PSVI-6 Effects of progesterone and matrix metalloproteinase inhibition on milk component synthesis in bovine mammary epithelial cells.

Abstract Mammary gland development and milk production are regulated by multiple factors, including hormonal regulation, cell-cell, and cell-extracellular matrix (ECM) interactions. Progesterone (PG), a major ovarian hormone, promotes cell proliferation and alveolar structure formation during pregnancy. It also activates metalloproteinase (MMP)-2 and MMP-9 to facilitate ductal branching through ECM degradation. Prolactin (PRL), a key lactation hormone, is essential for inducing milk-secretory differentiation in mammary epithelial cells (MECs). Following parturition, PRL inhibits MMP-2 and MMP-9 (MMP-2/9) activity and enhances blood-milk barrier function in MECs to prevent the leakage of milk components. Milk synthesis has been known to exclusively occur in MECs differentiated by PRL. However, a recent study suggested that PG might also contribute to milk synthesis by enhancing the expression of E-74 like factor 5 (Elf5), a key regulator of lactogenic differentiation in MECs. This study aimed to investigate the effects of PG and MMP-2/9 inhibition on milk component synthesis in primary bovine mammary epithelial cells (BMECs). BMECs were cultured in control cell culture media (CM) consisting of DMEM/F12 supplemented with 10% fetal bovine serum and 1% penicillin/streptocmycin. For two-dimensional (2D) and three-dimensional (3D) cell cultures, BMECs were seeded onto standard and Matrigel-coated cell culture plates, respectively. After reaching approximately 80% confluency, 2D and 3D BMECs were differentiated for 5 days using PRL-differentiation media (PRLM) or PG-differentiation media (PGM). While PGM increased the protein expression of Elf5 in BMECs (P < 0.05), it failed to induce milk component synthesis and reduced claudin-3 expression (P < 0.05). Cell culture supernatant collected from PGM showed enhanced MMP-2/9 activity (P < 0.05), leading to the laminin degradation. However, supplementation of PGM with 400 ng/mL MMP-2/9 inhibitor significantly decreased MMP-2/9 activity (P < 0.05). Additionally, MMP-2/9 inhibition and PGM treatment upregulated milk component (α-casein, β-casein, β-lactoglobulin, α-lactalbumin, and milk fats) production in BMECs (P < 0.05), reaching levels similar to PRLM. Claudin-3 expression also increased in PGM/MMP-2/9 inhibitor-treated cells (PGM+MMPi) (P < 0.05). Collectively, there findings demonstrated that PGM supplemented with MMP-2/9 inhibitor induces milk component production in 3D BMECs, to levels similar to those observed in PRLM. The key distinction between PRL and PG lies on their differential regulation of MMP-2/9 activity during differentiation, indicating that MMP-2/9 inactivation is a pivotal process for facilitating milk production in 3D BMECs. This study provides novel insights into the hormonal regulation of lactation, contributing to a deeper understanding of milk production mechanisms.