; UMAP, Uniform Manifold Approximation and Projection; WT, wild-type.

Figure 2.. Chrm3^−/− expands the population of basal cells and cycling cells.

(a) Upper: UMAP showing the eight clusters of the IFE, including four basal populations, two transition populations, one differentiated population, and a terminally differentiated population. Lower: Bar graph presenting the cellular composition of the WT and Chrm3^−/− IFE. (b) Expression of marker genes projected onto the UMAP to identify the basal cells (K14, K5, Col17a1, Itga6), differentiated cells (K10, K1, Klf4, Tgm3), and terminally differentiated cells (Lor). Red denotes high expression, and blue denotes low expression. (c) Heatmap showing the top 10 marker genes for each of the clusters defined in a. Blue boxes highlight some of the canonical marker genes. The complete marker gene list is shown in Supplementary Table S2. (d) Upper: cell cycle assignment projected onto the UMAP. Lower: bar graph showing the percentage of each cell cycle stage in the WT and Chrm3^−/− IFE. (e) A representative image of the epidermis WT and Chrm3^−/− mice staining with anti–Ki-67 antibody (bar = 20 μ m) as well as direct count of Ki-67–postive cells showing more positive cells in the basal layer of KO mice. P-value was calculated from two-tailed t-test. IFE, interfollicular epidermis; K, keratin; KO, knockout; Lor, loricrin; UMAP, Uniform Manifold Approximation and Projection; WT, wild-type.

Figure 3.. Differentiation is downregulated in the Chrm3^−/− IFE.

(a) Heatmap showing 120 of the differentially expressed genes between the WT and Chrm3^−/− IFE. The blue boxes highlight the genes related to keratinocyte differentiation. The complete gene list is shown in Supplementary Table S3. (b) GO analysis reveals that keratinocyte differentiation is downregulated in the Chrm3^−/− IFE. (c) GO analysis reveals that terms related to cell proliferation are upregulated in the Chrm3^−/− IFE. (d) Distribution patterns of K5- and K10-positive keratinocytes in the epidermis of WT and Chrm3^−/− mice visualized by confocal microscopy of skin samples stained with anti-K5 (red) and anti-K10 (green) antibodies (bar = 20 μ m) and quantitative analysis of the intensity of fluorescence produced by each antibody within IFE showing an increase in K5 and decrease in K10 expression in KO mice. p - value was calculated from two-tailed t-test. (e) Expression patterns of the six groups of genes defined in Lin et al. (2020) shown on the left and enrichment score and corresponding false discovery rate q-value obtained from GSEA shown on the right. GO, Gene Ontology; GSEA, Gene Set Enrichment Analysis; IFE, interfollicular epidermis; K, keratin; KO, knockout; WT, wild-type.

Figure 4.. Epidermal differentiation is downregulated in suprabasal cells and adhesion molecules are downregulated in Chrm3^−/− basal cells.

(a) Scoring of the cells in each differentiation stage on the basis of their expression level of six groups of genes defined in Lin et al. (2020) shows that the disturbance in the differentiation profile is most evident in the differentiated cells. P-value was calculated from t-test. ns: p > 0.05, *p ≤ 0.05, **p ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001. (b) Gene Ontology analysis on genes downregulated in Chrm3^−/− basal cells show enrichment for skin development and keratinocyte differentiation. Genes contributing to the enrichment of some of the terms are listed. The complete list of differentially expressed genes in Chrm3^−/− versus in WT basal cells is provided in Supplementary Table S5. (c) Adhesion molecules are downregulated in the Chrm3^−/− basal cells. P-value was calculated from t-test. ns: p > 0.05, *p ≤ 0.05, ** p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. ns, not significant; WT, wild-type.