Molecular and Physiological Functions of PACAP in Sweat Secretion

Abstract

Sweat plays a critical role in human body, including thermoregulation and the maintenance of the skin environment and health. Hyperhidrosis and anhidrosis are caused by abnormalities in sweat secretion, resulting in severe skin conditions (pruritus and erythema). Bioactive peptide and pituitary adenylate cyclase-activating polypeptide (PACAP) was isolated and identified to activate adenylate cyclase in pituitary cells. Recently, it was reported that PACAP increases sweat secretion via PAC1R in mice and promotes the translocation of AQP5 to the cell membrane through increasing intracellular [Ca²⁺] via PAC1R in NCL-SG3 cells. However, intracellular signaling mechanisms by PACAP are poorly clarified. Here, we used PAC1R knockout (KO) mice and wild-type (WT) mice to observe changes in AQP5 localization and gene expression in sweat glands by PACAP treatment. Immunohistochemistry revealed that PACAP promoted the translocation of AQP5 to the lumen side in the eccrine gland via PAC1R. Furthermore, PACAP up-regulated the expression of genes (Ptgs2, Kcnn2, Cacna1s) involved in sweat secretion in WT mice. Moreover, PACAP treatment was found to down-regulate the Chrna1 gene expression in PAC1R KO mice. These genes were found to be involved in multiple pathways related to sweating. Our data provide a solid basis for future research initiatives in order to develop new therapies to treat sweating disorders.

Keywords: DNA microarray; PACAP; aquaporin5 (AQP5); eccrine gland; sweat gland.

Figure 1

Analysis of PAC1R distribution in mouse eccrine gland. The WT mouse and PAC1R KO mouse skins were immunostained with PAC1R (red; A and C) and counter stained with DAPI (nuclei, B and D). Scale bar = 50 µm.

Figure 2

A 3D analysis of the AQP5 vesicles localization in mouse eccrine cells. Mouse skins were immunostained with AQP5 (green) and counter stained with DAPI (nuclei). (a,b): The white arrows highlight AQP5-immunoreactive vesicles in the cytoplasm of mouse skin. Scale bar = 10 µm.

Figure 3

Changes in the intracellular localization of AQP5 in WT mice and KO mice after PACAP addition. Mouse skins were immunostained with AQP5 (green) and counter stained with DAPI (nuclei). Representative confocal micrographs of mouse eccrine gland transfected with (A) 30 min after PACAP treatment, (B) 30 min after vehicle treatment, (C) 60 min after PACAP treatment, (D) and 60 min after vehicle treatment in WT mice. Representative confocal micrographs of mouse eccrine gland transfected with (E) 30 min after PACAP treatment, (F) 30 min after vehicle treatment, (G) 60 min after PACAP treatment, (H) and 60 min after vehicle treatment in PAC1R KO mice. Scale bar = 10 µm.

Figure 4

Differentially expressed genes in the WT mice and KO mice after the addition of PACAP at 30 and 60 min by DNA microarray analysis. Two DNA microarray chips were used for each sample condition, i.e., a dye-swap approach was used to label one set of RNAs by Cy3 and the other by Cy5 for each control and PACAP sample, respectively, as detailed in the Materials and Methods section. A 4 × 44-K format slide containing 4 chips is illustrated for the current study. The histograms show the up (red)- and down (blue)-regulated gene (≧/≦ 2.0/0.5-fold) numbers in each examined tissue. The gene expression data are publicly available under the GEO series accession number GSE223124 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE223124; accessed on 19 February 2023).

Figure 5

Top 20 genes expressions that were altered by PACAP in WT mice and PAC1R KO mice. Genes involved in contractile protein-related genes (gray), antimicrobial activity (yellow), blood coagulation-related genes (orange), and oxidative phosphorylation- and ATP production-related genes (green). The gene expression data are publicly available under the GEO series accession number GSE223124 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE223124; accessed on 19 February 2023).

Figure 6

Summary of the genes that may be involved in PACAP-induced membrane translocation of AQP5. Red, up-regulated genes; and blue, down-regulated genes. The gene expression data are publicly available under the GEO series accession number GSE223124 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE223124; accessed on 19 February 2023).