S100A9 Affects Milk Protein Content by Regulating Amino Acid Transporters and the PI3K-Akt, WNT, and mTOR Signaling Pathways.

Background: Calgranulin B (S100A9) was found to be strongly associated with milk protein percentage in dairy cattle in our previous genome-wide association study.

Methods: SNPs in S100A9 were identified via pooled sequencing, and genotyping of 1054 cows was performed individually using MassArray with MALDI-TOFMS technology. Association analyses between the S100A9 SNPs and five milk production traits were conducted using SAS 9.2 software. Functional studies of S100A9 were conducted using quantitative PCR, Western blot, CCK-8, and immunofluorescence assays.

Results: In the present study, we further verified that two SNPs in S100A9, g.17115387 C>A and g.17115176 C>A, were significantly associated with milk protein percentage. We found that S100A9 could affect the expressions of caseins CSN1S1, CSN2, and CSN3 in MAC-T cells by regulating the expressions of amino acid transporter genes. We investigated the effects of S100A9 on the PI3K-Akt, WNT, and mTOR pathways, which are well known to play important roles in mammary gland development and milk protein synthesis. Our results suggest that S100A9 regulates the expressions of the relevant genes in these pathways, and thus potentially influences the protein synthesis in the mammary gland.

Conclusions: This study demonstrates the important role of the S100A9 gene in the milk protein trait of dairy cattle and provides new insights into the molecular mechanism of milk protein content.