The current theory on the pathogenesis of prolactinomas includes the effects of endocrine hormone, abnormal expression of genes, and microRNA irregular expression[30, 31]. However, the exact pathogenesis of prolactinomas was still not clear. The gender difference in the occurrence of prolactinoma has long existed in the clinical, especially in the period of 20-30 years old, the incidence ratio of male and female even reached 1:10. The reason for this phenomenon may be the high estrogen level of women at this stage.[32] The role of estrogen is well established in promoting prolactinoma, DRD2 Knockout mice and estradiol-injected rats or mice are the most extensively acceptable prolactinoma animal models[10, 28, 29], Estradiol-injected mice was selected in the present study to clarify the role of estradiol in prolactinoma. ERs can regulate Toll-like receptor (TLR) signaling pathways in the immune system[33], Additionally, a previous study reported that estrogen promotes upregulation of TLR4/NF‑κB/p38MAPK activation via ERβ[24]. Based on these previously results, we suppose that estrogen-activated ERβ/TLR4/NF‑κB/p38MAPK pathway participated in the pathogenesis of prolactinomas.
TLR4 plays a fundamental role in pathogen recognition and activation of innate immunity, and is often overexpressed in malignant and tumour-infiltrating immune cells. TLR4 plays a crucial role in mesenchymal stem cell (MSC)induced inhibition of natural killer (NK) cell function. TLR4 ultimately activates the transcription factor NF-κB by linking to MyD88, which is required for the expression of cytokines, chemokines, and other stimulating molecules such as TNF-α, IL-1β, IL-6, IL-8, IL-12, and MIP 1α.[34]Various stimulators induce activation of the p38MAPK, an important kinase involved in cell proliferation and apoptosis.[35] The proliferation-relatedproteinsB-cell-specific Moloney murine leukemia virus insertion site1(BMI1) ,Bcl2, and Bax were determined in the pituitary gland via western blot. The BMI1 and Bcl2/Bax protein expression of estradiol-treated mice are increased significantly compared with WT mice. However, this effect reversed in estradiol-treated TLR4-/- mice. The results proved that estrogen-induced prolactinoma may be related to pituitary proliferation.
TLR4 is highly expressed in various kinds of tumor cells, and activation of TLR4 mediated p38MAPK promotes tumor growth and proliferation[36, 37]. Therefore, TLR4 can be a therapeutic target. In this study, immunohistochemistry analysis showed that ERβ and TLR4 were overexpressed, and TLR4 was co-located with PRL protein in human prolactinoma tissues by immunofluorescence analysis. The high expression and positive correlation of TLR4 and ERβ are first found in clinical samples of prolactinoma. Furthermore, TLR4 co-located with PRL, a marker of prolactinoma, laying a foundation for subsequent in vitro and in vivo studies.
Western blot results also showed that the expression of ERβ, TLR4 and PRL were increased in the pituitary tissues of estradiol-treated mice. Furthermore, TLR4 knockout significantly inhibited tumor overgrowth and PRL secretion, and ERβ, TLR4 and PRL protein expression in estradiol-induced mice. It was further verified that TLR4 is a potential target of prolactinomas. It is reported that estradiol and ERs regulate inflammatory pathways of innate immune cells, including dendritic cells and macrophages[33].As a classic protein regulating innate immunity and inflammation, TLR4 has many interactions with estrogen[38]. Therefore, excessive estrogen perhaps was a key cause that influenced immune and inflammatory processes in the body. The TLR4 activation effect of estradiol have been proved in vitro and in vivo experiments in our study, thus we have sufficient evidence to verify the role of TLR4 in estradiol-mediated prolactinoma. In order to verify the role of estradiol in the ERβ/TLR4/NF-κB/p38MAPK pathway in prolactinoma, the effects of inhibition and activation of ERβ on TLR4/NF-κB/p38MAPK pathway and PRL expression were investigated in MMQ cells in vitro. MMQ cells were treated with estradiol and fulvestrant, which can activate or inhibit the protein expression of ERβ/TLR4/NF-κ B/p38MAPK pathway and PRL expression. Meanwhile, the expression of TLR4/NF-κB/p38MAPK pathway was inhibited after the transfection knockdown of TLR4, and the expression of ERβ was also decreased, which indicate that there may be an interaction between TLR4 and ERβ. Then TLR4 protein directly binding to ERβ protein were found by immunoprecipitation, these results indicate that both TLR4 and ERβ play important roles in mediating the occurrence of prolactinoma. In addition, the expression of p38MAPK and PRL were increased significantly after treatment with LPS and E2 than with the single LPS or E2, which proves that the interaction of TLR4 and ERβ can synergistically regulate prolactin expression and tumor growth.
Our study first reported the role of the TLR4/NF-κB/p38MAPK pathway in prolactinoma and TLR4 knockout inhibited the proliferation and secretion of prolactin in prolactinoma mice effectively. The limitation of this study is that only paraffin specimens of human prolactinoma tissues were obtained, and more detailed clinical data were not obtained, such as the relationship between tumor size and TLR4 positive expression in tissues. Moreover, drugs targeting inhibition of TLR4/p38MAPK in pituitary tissue for prolactinoma have not been developed, further research is needed. in conclusion, previous studies of our group demonstrated that MAPK14 and NLRP3 knockout can inhibit the proliferation and secretion of prolactin in prolactinoma. Therefore, this study focused on exploring the role of their upstream protein TLR4 in prolactinoma and whether estradiol-mediated prolactinoma genesis is mediated by TLR4/NF-κB/p38MAPK pathway. We reveal the correlation between the interacting ERβ and TLR4 signaling pathways. In addition, we demonstrated that E2 or LPS promoted PRL expression and prolactin tumor proliferation through the p38MAPK pathway. Further research on drugs targeting estrogen receptor and TLR4/NF-κB/p38MAPK pathway is the focus of our research, and new drug development based on these targets may provide new treatment strategies for prolactinoma.