Involvement of NADPH oxidase and NF-κB activation in CXCL1 induction by vascular endothelial growth factor in human endometrial epithelial cells of patients with adenomyosis

• VEGF and CXCL1 are expressed in the endometrium specimens with adenomyosis.
• VEGF is an effective CXCL1 inducer in human endometrial epithelial cells.
• The VEGFR, p47 phox NADPH and NF-κB pathways are involved in VEGF-induced CXCL1 expression.
• The released CXCL1 by HEECs is required for attracting vascular endothelial cell migration.

Chemokines were known to participate in inflammation and angiogenesis but have been recently recognized to be involved in embryonic implantation and endometrium-related pathologies. Among these chemokines, the CXC chemokines, such as CXCL1, have potential roles to work as biomarkers to identify patients with uterine adenomyosis. In this study, human endometrial epithelial cells (HEECs) were derived from patients’ endometrium with adenomyosis. The inductive effects of CXCL1 production by various mediators/growth factors were investigated in the HEECs. Of the tested mediators, VEGF was found to be the most effective. The immunohistochemistry and RT-PCR analysis revealed a positive staining for VEGF and CXCL1 at the epithelium and the presence of CXCL1 in the human endometrium specimens, respectively. The CXCL1 induction by VEGF could be reduced by the antagonist for VEGF receptor (VEGFR), and by the inhibitors for NADPH oxidase and NF-κB signaling pathway. However, it was not affected by sex hormones and the inhibitors for MAPKs, PI-3K, protein kinase A and C. In parallel, VEGF induced p47 phox NADPH oxidase activation, IκBα phosphorylation, NF-κB translocation and NF-κB-DNA complex formation in the HEECs. Moreover, the CXCL1 released by the HEECs with VEGF stimulation attracted vascular endothelial cell migration. Taken together, we show that VEGF and CXCL1 are expressed in epithelium of the endometrium with adenomyosis and demonstrate here for the first time that VEGF is capable of inducing CXCL1 expression in HEECs through VEGFR, p47 phox NADPH oxidase and NF-κB signaling pathway, which is functionally required for attracting vascular endothelial cell migration.

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