Canonical prolactin signaling and global mRNA expression in the skin of Holstein heifers carrying the SLICK1 allele of the prolactin receptor gene.
The SLICK1 allele of the prolactin receptor gene (SLICK1) is associated with a short hair coat and thermotolerance in cattle. SLICK1 includes a single base pair deletion that creates a premature stop codon and prevents transcription of 120 amino acids involved in JAK/STAT signaling. It is unknown if SLICK1 modifies JAK/STAT signaling or the transcriptional response to prolactin. To investigate PRLR-associated signaling pathways in heterozygous SLICK1+/- Holsteins (slick), we performed immunohistochemistry on skin explants obtained from slick (n=5) and non-slick (n=6) heifers to evaluate phosphorylated (p)STAT1, pSTAT3, and pSTAT5 immunoreactivity (pSTAT1+, pSTAT3+, pSTAT5+) in hair follicles (HF) and sweat glands (SG). In slick skin, more HF had no pSTAT3 immunoreactivity compared to non-slick skin. No difference was found for the proportion of pSTAT1+ or pSTAT5+ HF, nor the proportion of pSTAT1+ and pSTAT3+ SG between genotypes. Within immunoreactive HF and SG, there were no differences between genotypes in the proportion of pSTAT1+, pSTAT3+, or pSTAT5+ cells in HF, or pSTAT1+ and pSTAT3+ cells in SG. Next, we investigated pSTAT3 immunoreactivity and the transcriptome of skin explants after exposure to prolactin in vitro. Skin explants from slick (n=6) and non-slick (n=6) heifers were cultured for 36 h in the presence of 50 ng/mL recombinant ovine prolactin, bisected, and each half underwent immunohistochemistry for pSTAT3 or RNA sequencing. No difference was found between genotypes in the proportion of pSTAT3+ HF or SG, nor the proportion of pSTAT3+ cells within HF or SG. RNA was poly-A enriched and sequenced using Novaseq6000 (Illumina) and 221,342,181 reads were mapped to the bovine genome (bosTau 9). The DESeq package was used to determine the differentially expressed genes (DEG) with P<0.01 and fold-change>1.5. There were 87 upregulated and 79 downregulated DEG in slick compared to non-slick skin. Ingenuity Pathway Analysis identified IL-17, leukocyte extravasation, and wound healing as upregulated signaling pathways, as well as activation of TNF, IL-1ß, OSM, IFN?, IL-17a, and IL-1R and inhibition of SHH and BMP4 upstream of the DEG. Analysis of genomic regions within ± 2 kb of all DEG's respective transcription start sites revealed enrichment of three binding sites for the OCT1 transcription factor. In conclusion, our results suggest differences in local immune regulation, hair growth inhibition, and tissue remodeling in slick skin. Overall design: Skin biopsies (6mm^2) were collected from half-sister heifers that were sired by one of two heterozygous slick bulls. Skin explants were cultured in the presence of ovine recombinant prolactin for 36 hours. Gene expression analysis of RNA-seq data from the cultured skin explants was used to determine differentially expressed genes between genotypes.