Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare dermal transcriptomes of control group (wild-type IMQ-untreated C57/BL6 mice), model group (wild-type IMQ-treated C57/BL6 mice) and XYF group (model group mice treated by XYF). Methods: Dermal mRNA profiles of control group, model group and XYF group mice were generated by deep sequencing. Total RNA samples were prepared from intact dermal ear sheets of mouse ear skin from two mice. Double-strand cDNA was generated from equal amounts of total RNA by following TruSeq8 RNA Library Prep Kit v2 (#RS-122-2001/2002, Illumina). The cDNA libraries were sequenced using Illumina Hi-seq2500. STAR software was utilized for sequence alignment between the preprocessing sequence and reference genome sequence of mice downloaded from the Ensembl database(Mus_musculus.GRCm38.90,ftp://ftp.ensembl.org/pub/release-90/gtf/mus_musculus/ Mus_musculus.GRCm38.90.chr.gtf.gz). Transcript assembly of mRNA sequencing data was performed by StringTie software. The expression levels for each of the genes were normalized to FPKM (fragments per kilobase of transcript per million fragments mapped). DESeq 2 was applied to conduct the analysis of differentially expressed genes (DEG). The cutoffs of DEG method were determined as the P value < = 0.05, the FDR value < = 0.05 and the Fold Change (FC) value > = 2. Results: Heatmap analysis of differentially expressed genes (DEG) revealed distinct transcriptomes between different groups, which displayed a partial reversal of pathological gene-expressing modifications by the treatment of XYF. Detailed comparison identified shared DEGs of opposite deviations between control group versus model group and model group versus XYF group. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis uncovered the dermis from disease-model group was greatly disturbed in cytokine-cytokine receptor interaction, chemokine signaling pathways, IL-17 signaling pathway, TNF signaling pathway, Wnt signaling pathway and PI3K-Akt signaling pathway. As for XYF therapy, several metabolic pathways, Calcium signaling, cAMP signaling pathway and cGMP-PKG signaling pathway were affected within the dermis. Further Gene Set Enrichment Analysis (GSEA) exposed multiple metabolic pathways, which involved ether lipid metabolism, alpha linolenic acid metabolism, arachidonic acid metabolism, linoleic acid metabolism, glycerophospholipid metabolism, glycosaminoglycan biosynthesis heparan sulfate, along with VEGF signaling and cell cycle were considerably suppressed in the dermal tissue when given XYF. Conclusions: XYF might regulate various inflammatory signaling, metabolic processes and microvascular formation to influence γδT cell biology.

Project description:Calcium and 1,25-dihydroxyvitamin D3 (1,25D3), through the actions of their respective receptors, the Ca2+-sensing receptor (CaSR) and the vitamin D receptor (VDR), potentiate keratinocyte differentiation. VDR regulates epidermal keratinocyte proliferation and differentiation by modulating gene transcription, whereas the CaSR, a member of the family C G-protein coupled receptor, calcium mobilizes intracellular calcium and induces the formation of cell-cell junctions. 1,25D3 augments the sensitivity of the prodifferentiating actions of calcium by increasing the expression of CaSR. CaSR- and VDR-deficient keratinocytes share common characteristics such as abnormal intercellular adhesion and sphigolipid metabolism. Both CaSR and VDR are abundantly expressed in epidermal stem cell populations including CD34 expressing bulge keratinocytes in hair follicles and basal cells in interfollicular epidermis. To delineate the role of CaSR- and VDR-dependent pathways in regulating epidermal development and functions in physiological state, we generated conditional CaSR-null and VDR-null mice, where Casr and VDR gene was removed from keratinocytes. Keratinocyte-specific CaSR-null and VDR-null mice manifest distinct phenotypes. CaSR-null mice display defective epidermal permeability barrier function due to aberrant keratinocyte differentiation. VDR-null mice develop alopecia after completing the first hair follicle cycling. Concurrent ablation of CaSR and VDR genes in keratinocytes delays rate of wound repair and increases incidence of skin tumor formation to a greater extent than deletion of the CaSR or VDR alone, indicative of synergistic effects of calcium and 1,25D3 signaling. Gene expression profiles and subsequent pathway analysis on the epidermis derived from 5-day-old neonates revealed that ablation of CaSR or VDR increased expression of genes associated with cancer progression and metastasis. Deletion of VDR markedly inhibited signaling pathways that regulate hair development. Furthermore, concurrent ablation of CaSR and VDR significantly suppressed calcium, VDR, Wnt/b-catenin signaling, as well as epithelial adherence junction signaling to maintain appropriate keratinocyte adhesion. These results indicated the interplay of calcium/CaSR and 1,25D3/VDR signaling in keratinocyte proliferation and differentiation, and their importance in maintaining normal epidermal adhesion and functions. n=3 CON and KO (each sample contain RNA isolated from neonatal epidermis separated from 3 mice)

Project description:Calcium and 1,25-dihydroxyvitamin D3 (1,25D3), through the actions of their respective receptors, the Ca2+-sensing receptor (CaSR) and the vitamin D receptor (VDR), potentiate keratinocyte differentiation. VDR regulates epidermal keratinocyte proliferation and differentiation by modulating gene transcription, whereas the CaSR, a member of the family C G-protein coupled receptor, calcium mobilizes intracellular calcium and induces the formation of cell-cell junctions. 1,25D3 augments the sensitivity of the prodifferentiating actions of calcium by increasing the expression of CaSR. CaSR- and VDR-deficient keratinocytes share common characteristics such as abnormal intercellular adhesion and sphigolipid metabolism. Both CaSR and VDR are abundantly expressed in epidermal stem cell populations including CD34 expressing bulge keratinocytes in hair follicles and basal cells in interfollicular epidermis. To delineate the role of CaSR- and VDR-dependent pathways in regulating epidermal development and functions in physiological state, we generated conditional CaSR-null and VDR-null mice, where Casr and VDR gene was removed from keratinocytes. Keratinocyte-specific CaSR-null and VDR-null mice manifest distinct phenotypes. CaSR-null mice display defective epidermal permeability barrier function due to aberrant keratinocyte differentiation. VDR-null mice develop alopecia after completing the first hair follicle cycling. Concurrent ablation of CaSR and VDR genes in keratinocytes delays rate of wound repair and increases incidence of skin tumor formation to a greater extent than deletion of the CaSR or VDR alone, indicative of synergistic effects of calcium and 1,25D3 signaling. Gene expression profiles and subsequent pathway analysis on the epidermis derived from 5-day-old neonates revealed that ablation of CaSR or VDR increased expression of genes associated with cancer progression and metastasis. Deletion of VDR markedly inhibited signaling pathways that regulate hair development. Furthermore, concurrent ablation of CaSR and VDR significantly suppressed calcium, VDR, Wnt/b-catenin signaling, as well as epithelial adherence junction signaling to maintain appropriate keratinocyte adhesion. These results indicated the interplay of calcium/CaSR and 1,25D3/VDR signaling in keratinocyte proliferation and differentiation, and their importance in maintaining normal epidermal adhesion and functions. n=3 CON and DKO (each sample contain RNA isolated from neonatal epidermis separated from 3 mice)

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Next Generation Sequencing Facilitates Quantitative Analysis of Epidermal Transcriptomes of Imiquimod (IMQ)-Induced Psoriasis-Like Dermatitis Treated by Xiao-Yin-Fang (XYF) Therapy

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