O-73: CALPAIN5 expression is regulated by HOXA10 in human endometrial cells and down regulated in endometriosis

Abstract

CALPAIN5 (CAPN5) is member of the calpain-like cystein protease family and has been implicated in the regulation of a variety of cellular functions, including differentiation and apoptosis. Our lab conducted a microarray screen that had identified CAPN5 as target of HOXA10 transcriptional control in endometrium. Here we demonstrate regulated CAPN5 expression in endometrium, decidua and endometriosis. Analysis of CAPN5 expression in human reproductive tissue and its regulation in endometrial cell lines. Ten endometrial biopsies (5 proliferative phase and 5 secretory phase) were obtained from fertile controls and 3 first trimester decidual samples were obtained at the time of elective termination. Histologically confirmed biopsies of endometriosis were obtained from 10 women at the time of laparoscopy. All women were in the reproductive age, had regular menstrual cycle, and were not using hormonal medication. Immunohistochemistry (IHC) was used to identify CAPN5 protein in eutopic and ectopic endometrium as well as first trimester decidua. The human endometrial stromal cell line, HESC, the human endometrial epithelial cell line, Ishikawa were transfected with either 4.0μg pcDNA/HOXA10 or 20μM HOXA10 siRNA; transfection with empty pcDNA vector or nonspecific siRNA served as respective controls. Cells were transfected in quadruplicate at 60-70% confluence. Forty eight hours after the transfection total RNA was isolated. qRT-PCR was performed in duplicated to determine expression levels of HOXA10 and CAPN5 in each group. IHC was performed with CAPN5 polyclonal anti-body. IHC results were assessed by 2 evaluators blinded to the study groups, and H-SCORES were determined for the stroma, glands and decidua. Statistical analysis was performed using Mann-Whitney test for H-SCORE and ANOVA with Bonferroni pos-test for qRT-PCR. CAPN5 was expressed in endometrial stromal and glandular cells throughout the menstrual cycle. CAPN 5 was also expressed in first trimester decidual cells at higher levels than secretory phase stromal cells (p<0.05). The regulatory relationship between HOXA10 and CAPN5 was established by transient transfection analysis. CAPN5 gene expression increased 11-fold (p<0.05) after pcDNA/HOXA10 transfection of the HESC, and decreased 23-fold (p<0.05) after HOXA10 siRNA treatment. CALPAIN5 was not regulated by HOXA10 in Ishikawa cells. There was a decrease in CAPN5 expression in both stromal and glandular cells in endometriosis to 50% of that seen in fertile controls. (p<0.05). CAPN5 is expressed in normal endometrium and at upregulated in decidua. CAPN5 expression is decreased in endometriosis compared to eutopic endometrium. CAPN5 expression is regulated by HOXA10. The previously demonstrated decreased HOXA10 expression in endometriosis is a likely molecular mechanism by which CAPN5 expression is concomitantly decreased. CAPN5 is a novel regulatory molecule potentially involved in the pathogenesis if endometriosis. Decreased CAPN5 in endometriosis may lead to reduced apoptosis and contribute to the increased proliferative potential of this disease.