76 Transcriptional profiles and signaling pathways associated with the SLICK1 allele of the prolactin receptor gene in Holstein cattle

Prolactin is a hormone recognised for its roles in lactation, reproduction, hair growth, and immune processes. Mutations in the prolactin receptor gene (PRLR), termed ‘SLICK’ alleles, are associated with a short hair phenotype and improved thermotolerance in cattle. The SLICK1 allele, a single base pair deletion, introduces a premature stop codon and prevents transcription of 120 amino acids of the intracellular tail of PRLR, including several tyrosine residues. In response to prolactin binding to PRLR, STAT molecules bind to these tyrosines where they are activated via phosphorylation by JAK2, resulting in changes in transcription of target genes. It is unknown whether the presence of the SLICK1 allele and the shortening of PRLR modifies JAK/STAT signaling. To investigate PRLR-associated signaling pathways in Holsteins harboring SLICK1 (slick), we obtained skin biopsies from slick (n = 6) and nonslick (n = 6) half-sister heifers and cultured them for 36 h in the presence of 50 ng/mL recombinant ovine prolactin. After culture, explants were bisected and each half was subjected to either immunohistochemistry (n = 6 slick, n = 4 nonslick) using a rabbit mAb targeting pSTAT3 or RNA sequencing (n = 6 slick, n = 6 nonslick). The proportion of hair follicles immunoreactive for pSTAT3 (pSTAT3+) and the proportion of immunoreactive cells within positive follicles between genotypes were analysed by one-way ANOVA using SAS version 9.4. The RNA was extracted and poly-A enriched before sequencing using a NovaSEqn 6000 (Illumina) with 150 bp paired ends. In total, 221 342 181 reads were mapped to the bovine reference genome (bosTau 9). The DESeq package for R was used to determine the differentially expressed genes (DEG) based on P < 0.01 and fold change >1.5. Analysis of gene ontology and biological functions was performed with the Ingenuity Pathway Analysis software. No difference was found between genotypes in the proportion of hair follicles with pSTAT3+ (P = 0.6), nor in the proportion of pSTAT3+ cells within pSTAT3+ follicles (P = 0.9). Exposure to prolactin resulted in 87 up-regulated and 79 down-regulated DEG in slick compared to nonslick skin. Functional analysis identified IL-17, leukocyte extravasation, and wound healing as up-regulated canonical signaling pathways in slick skin. Upstream analysis indicated activation of the immune regulators TNF, IL-1β, OSM, IFNγ, IL-17α, and IL-1R and down-regulation of the hair growth regulators SHH and BMP4 in slick skin. Validation of DEG by reverse-transcription quatntitative polymerase chain reaction confirmed CSF3 as up-regulated 3-fold and MMP12 as down-regulated 3.5-fold in slick skin (P = 0.03). In conclusion, activation of immune-related pathways and down-regulation of hair growth regulators could indicate differences in local immune regulation and inhibition of hair growth and tissue remodeling in the skin of Holsteins carrying the SLICK1 allele. These changes are unlikely to be downstream of STAT3 signaling as we did not observe a difference in STAT3 activation in response to prolactin in the hair follicles of these animals.

Funding was provided by the Holstein Association USA Research Grant Program.