A novel role for the AAA ATPase spastin as a HOXA10 transcriptional corepressor in Ishikawa endometrial cells.

Homeobox A10 (HOXA10), a transcription factor required for uterine development and embryo receptivity, functions downstream of estrogen and progesterone in uterine endometrium. HOXA10 represses endometrial expression of empty spiracles homeobox 2 (EMX2), the human ortholog of Drosophila empty spiracles. The ATPases associated with various cellular activities (AAA) ATPase spastin has a well-characterized role in neurotransmitter trafficking. In this study, we characterize a novel role of spastin in transcriptional regulation. We identified spastin as a novel component of the HOXA10 transcriptional complex in Ishikawa nuclear extracts by immunoprecipitation and mass spectrophotometry. Using EMX2 as a model endometrial HOXA10 target gene, we show that the HOXA10-spastin corepressor complex bound the EMX2 promoter in chromatin immunoprecipitation assays. HOXA10 has been previously shown to repress endometrial EMX2 expression. We further observed that, although cotransfection of HOXA10 and spastin continued to repress endometrial EMX2-luciferase expression, the repression was reversed when spastin small interfering RNA was cotransfected with HOXA10. Mutations in the nuclear localization signal sequences of spastin abrogated not only its nuclear translocation but also its colocalization with HOXA10 as well as reversed EMX2-luciferase repression. Here, we describe a novel role for the AAA ATPase spastin in Ishikawa cells as a HOXA10 corepressor of EMX2. Uterine EMX2 levels are inversely related to embryo implantation rates. HOXA10 acts downstream of progesterone and has been shown to facilitate embryo implantation through regulation of endometrial EMX2 expression. Endometrial spastin, therefore, likely has a novel function downstream of estrogen and progesterone in implantation biology as a cofactor of HOXA10.

HOXA10 inhibits Kruppel-like factor 9 expression in the human endometrial epithelium.

Kruppel-like factor 9 (KLF9) is a zinc finger transcription factor that regulates estrogen and progesterone action by modulating the activity of progesterone receptor (PGR). The transition from proliferative to secretory endometrial epithelium involves loss of estrogen receptor/PGR expression and loss of direct response to sex steroids. HOXA10 partially mediates progesterone responsiveness in the endometrium. Here, we demonstrate that HOXA10 directly regulates KLF9 in endometrial epithelial cells and not in stromal cells. Immunohistochemistry performed on endometrial tissue obtained from normal, reproductive-age women revealed that KLF9 expression was decreased in the secretory phase of the menstrual cycle compared to the proliferative phase. In vitro, HOXA10 transfection of human endometrial epithelial cells (Ishikawa), but not stromal cells (HESC), resulted in a greater than 50% decrease in KLF9 mRNA and protein expression. Reporter constructs driven by the KLF9 promoter were repressed by cotransfection with HOXA10. Electrophoretic mobility shift assay was used to demonstrate direct binding of HOXA10 to the KLF9 promoter. Targeted mutation of the HOXA10-binding site in the KLF9 promoter resulted in loss of HOXA10 binding and loss of repression by HOXA10 in reporter assays. HOXA10 directly and selectively repressed KLF9 expression in endometrial epithelial cells. HOXA10 repression of KLF9 likely contributes to the loss of sex steroid responsiveness in secretory-phase endometrial epithelium.

3-Phosphoglycerate dehydrogenase expression is regulated by HOXA10 in murine endometrium and human endometrial cells.

3-Phosphoglycerate dehydrogenase (PHGDH, 3-PGDH) is an enzyme necessary for de novo l-serine biosynthesis. HOXA10 expression is required for endometrial receptivity; however, few target genes of HOXA10 regulation are known. Using a microarray we identified Phgdh as a target of HOXA10 regulation in murine endometrium and confirmed this regulatory relationship in human endometrial cells. PHGDH was downregulated 2.0-fold by HOXA10 and upregulated 4.4-fold by HOXA10 antisense in vivo. In human endometrial cells, real-time PCR results show that pcDNA3.1/HOXA10 transfection decreased PHGDH mRNA expression to 40% of pretreatment level (P<0.05), while PHGDH mRNA expression was increased 2.1-fold (P<0.05) by HOXA10 siRNA. Western blot results confirmed the regulatory relationship in both primary human endometrial stromal and epithelial cells, as well as in human endometrial stromal cells and Ishikawa cells. In human cycling endometrial tissue, immunohistochemical results showed that PHGDH expression is relatively high in the proliferative phase in glandular cells and lower in the secretory phase. Here we report novel expression and regulation of PHGDH in murine and human endometrium. PHGDH is expressed in both endometrial epithelial and stromal cells. HOXA10 represses endometrial PHGDH expression. PHGDH is necessary for serine biosynthesis, which serves as a substrate for protein synthesis. One mechanism by which HOXA10 regulates cellular differentiation may involve limiting protein synthesis in the secretary phase.

Thrombin and interleukin-1beta decrease HOX gene expression in human first trimester decidual cells: implications for pregnancy loss.

Bleeding or inflammation in early pregnancy may result in pregnancy loss or defective implantation. Their effect on HOX gene expression in first trimester decidua is unknown. Bleeding results in thrombin generation, although infection or inflammation results in production of cytokines typified by Interleukin-1beta (IL-1beta). First trimester decidual cells were pretreated with 17beta estradiol (E(2)), medroxyprogesterone acetate (MPA) or both and subsequently treated with thrombin or IL-1beta. Affymetrix microarray analysis was used to assess the expression of all HOX genes and confirmed using real-time RT-PCR. E(2) or MPA treatment resulted in significant increases in HOXA10 and HOXA11. Subsequent treatment with thrombin resulted in diminished expression of HOXA10 and HOXA9. Treatment with IL-1beta resulted in decreased expression of HOXA1, 3, 9, 10 and 11. HOXA10 expression was reduced by 70% after thrombin treatment (P = 0.018) and by 90% after IL-1beta treatment (P = 0.004). HOXA11 mRNA expression was decreased by 88% after IL-1beta treatment (P < 0.001), but not by thrombin treatment. Decidua was collected at the time of elective termination of pregnancy (n = 10) or surgical treatment of spontaneous pregnancy loss (n = 10). Real-time PCR and western analysis demonstrated decreased HOXA10 and HOXA11 RNA and protein expression in the decidua of spontaneous pregnancy loss compared with that of viable pregnancies. In conclusion, multiple HOX genes are expressed in decidual cells and inhibited by thrombin and IL-1beta. Since HOXA10 and HOXA11 are known to be necessary for successful pregnancy, these findings suggest a molecular mechanism by which bleeding or inflammation may affect pregnancy outcome.

Vascularization and expression of angiogenic factors in partial and complete molar pregnancies.

OBJECTIVE: To determine the microvessel density (MVD) at the implantation site of normal placenta (NP) and molar pregnancies and to correlate MVD with clinical data and underlying angiogenic factors. STUDY DESIGN: Immunolocalization of CD31, vascular endothelial growth factor and angiopoietin 1 and 2 were performed on NPs, nonpersistent partial moles, persistent partial moles (PPM), nonpersistent complete moles and persistent complete moles (PCM). RESULTS: Significant differences were identified in the MVD between NP and complete mole (CM), and PM and CM (p < 0.001 and p < 0.035, respectively). MVD in PPM and PCM was significantly higher (p = 0.036 and p < 0.001, respectively) when compared to NP. MVD > 100 per high-power field was associated with an increased risk of persistence (p < 0.04). MVD showed a strong correlation with immediate postevacuation hCG levels (p < 0.03). Angiopoietin 2 staining was more heterogeneous, with lower overall expression in molar pregnancies as compared to more homogeneous expression in NP (p < 0.05). CONCLUSION: MVD is highly correlated with hCG levels, suggesting that hCG may act as an angiogenic factor during implantation of molar pregnancy. MVD at the implantation site may be associated with excessive trophoblastic proliferation or reflect high hCG levels, which places patients at increased risk of persistent neoplasia.

Claudin-4 overexpression in epithelial ovarian cancer is associated with hypomethylation and is a potential target for modulation of tight junction barrier function using a C-terminal fragment of Clostridium perfringens enterotoxin.

BACKGROUND: Claudin-4, a tight junction (TJ) protein and receptor for the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), is overexpressed in epithelial ovarian cancer (EOC). Previous research suggests DNA methylation is a mechanism for claudin-4 overexpression in cancer and that C-CPE acts as an absorption-enhancing agent in claudin-4-expressing cells. We sought to correlate claudin-4 overexpression in EOC with clinical outcomes and TJ barrier function, investigate DNA methylation as a mechanism for overexpression, and evaluate the effect of C-CPE on the TJ. METHODS: Claudin-4 expression in EOC was quantified and correlated with clinical outcomes. Claudin-4 methylation status was determined, and claudin-4-negative cell lines were treated with a demethylating agent. Electric cell-substrate impedance sensing was used to calculate junctional (paracellular) resistance (Rb) in EOC cells after claudin-4 silencing and after C-CPE treatment. RESULTS: Claudin-4 overexpression in EOC does not correlate with survival or other clinical endpoints and is associated with hypomethylation. Claudin-4 overexpression correlates with Rb and C-CPE treatment of EOC cells significantly decreased Rb in a dose- and claudin-4-dependent noncytotoxic manner. CONCLUSIONS: C-CPE treatment of EOC cells leads to altered TJ function. Further research is needed to determine the potential clinical applications of C-CPE in EOC drug delivery strategies.

Thrombin and interleukin-1beta regulate HOXA10 expression in human term decidual cells: implications for preterm labor.

CONTEXT: Preterm delivery is commonly caused by intraamniotic infection with expression of proinflammatory cytokines (IL-1beta) or by abruption resulting in generation of decidual thrombin. Although human parturition is not preceded by overt progesterone withdrawal, progesterone resistance likely leads to labor. The uteri of Hoxa10(-/-) mice demonstrate progesterone resistance; several genes, including prostaglandin receptors, are inappropriately regulated in response to progesterone. OBJECTIVE: We hypothesized that IL-1beta or thrombin would decrease HOXA10 expression, contributing to the progestin-resistant environment. We analyzed expression of HOX genes and their regulation by IL-1beta or thrombin in decidual cells. DESIGN AND SETTING: We conducted an in vitro experiment at an academic medical center. INTERVENTION: Term decidual cells were treated with estradiol (E(2)) or E(2) plus medroxyprogesterone acetate followed by addition of thrombin or IL-1beta. MAIN OUTCOME MEASURE: HOX mRNA was evaluated by microarray and confirmed by quantitative RT-PCR. Protein expression was detected using immunohistochemistry and Western analysis. RESULTS: HOXA9, HOXA10, and HOXA11 were expressed in decidual cells and regulated by IL-1beta and thrombin. HOXA10 was further analyzed because of its association with progesterone responsiveness. After E(2) treatment, IL-1beta and thrombin decreased HOXA10 mRNA by 94 and 81%, respectively. After E(2) plus medroxyprogesterone acetate treatment, IL-1beta and thrombin resulted in an 86 and 72% decrease in HOXA10 mRNA, respectively. A similar decrease was noted in HOXA10 protein expression. CONCLUSION: The expression of HOXA10 protein at term indicates that it may have a role in maintaining decidual cell phenotype and pregnancy. The dramatic decrease of HOXA10 in response to IL-1beta or thrombin may contribute to progestin resistance in preterm labor, mimicking progesterone resistance seen in Hoxa10(-/-) mice.

HOXA10, Pbx2, and Meis1 protein expression in the human endometrium: formation of multimeric complexes on HOXA10 target genes.

HOXA10 is a transcription factor necessary for embryonic uterine development and for adult endometrial receptivity. The three-amino acid loop extension family of cofactors, including Pbx and Meis, provide HOX target gene specificity in development and myeloid differentiation. Here we demonstrate the expression of Pbx and Meis family cofactors in the human endometrium and their interaction with HOXA10. Using immunohistochemical analysis, we found that Pbx2 and Meis1, but not Pbx1, Pbx3, or Meis2, were expressed in human endometrium. HOXA10, Pbx2, and Meis1 were expressed in the stroma throughout the menstrual cycle. The glandular expression of HOXA10 and Meis1 was menstrual cycle stage specific, whereas glandular Pbx2 expression did not vary. Pbx2, but not Meis1, was expressed in Ishikawa cells. EMSA demonstrated HOXA10-Pbx2 binding as a heterodimer to an enhancer of the EMX2 gene, a known target of HOXA10 regulation. Ablation of the Pbx binding site, but not ablation of the HOXA10 binding site in EMX2, resulted in loss of dimer binding. Based on the observed expression and binding patterns of Pbx2, Meis1, and HOXA10, it is likely that heterodimeric and trimeric complexes involving these proteins determine HOXA10 target gene specificity. Enhanced target gene specificity imparted by multimer binding is likely necessary for HOXA10-mediated endometrial receptivity.