The molecular basis of chemokine action in human endometrium.

The function of the human endometrium is to accept the implanting embryo and provide an environment for its subsequent growth and development by forming the maternal side of the placenta. The endometrium undergoes rapid cyclic changes of cell proliferation, differentiation and renewal and briefly becomes receptive to the embryo allowing its attachment and subsequent invasion during the implantation window. These processes are controlled systemically by steroid hormones which are thought to activate and control local molecular effectors including chemotatic cytokines known as chemokines. Chemokines are known to recruit leukocytes by chemotaxis, but also have a wider multifunctional in the control of cell function.Limited studies have shown expression of the chemokine ligand CXCL12 and its receptor CXCR4 in the human endometrium. However, little is known about the precise role of these molecules in endometrial function. To investigate this further, real-time RT-PCR was used to measure the expression of CXCL12 and CXCR4 mRNA in endometrial biopsies throughout the menstrual cycle. Both CXCR4 and CXCL12 were expressed by the endometrium at all stages of the cycle. Expression of CXCL12 mRNA was relatively low and did not change throughout the menstrual cycle. However, CXCR4 mRNA significantly increased during the early proliferative phase of the cycle, with a second slight increase during the mid secretory phase.Expression of CXCL12 and CXCR4 in cultured human endometrial epithelial and stromal cells was investigated using real-time RT-PCR used to measure levels of CXCL12 and CXCR4 mRNA expression. CXCL12 expression was found to be higher in stromal cells compared to epithelial cells, while expression of CXCR4 was highest in the endometrial epithelial cells. Expression of CXCR4 mRNA was also investigated in Ishikawa and HEC-1-B cell lines. Both cell lines were found to express CXCR4 mRNA, though levels were higher in Ishikawa cells compared to HEC-1-B cells, which was more comparable to their primary epithelial cell counterparts.Although the functions of CXCL12 in the endometrium remain unknown, CXCL12 has been shown to increase the expression of interleukin 6 (IL6), interleukin8 (IL8), matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9) and vascular endothelial growth factor A (VEGFA) in various non-endometrial cell types. As all of these factors have previously been shown to be of potential importance to endometrial function, their basal mRNA expression was quantified in primary epithelial and stromal endometrial cells and Ishikawa and HEC-1-B cell lines using real-time RT-PCR. To investigate effects of CXCL12 on these endometrial factors, all four endometrial cell types were incubated with CXCL12 and the expression of IL6, IL8, MMP2, MMP9 and VEGFA was measured using real-time RT-PCR. Basal levels of IL8 and MMP9 mRNA were found to be significantly higher in endometrial epithelial cells in comparison to stromal cells, while VEGFA was significantly higher in stromal cells in comparison to epithelial cells. Expression of IL6, IL8, MMP2 and MMP9 was several orders of magnitude lower in the Ishikawa and HEC-1-B cell lines in comparison to their primary epithelial counterparts suggesting that these cell lines are not good models to investigate endometrial function. Incubation with CXCL12 had no significant effect on the expression of any of these factors, in primary epithelial, stromal, Ishikawa or HEC-1-B cells, which suggests CXCL12 may not regulate their expression in the endometrium.To investigate the potential wider effects of CXCL12 on the expression of endometrial factors, a genome-wide microarray expression analysis was carried out in Ishikawa cells. Incubation of these cells with CXCL12 resulted in the identification of fifty one mRNA transcripts that were shown to be significantly and reproducibly up-regulated by CXCL12. Fourteen of these transcripts were produced by unknown genes, but thirty seven transcripts could be identified and potential functions attributed to them. Several of these genes have previously been shown to be of potential importance to endometrial function or to be known components of the CXCR4 signaling pathways.Overall, the results of this study have increased our knowledge of the expression and cellular distribution of CXCL12 and CXCR4 in the endometrium. This study has also identified several potential roles for CXCL12 and CXCR4 in endometrial function which would warrant further investigation.