Signaling and Signal Transduction in Endocrine Tissues
(T764) BLOCKING OF THE ENDOMETRIAL CHORIONIC GONODOTROPIN RECEPTOR (CG-R) BY SMALL INTERFERING RNA (siRNA).
Cameo, Paula1, Strakova, Zuzana1, Fazleabas, Asgerally1, 1 University of Illinois at Chicago, Chicago, IL
ABSTRACT- Chorionic gonadotropin (hCG) is the major embryonic signal involved in the interaction between the blastocyst and the receptive endometrium during implantation in the primate. We have previously demonstrated that in addition to rescuing the corpus luteum CG also induces changes in morphology and gene expression in the endometrium. Multiple mRNA transcripts for CG-R have been detected in human gonadal tissues. In the endometrium, CG-R like immunoreactivity has been demonstrated in several species like pig, rat, rabbit, baboon and humans. The objective of this study was to block CG-R expression in epithelial and stromal endometrial cells using siRNA and determine if the CG induced signal transduction cascade can be inhibited. Methodology: A human endometrial epithelial cell line (HES) and human uterine fibroblasts (HuF) were cultured and transfected with synthetic siRNA for the CG-R. A scrambled siRNA was used as a control. CG-R expression levels were analyzed by western blot using a CGR antibody made against a synthetic peptide, corresponding to the extracellular domain of the human CG-R. Inhibition of CG-R expression was confirmed by stimulation with CG following siRNA transfection. Cell lysates were analyzed for extracellular signal-regulated kinase (ERK 1/2) (BOR 2003;68:457-64) and p-38 MAP kinase phosporylation. Results: CG-R was partially blocked by siRNA transfection both in HES and HuF cells. We observed a decrease in CG-R expression by western blot 72 hours after siRNA trasfection. Phosphorylation of ERK1/2 and p-38 by CG were also inhibited in siRNA transfected HES and HuF cells respectively. Scrambled siRNA had no effect on CG-R expression. Conclusion: Inhibition of CG-R expression in both endometrial epithelial and stromal cells by siRNA transfection directly inhibits the signal transduction cascade that is activated by CG in these cells. These studies imply that inhibition or insufficient expression of the CG-R in the endometrium may affect embryo-maternal communications during the establishment of pregnancy. (Supported by NIH HD 42280 am TW 00671).
KEY WORDS: CG-R, siRNA