The Role Of Focal Adhesion Kinase In Endometriosis And Its Regulation In Endometrial Stromal Cells By 17β-estradiol And Progesterone

Endometriosis,a common gynecological disease,is defined as the presence of endometrial glands and stroma outside of the normal location in the uterus,mainly in the pelvis.The pathogenesis of this enigmatic disorder still remains controversial despite multiple theories regarding the etiology exist.Among them,the implantation hypothesis of retrograde menstruation suggested by Sampson is the most widely accepted one.However,the implantation theory doesn't explain that adhesion and persistence of endometriotic lesions in the pelvis only happen in approximately 10%-15%of women in reproductive age while the retrogradation of endometrial tissues via fallopian tubes during menstruation is a quasi-universal phenomenon.It is assumed that additional factors favor the adhesion and growth of endometrial cells that spill into the peritoneal cavity.Some studies indicate that eutopic endometrium of women affected by endometriosis is abnormal compared with that of healthy women.Although the morphology of eutopic endometrium from women with endometriosis is similar to the normal,its physiology and biochemistry are different. And until now,the discrimination has been found in gene expression,apoptosis,cell adhesion molecules,invasion and angiogenesis.The endometrial cells from women with endometriosis have a predisposition to adhere and grow outside of the uterus. The adhesion of retrograde endometrial cells to the extracellular matrix is one of the first necessary stages in the framework of the implantation theory.As reported before,a number of factors take part in the process of cell adhesion and a major role is played by interaction with the extracellular matrix.Among them, Focal adhesion kinase(FAK)is an intracellular signaling protein residing at sites of attachment of the cell to the underlying extracellular matrix and associating with integrin receptors and other molecules to the site of this interaction to form a signaling complex that transmits signals from the extracellular matrix to the cell cytoskeleton.As a non-receptor tyrosine kinase protein that was originally isolated from Rous sarcoma virus(v-src)transformed chicken embryo fibroblasts,FAK controls a number of biological processes through the binding sites for many signaling proteins.But no rearch has focused on the relationship of FAK and endometriosis before.The present study focused on the role of focal adhesion kinase in the pathogenesis and progress of endometriosis and its mechanism of regulation by estrogen and progesterone.Cell cultures,immunohistochemical technique,western blotting and RT-PCR were used in this study.The study was divided into four parts:1.the expression of FAK in eutopic endometrium of endometriosis;2.the expression of FAK in ectopic endometrium of endometriosis;3.the relationship of FAK and the biological behavior of endometrial stromal cells from endometriosis;4.the regulation of FAK by estrogen and progesterone. Part one The expression of FAK in eutopic endometrium of women with endometriosis and its clinicpathologically significanceObjective:To investigate whether FAK expression is altered in eutopic endometrium of women with endometriosis.Methods:FAK protein and mRNA expression was assessed by immunohistochemistry,western blotting analysis and RT-PCR.Results:1.FAK immunoreactive staining was present in the cytoplasm of glandular epithelial and stromal cells in both women with and without endometriosis. 2.The anti-FAK antibody detected a band at 125kDa in protein extracts from all endometrial tissues.After normalizing each band of FAK withβ-actin from different samples,we found that at secretory phase the average level of FAK expression of women with endometriosis(0.32±0.04)was significantly higher than that of control(0.29±0.05)(P＜0.05).But no significant difference was found between the two groups at proliferative phase(P＞0.05).3.Semi-quantitative analysis showed that FAK mRNA expression level in secretory endometrial samples of women with endometriosis(0.67±0.12)was significantly higher than that of control(0.60±0.11) (P＜0.05).There was no significant difference between the two groups at proliferative phase(P＞0.05).4.Only in secretory endometrial samples,there was a positive correlation between expression of FAK protein and mRNA and disease stage(r²=0.437,0.440,respectively.P=0.022,0.021,respectively),as well as pelvic pain and FAK protein and mRNA expression(r²=0.244,0.248,respectively. P=0.023,0.012,respectively).But no correlation was found between FAK expression and fertility problem,dysmenorrhea and average diameter of ovarian endometriotic cyst(P＞0.05).5.A positive correlation between serum E₂ level and FAK protein expression at proliferative phase(n=50,r²=0.141,P=0.003).And at secretory phase we observed a positive correlation between FAK protein expression and the ratio of E₂ to P.However,there was no correlation between FAK protein expression and the serum level of E₂ or P respectively at secretory phas(n=52, r²=0.117,P=0.013).Part two Expression of FAK in ovarian endometriotic tissues of women with ovarian endometriosisObjective:To investigate whether FAK was expressed in ovarian endometriotic tissues of women with endometriosis.Methods:FAK protein and mRNA expression was assessed by immunohistochemistry,western blotting analysis and RT-PCR.Results:1.FAK immunoreactive staining was present in the cytoplasm of epithelial and stromal cells in ovarian endometriotic tissues.2.After normalizing each band of FAK withβ-actin from different samples,FAK expression in ovarian endometriotic tissues(0.76±0.12)was significantly higher than that of normal endometrium(0.66±0.11)(P＜0.05).3.Semi-quantitative analysis showed that FAK mRNA expression in ovarian endometriotic tissues(0.71±0.11)was significantly higher than that of normal endometrium(0.60±0.14)(P＜0.01).4.FAK protein and mRNA level in ovarian endometriotic tissues of women with endometriosis was increased as disease stage progress(P＜0.05).Compared with women without pelvic pain,FAK protein and mRNA expression was significantly increased in cases with pelvic pain(P＜0.05).Elevation of FAK protein and mRNA expression was also associated with average diameter of ovarian endometriotic cyst(P＜0.05,). Part three The relationship of FAK and the biological behavior of endometrial stromal cells from endometriosisObjective:To investigate the relationship of FAK protein level and capability ofthe proliferation,adhesion and migration of endometrial stromal cells from women with endometriosis.Methods:FAK protein expression was assessed by western blotting analysis. The capability of proliferation,adhesion and metastasis of endometrial stromal cells were assessed by MTT,adhesion assay and migration assay.Result:1.By MTT assay,the growth rate,rate of adhesion and the migrated distance of endometrial stromal cells from endometriosis(0.37±0.06,37%±7%, 0.78±0.09mm,respectively)is significantly higher than the control group(0.30±0.05, 30%±6%,0.67±0.07mm,respectively)(P＜0.05).2.In endometrial stromal cell from endometriosis,FAK protein level is related with the cell behavior:proliferation, adhesion and migration(r²=0.745,0.696,0.708;P=0.001,0.004,0.003).Part four The role of 17β-estradiol and progesterone in the regnlation of FAK expression in endometrial stromal cellsObjective:to investigate the effects of 17β-estradiol and progesterone on the expression of FAK in cultured endometrial stromal cells and whether there is alteration in the reaction of endometrial stromal cells from women with endometriosis.Methods:FAK protein and mRNA expression was assessed by westem blotting analysis and RT-PCR. Results:1.Elevated expression of FAK mRNA and protein was seen in the cultured endometrial stromal cells treated with 17β-estradiol,and the expression is higher as the tensity of 17β-estradiol and treated time progressed.But after 10^-7nmol/L and 24h,the expression of FAK became lowed down.Progesterone alone didn't induce the up-regulation of FAK mRNA and protein(P＞0.05).But FAK protein and mRNA expression in endometrial stromal cells treated by 17β-estradiol and progesterone(0.38±0.04,0.36±0.05)is lower than those treated by 17β-estradiol alone(0.56±0.05,0.47±0.06)(P＜0.01).2.In endometrial stromal cells from women with endometriosis,the level of up-regulation by 17β-estradiol (0.42±0.05,0.32±0.04)is significantly higher than those from women without(0.33±0.06,0.23±0.04)(P＜0.01).And progesterone could inhibit the up-regulation induced by 17β-estradiol(P＞0.05).Conclusion:1.FAK immunoreactive staining was present in the cytoplasm of glandular epithelial and stromal cells in eutopic endometrium.Expression of FAK was increased in secretory endometrium of women with endometriosis,and the elevation of FAK expression was associated with disease stage and pelvic pain, which might be related to the pathogenesis and progress of endometriosis.2.FAK immunoreactive staining was present in the cytoplasm of glandular epithelial and stromal cells in ectopic endometriotic tissue.Its expression level was associated with the clinicopathology,which might be related to the progress of endometriosis.3.The capability of proliferation,adhesion and migration of endometrial stromal cells from women with endometriosis is higher than those from women without.The capability of proliferation,adhesion and migration of end0metrial stromal cells from endometriosis is related with the FAK protein level,which suggests that FAK plays a role in the pathogenesis and progress of endometriosis.4.In normal endometrial stromal cells 17β-estradiol could up-regulate FAK level, and the up-regulation was inhibited by the addition of progesterone.But progesterone alone had no effect on FAK expression.5.Endometrial stromal cells from endometriosis were more over-sensitive to 17β-estradiol and resisted to progesterone,which up-regulated the expression of FAK and contributed to the pathogenesis and progress of endometriosis.