Lipocalin-2 (Lcn-2) is an acute-phase protein that has been implicated in diverse physiological processes in mice, including: apoptosis, ion transport, inflammation, cell survival, and tumorigenesis. This study characterized the biological activity of Lcn-2 in human endometrial carcinoma cells (RL95-2). Exposure of RL95-2 cells to Lcn-2 for >24 h reduced Lcn-2-induced cell apoptosis, changed the cell proliferation and up-regulated cytokine secretions, including: interleukin-8 (IL-8), inteleukin-6 (IL-6), monocyte chemotatic protein-1 (MCP-1) and growth-related oncogene (GRO). However, IL-8 mRNA and protein levels were dramatically increased in Lcn-2-treated RL95-2 cells. To determine the IL-8 effect on Lcn-2-treated RL95-2 cells was our major focus. Adding recombinant IL-8 (rIL-8) resulted in decreased caspase-3 activity in Lcn-2-treated cells, whereas the addition of IL-8 antibodies resulted in significantly increased caspase-3 activity and decreased cell migration. Data indicate that IL-8 plays a crucial role in the induction of cell migration. Interestingly, Lcn-2-induced cytokines, secretion from RL95-2 cells, could not show the potent cell migration ability with the exception of IL-8. We conclude that Lcn-2 triggered cytokine secretions to prevent RL95-2 cells from undergoing apoptosis and subsequently increased cell migration. We hypothesize that Lcn-2 increased cytokine secretion by RL95-2 cells, which in turn activated a cellular defense system. This study suggests that Lcn-2 may play a role in the human female reproductive system or in endometrial cancer.

Lipocalin 2 (LCN2) is an induced stressor that promotes the epithelial-mesenchymal transition (EMT). We previously demonstrated that the development of endometriosis in mice correlates with the secretion of LCN2 in the uterus. Here, we sought to clarify the relationship between LCN2 and EMT in endometrial epithelial cells and to determine whether LCN2 plays a role in endometriosis. Antibodies that functionally inhibit LCN2 slowed the growth of ectopic endometrial tissue in a mouse model of endometriosis, suggesting that LCN2 promotes the formation of endometriotic lesions. Using nutrient deprivation as a stressor, LCN2 expression was induced in cultured primary endometrial epithelial cells. As LCN2 levels increased, the cells transitioned from a round to a spindle-like morphology and dispersed. Immunochemical analyses revealed decreased levels of cytokeratin and increased levels of fibronectin in these endometrial cells, adhesive changes that correlate with induction of cell migration and invasion. Lcn2 knockdown also indicated that LCN2 promotes EMT and migration of endometrial epithelial cells. Our results suggest that stressful cellular microenvironments cause uterine tissues to secrete LCN2 and that this results in EMT of endometrial epithelial cells, which may correlate with the development of ectopic endometriosis. These findings shed light on the role of LCN2 in the pathology of endometrial disorders.

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The 24p3 protein is a 25 kDa glycoprotein that is secreted into the uterine fluid during the proestrous phase of mice. We assessed the effects on spermatozoa motility and on the functions of mouse spermatozoa using the computer-assisted sperm analysis method, cytochemical staining and detection of the protein tyrosine phosphorylation pattern. Compared with the control cells, sperm motility was stimulated by the addition of 24p3 protein into the medium. Introducing 24p3 protein enhanced progressive motility but did not promote the appearance of hyperactivated movement. The presence of 24p3 protein in the medium did not allow the cells to undergo the capacitated protein tyrosine phosphorylation pattern and acrosome reaction. The tyrosine phosphorylation pattern shows phosphoproteins in the range of Mr 50,000-106,000 correlated with the sperm progressive motility after the addition of 24p3 protein into the medium. Using flow cytometry, we assessed the changes in the intracellular pH and measured the intracellular cAMP concentration with an immunodetection kit. The results indicated that the elevation in intracellular pH from 6.67 to 6.89, increase of intracellular cAMP accumulation, and protein tyrosine phosphorylation might be the factors in enhancement of sperm motility as the 24p3 protein bound to the spermatozoa. The 24p3 protein may have a role in regulating flagellar motility.