Matrix metalloproteinases (MMPs) are a multigene family of zinc-dependent endopeptidases enzymes that degrade the extracellular matrix (ECM) [1, 2]. Until now, 26 different MMPs have been confirmed in humans. Based on substrate specificity and domain homologies, MMPs were divided into six groups: collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs, and other non-classified MMPs . Typical MMP consists of several conserved domains including pre-domain, propeptide, catalytic domain, and hemopexin domain. Among these domains, the catalytic domain consists of 170 amino acids (AAs) and contains a conserved three histidine sequence which is required for zinc chelation and plays an important role in MMP activity .
Given their role in the degradation of the extracellular matrix, MMPs, and the plasminogen activator (PA) system may play also a critical role in the extensive remodeling that occurs in the bovine mammary gland during development, lactation, and involution . The mammary gland undergoes many changes in structure and function, including cyclic expansions corresponding to the hormonal changes induced by the estrous/menstrual cycle, as well as the dramatic changes that occur during pregnancy, lactation, and involution during the lifetime of the female . MMPs play a pivotal role in mammary ductal morphogenesis, angiogenesis, and glandular tissue architecture remodeling . For example, remodeling of the mammary tissue is also linked with the destruction of the lobular-alveolar structure of the gland and the extracellular matrix during mammary involution . At the same time, the metalloproteinase (MMP) system is activated and induces the proteolysis of extracellular matrix components .
Among MMPs, MMP9 has been shown to regulate many cellular processes, such as tissue repair, angiogenesis, apoptosis, cell migration, and wound healing . In the mammary gland, MMP9 preferentially degrades ECM during angiogenesis and tissue remodeling. The previous study indicated that MMP9 activity is regulated at three levels: gene transcription and mRNA stability, enzymatic form activation, and inhibition by endogenous inhibitors such as tissue inhibitors of metalloproteinases (TIMPs) . The MMP9 gene can be induced by a variety of oncogene products, cytokines, mitogens, and phorbol ester . These stimulators can upregulate the expression of MMP9 by modulating the activation of transcription factors such as NF-κB and AP-1 through the PI3K/AKT and MAPK signaling pathways. It is well known that human MMP9 promoter contains cis-acting regulatory elements for transcription factors including an NF-κB site (located at -600bp), an SP-1 site (located at -558bp), and two AP-1 sites (located at -79bp and -533bp) .
Phosphatidylinositol-3-kinase is an intracellular protein with catalytic activity, which can regulate the phosphorylation of downstream Akt and constitutes the PI3K/Akt signaling transduction pathway . The PI3K/AKT signaling pathway plays an important role in the regulation of cell survival, growth, proliferation, angiogenesis, and metabolism. In MDA-MB-231 cells, CoQ0 suppressed MMP-9 protein expression by inhibiting the PI3K/AKT/NF-κB pathway . PA effectively suppressed human breast cancer MDA-MB-231 cell migration, invasion, and cell motility by abating the expression of NF-κB and AP-1 to reduce the expression of MMP-9 through ERK/PI3K/Akt/mTOR signaling pathway . MMP9 secretion is mediated by 12-LOX in PC-3 cells via the activation of the PI3K/AKT/NF-κB signaling pathway .
However, the molecular mechanism governing MMP9 expression in dairy cow mammary epithelial cells has been poorly understood. In the current study, we aimed to investigate the mechanism by which PI3K/AKT/mTOR pathway is involved in NF-κB activation and MMP9 expression. We hypothesized that the PI3K/AKT/mTOR signaling pathway regulates MMP9 expression through NF-κB (P65) in mammary epithelial cells of dairy cows. To test our hypothesis, we evaluated the effects of LY294002, Rapamycin, and Celastrol on NF-κB (P65) nuclear translocation and MMP9 expression in mammary epithelial cells. We then investigated the crucial role of NF-κB(P65) in the regulation of the MMP9 promoter activity.