Transcriptomic Analysis Reveals Candidate Ligand-Receptor Pairs and Signaling Networks Mediating Intercellular Communication between Hair Matrix Cells and Dermal Papilla Cells from Cashmere Goats

Abstract

Hair fiber growth is determined by the spatiotemporally controlled proliferation, differentiation, and apoptosis of hair matrix cells (HMCs) inside the hair follicle (HF); however, dermal papilla cells (DPCs), the cell population surrounded by HMCs, manipulate the above processes via intercellular crosstalk with HMCs. Therefore, exploring how the mutual commutations between the cells are molecularly achieved is vital to understanding the mechanisms underlying hair growth. Here, based on our previous successes in cultivating HMCs and DPCs from cashmere goats, we combined a series of techniques, including in vitro cell coculture, transcriptome sequencing, and bioinformatic analysis, to uncover ligand-receptor pairs and signaling networks mediating intercellular crosstalk. Firstly, we found that direct cellular interaction significantly alters cell cycle distribution patterns and changes the gene expression profiles of both cells at the global level. Next, we constructed the networks of ligand-receptor pairs mediating intercellular autocrine or paracrine crosstalk between the cells. A few pairs, such as LEP-LEPR, IL6-EGFR, RSPO1-LRP6, and ADM-CALCRL, are found to have known or potential roles in hair growth by acting as bridges linking cells. Further, we inferred the signaling axis connecting the cells from transcriptomic data with the advantage of CCCExplorer. Certain pathways, including INHBA-ACVR2A/ACVR2B-ACVR1/ACVR1B-SMAD3, were predicted as the axis mediating the promotive effect of INHBA on hair growth via paracrine crosstalk between DPCs and HMCs. Finally, we verified that LEP-LEPR and IL1A-IL1R1 are pivotal ligand-receptor pairs involved in autocrine and paracrine communication of DPCs and HMCs to DPCs, respectively. Our study provides a comprehensive landscape of intercellular crosstalk between key cell types inside HF at the molecular level, which is helpful for an in-depth understanding of the mechanisms related to hair growth.

Keywords: cashmere goat; dermal papilla cells; hair follicle; hair matrix cells; intercellular crosstalk; ligand-receptor pair; signaling axis.

Figure 1

Coculture alters the cell cycle distribution pattern of goat DPCs and HMCs. (a,b) Representative figures of flow cytometry analysis and the percentages of G1, S, and G2/M phases in the cell cycle of monocultured and cocultured DPCs. (c,d) Representative figures of flow cytometry analysis and the percentages of three phases in the cell cycle of monocultured and cocultured HMCs. Results shown are the mean ± sd of three replicates in each group. DPCs, dermal papilla cells; HMCs, hair matrix cells; DPCsCO, dermal papilla cells cocultured; HMCsCO, hair matrix cells cocultured. Statistical significance between two groups was determined by a two-tailed student’s t-test: * p < 0.05, ** p < 0.01.

Figure 2

Comprehensive analysis of the transcriptomic profiles of monocultured and cocultured goat cells at a global level. (a) Violin plot showing the distribution of transcript abundances measured by FPKM value in four groups; (b–d) Principal component analysis (PCA), sample clustering analysis, and Pearson’s correlation analysis of four groups using transcriptome data; (e) Boxplots showing the counts of differentially expressed genes (DEGs) in three groups; (f,g) Volcano plot displaying the status of gene expression in DPCsCO versus DPCs and HMCsCO versus HMCs, respectively; (h,i) Veen graphs showing the identification of 516 and 181 signature genes of DPCs and HMCs, respectively. Data was analyzed from three biological replicates in each group.

Figure 3

Expression patterns of genes involved in key biological processes in hair growth. (a) Heatmap showing the expression patterns of keratins (KRTs) related to hair composition; (b) Heatmap of the expression pattern of genes regulating cellular apoptosis in cells; (c) Heatmap displaying the relative abundances of genes encoding DNA methylases and demethylases; (d) Heatmap characterizing the relative expressions of genes encoding enzymes, receptors, and inhibitors related to prostaglandin synthesis and signaling transduction.

Figure 4

Identification of core ligands, receptors, and transcriptional factors affected by coculture of goat DPCs and HMCs. (a) Boxplots showing the counts of ligands, receptors, and transcription factors (TFs) identified from each group. (b–d) Veen graph and heatmap displaying the key ligands, receptors, and TFs in goat DPCs found by overlapping the differentially expressed genes from DPCsCO versus DPCs and HMCsCO versus DPCsCO; (e) Interaction network of TFs detected in (d); (f–h) Veen graph and heatmap showing the key ligands, receptors, and TFs in goat HMCs discovered by overlapping the differentially expressed genes from HMCsCO versus HMCs and HMCsCO versus DPCsCO.

Figure 5

Ligand-receptor pairs mediate the autocrine signaling loop in goat DPCs and HMCs. (a,b) Circos plots exhibiting the ligand-receptor pairs mediating the autocrine signaling in goat DPCs constructed by differentially expressed ligands and receptors from DPCsCO versus DPCs group and HMCsCO versus DPCsCO, respectively; (c,d) Circos plots displaying the ligand-receptor pairs mediating the autocrine signaling in goat HMCs built by the differentially expressed ligands and receptors from HMCsCO versus HMCs group and HMCsCO versus DPCsCO, respectively. The width of the edge corresponds to the value of CS.

Figure 6

Ligand-receptor pairs mediate the paracrine signaling-linked intercellular crosstalk between goat DPCs and HMCs. (a) Ligand-receptor pairs mediating the intercellular communication identified from upregulated ligands in DPCsCO in DPCsCO versus DPCs group and upregulated receptors from HMCsCO versus HMCs group; (b) Ligand-receptor pairs mediating signal from DPCs to HMCs identified by upregulated ligands in DPCsCO in HMCsCO versus DPCsCO group, and upregulated receptors in HMCsCO in HMCsCO versus DPCsCO group; (c) Ligand-receptor pair mediating signaling from HMCs to DPCs identified by similar strategy in (a); (d) Ligand-receptor pairs linking signaling from HMCs to DPCs identified by similar strategy in (b).

Figure 7

Ligand-receptor-signaling protein-TFs axis mediating the paracrine intercellular communication from goat DPCs to HMCs using differentially expressed genes from DPCsCO versus DPCs and HMCsCO versus HMCs as CCCExplorer input.

Figure 8

Ligand-receptor-signaling protein-TFs axis mediating the paracrine intercellular communication from goat DPCs to HMCs using differentially expressed genes between DPCsCO versus HMCsCO as CCCExplorer input.

Figure 9

Treatment of goat DPCs using recombinant IL1A and leptin alters cellular status. (a) Relative abundances of IL1A, IL1R1, and IL1RN in four cell types; (b) Relative mRNA expression of IL1A, IL1R1, FGF7, and LEPR in goat DPCs treated with 0, 1, and 10 ng/mL recombinant IL1A protein (n = 3); (c) Cell viability of goat DPCs treated with 0 or 10 ng/mL recombinant IL1A protein (n = 6); (d) Relative abundances of LEP and LEPR in four cell types; (e) Cell viability of goat DPCs treated with recombinant leptin protein (0 or 100 ng/mL), JAK inhibitor (WP1066, 2.5 μM), or the combination of recombinant leptin protein (100 ng/mL) and JAK inhibitor (2.5 μM), n = 6; (f) Cellular apoptosis of goat DPCs in four groups indicated by Hoechst 33,342 staining, with the arrow head displaying the condensed cell nucleus of cells that undergo apoptosis (n = 3); (g) The ratio of apoptotic cells in four groups measured by the percentage of condensed cell nuclei. **, p < 0.01.