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.

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.