UC Davis

Prolactin is a hormone best recognized for its roles in lactation and reproduction, although it has important immunomodulatory roles, particularly in the context of stress. Several mutations in the prolactin receptor gene have been identified in cattle that are associated with a short hair phenotype and improved thermotolerance in hot environments. Among these mutations, the SLICK1 allele is a single base pair deletion resulting in a frameshift mutation that introduces a premature stop codon, preventing transcription of 120 amino acids of the prolactin receptor intracellular tail. The shortened prolactin receptor tail lacks tyrosine residues that are docking sites for signal transducers and activators of transcription (STAT) molecules. The canonical pathway utilized by the prolactin receptor is JAK2/STAT5, although other STAT proteins such as STAT1 and STAT3 are also involved. It is unknown whether the presence of the SLICK1 allele could modify the functioning of JAK/STAT signaling pathways. To investigate prolactin receptor-associated signaling pathways in cattle carrying the SLICK1 allele, we performed immunohistochemistry in skin biopsies obtained from slick and non-slick heifers to evaluate the presence and abundance of phosphorylated (activated) STAT1, STAT3, and STAT5 in hair follicles and sweat glands. The presence of pSTAT3 was detected less often (P = 0.03) in hair follicles of slick (42.5% ± 10.7%; n=5) compared to non-slick heifers (79.5% ± 9.7%; n=6). No difference between genotypes was found for the presence of immunoreactivity for pSTAT1 (slick = 37.9% ± 11.3%; non-slick = 43.9% ± 10.3%; P = 0.70) or pSTAT5 (slick = 8.9% ± 7.1%; non-slick = 15% ± 6.5%; P = 0.52) in hair follicles. Additionally, presence of immunoreactivity for pSTAT1 (slick = 47.5% ± 21.8%; non-slick = 91.7% ± 25.2%; P = 0.24) and pSTAT3 (slick = 33.3% ± 24.3%; non-slick = 91.7% ± 24.3%; P = 0.16) in sweat glands did not differ between genotypes. Immunoreactivity for pSTAT5 was only detected in one slick sweat gland and zero non-slick sweat glands and therefore was not stat analyzed. When immunoreactive structures were detected, no difference was found regarding the proportion of cells positive for pSTAT1 (slick = 2.9% ± 2%; non-slick = 3.5% ± 1.9%; P = 0.84), pSTAT3 (slick = 6.4% ± 3.1%; non-slick = 8.7% ± 2.8%; P = 0.60), or pSTAT5 (slick = 0.5% ± 0.8%; non-slick = 1.5% ± 0.7%; P = 0.37) in hair follicles, or for pSTAT1 (slick = 11% ± 5.3%; non-slick = 12% ± 6.2%; P = 0.91) and pSTAT3 (slick = 18% ± 12.8%; non-slick = 16.7% ± 12.9%; P = 0.94) in sweat glands. Since prolactin signaling through its canonical receptor affects gene transcription, we investigated the global transcriptional response of skin explants from heifers carrying the SLICK1 allele and non-slick half-sisters after exposure to prolactin in vitro. Skin explants were subjected to RNA sequencing and the resulting data were analyzed using Ingenuity Pathway Analysis software. Bioinformatic analysis revealed that among the canonical signaling pathways identified in the expanded dataset were IL-17 signaling (P = 3.24e-3, z-score = 2.24), leukocyte extravagation signaling (P = 5.75e-4, z-score = 2.00), and wound healing signaling pathway (P = 2.29 e-3, z-score = 0.82). Upstream analysis identified differentially activated upstream regulators including TNF (P = 1.23 e-5, z-score = 3.38), IL-1β (P = 8.71e-3, z-score = 3.11), OSM (P = 1.49e-2, z-score = 2.73), IFNγ (P = 6.27e-3, z-score = 2.60), IL-17α (P = 6.69e-3, z-score = 2.40), IL-1R (P = 1.90e-5, z-score = 2.20), SHH (P = 5.24e-3, z-score = -2.18), and BMP4 (P = 4.12e-4, z-score = -2.18). Our results indicate that STAT3 is phosphorylated less often in the hair follicles of the skin of heifers carrying the SLICK1 allele. Additionally, the differential activation of immune-related genes and pathways could indicate differences in local immune regulation in the skin of SLICK1-carrier heifers.