Rab11a drives adhesion molecules to the surface of endometrial epithelial cells.

STUDY QUESTION: Is Rab11a GTPase, a regulator of intracellular trafficking, of significance in endometrial functions? SUMMARY ANSWER: Rab11a is an important component of the cascades involved in equipping the endometrial epithelium (EE) with 'adhesiveness' and 'cohesiveness'. WHAT IS KNOWN ALREADY: Cell adhesion molecules (CAMs) have been investigated extensively for modulation in their endometrial expression during the peri-implantation phase. However, the mechanisms by which CAMs are transported to the EE surface have not received the same attention. Rab11a facilitates transport of specific proteins to the plasma membrane in endothelial cells, fibroblasts, embryonic ectodermal cells, etc. However, its role in the transport of CAMs in EE remains unexplored. STUDY DESIGN, SIZE, DURATION: In-vitro investigations were directed towards deciphering the role of Rab11a in trafficking of CAMs (integrins and E-cadherin) to the cell surface of Ishikawa, an EE cell line. Towards this, Rab11a stable knockdown (Rab-kd) and control clones of Ishikawa were generated. JAr (human trophoblastic cell line) cells were used to form multicellular spheroids. Pre-receptive (n = 6) and receptive (n = 6) phase endometrial tissues from women with proven fertility and receptive phase (n = 6) endometrial tissues from women with unexplained infertility were used. PARTICIPANTS/MATERIALS, SETTING, METHODS: Rab-kd and control clones were used for in-vitro assays. Live cells were used for biotinylation, JAr spheroid assays, flow cytometry, trans-epithelial electrical resistance assays and wound-healing assays. Lysosome and Golgi membranes were isolated by ultracentrifugation. Confocal microscopy, immunoblotting, qRT-PCR and immunohistochemistry were employed for assessing the expression of Rab11a, integrins and E-cadherin. MAIN RESULTS AND THE ROLE OF CHANCE: shRNA-mediated attenuation of Rab11a expression led to a significant (P < 0.01) decline in the surface localization of αVß3 integrin. Cell surface protein extracts of Rab-kd clones showed a significant (P < 0.05) reduction in the levels of αV integrin. Further, a significant (P < 0.01) decrease was observed in the percent JAr spheroids attached to Rab-kd clones, compared to control clones. Rab-kd clones also showed a significant (P < 0.001) decline in the total levels of E-cadherin. This was caused neither by reduced transcription nor by increased lysosomal degradation. The role of Rab11a in maintaining the epithelial nature of the cells was evident by a significant increase in the migratory potential, presence of stress-fibres and a decrease in the trans-epithelial resistance in Rab-kd monolayers. Further, the levels of endometrial Rab11a and E-cadherin in the receptive phase were found to be significantly (P < 0.05) lower in women with unexplained infertility compared to that in fertile women. Taken together, these observations hint at a key role of Rab11a in the trafficking of αVß3 integrin and maintenance of E-cadherin levels at the surface of EE cells. LARGE-SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The in-vitro setting of the study is a limitation. Further immunohistochemical localizations of Rab11a and CAMs were conducted on a limited number of human endometrial samples. WIDER IMPLICATIONS OF THE FINDINGS: Rab11a-mediated trafficking of endometrial CAMs in EE cells can be explored further for its potential as a target for fertility regulation or infertility management. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Indian Council of Medical Research (ICMR), the Department of Science and Technology (DST), the Council of Scientific and Industrial Research (CSIR), Government of India. No competing interests are declared.

Androgen receptor as a regulator of ZEB2 expression and its implications in epithelial-to-mesenchymal transition in prostate cancer.

Zinc finger E-box-binding protein 2 (ZEB2) is known to help mediate the epithelial-to-mesenchymal transition, and thereby it facilitates cancer metastasis. This study was initiated to explore whether ZEB2 expression differs in prostate cancer (PCa, n=7) and benign prostatic hyperplasia (BPH, n=7) tissues. In PCa tissues, the levels of both immunoreactive ZEB2 and androgen receptor (AR) were found to be significantly higher (P<0.05) when compared with BPH tissues. Co-regulation of AR and ZEB2 prompted us to investigate the role of androgenic stimuli in ZEB2 expression. ZEB2 expression was found to be significantly (P<0.05) upregulated after androgen stimulation and downregulated following AR silencing in LNCaP cells, an androgen-dependent PCa cell line. This finding suggested AR as a positive regulator of ZEB2 expression in androgen-dependent cells. Paradoxically, androgen-independent (AI) cell lines PC3 and DU145, known to possess low AR levels, showed significantly (P<0.05) higher expression of ZEB2 compared with LNCaP cells. Furthermore, forced expression of AR in PC3 (PC3-AR) and DU145 (DU-AR) cells led to reductions in ZEB2 expression, invasiveness, and migration. These cells also exhibited an increase in the levels of E-cadherin (a transcriptional target of ZEB2). Co-transfection of AR and ZEB2 cDNA constructs prevented the decline in invasiveness and migration to a significant extent. Additionally, ZEB2 downregulation was associated with an increase in miR200a/miR200b levels in PC3-AR cells and with a decrease in miR200a/miR200b levels in AR-silenced LNCaP cells. Thus, AR acts as a positive regulator of ZEB2 expression in androgen-dependent cells and as a negative regulator in AI PCa cells.

High mobility group box 1 (HMGB1) protein in human uterine fluid and its relevance in implantation.

STUDY QUESTION: Does a differential abundance of high mobility group box 1 (HMGB1) protein in uterine fluid (UF) have a functional significance? SUMMARY ANSWER: In rats, an excess of HMGB1 in UF during the receptive phase is detrimental to pregnancy. WHAT IS KNOWN ALREADY: The identification of constituents of the human uterine secretome has been a subject of renewed interest, due to the advent of high throughput proteomic technologies. Proteomic-based investigations of human UF have revealed the presence of several proteins such as mucins, host defense proteins S100, heat shock protein 27 and haptoglobin, etc. The present study reports on the presence of HMGB1, a nuclear protein, in human UF. Activated macrophages/monocytes, natural killer cells, mature dendritic cells, pituicytes and erythroleukemic cells are also known to secrete HMGB1. Existing data suggest that extracellular HMGB1 plays a role in inflammation. STUDY DESIGN, SIZE, DURATION: The human part of this study was cross-sectional in design. UF and endometrial tissues were collected from regularly cycling women in the early secretory (i.e. pre-receptive phase, Day 2 post-ovulation, n = 7) or secretory phase (i.e. receptive phase, Day 6 post-ovulation, n = 7) of their menstrual cycles. Samples were also collected from cycling rats in the proestrous (n = 8) or metestrous (n = 8) phase of their estrous cycles. Uteri were also collected from HMGB1-treated pregnant (n = 7) and untreated pseudo-pregnant (n = 7) rats and from pregnant rats at Day 3-5 post-coitum (p.c.) (n = 18, 3 each for six-time points). PARTICIPANTS/MATERIALS, SETTING, METHODS: In each group of human samples, four samples were used for isobaric tag for relative and absolute quantification (iTRAQ) analysis and three samples were used for immunoblotting experiments to determine the abundance of HMGB1 in pre-receptive and receptive phase UF samples. HMGB1 levels in rat UF and endometrial tissue samples were estimated by ELISA and immunohistochemical studies, respectively. The expression of inflammation-associated molecules, such as nuclear factor kappa B (NFκB), receptor for advanced glycation end products (RAGEs), tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), was analyzed by immunohistochemistry in HMGB1-treated and pseudo-pregnant rats. MAIN RESULTS AND THE ROLE OF CHANCE: HMGB1 was identified as one of the differentially abundant proteins in the list generated by 8-plex iTRAQ analysis of receptive and pre-receptive phase UF samples. In both humans and rats, secreted and cellular levels of HMGB1 showed a similar pattern, i.e. significantly (P < 0.05) lower abundance in the receptive phase compared with that in the pre-receptive phase. A significant (P < 0.05) decline was also observed in the endometrial expression of HMGB1 on the day of implantation in pregnant rats. Exogenous administration of recombinant HMGB1, on Day 3 p.c., led to pregnancy failure, whereas administration of recombinant leukemia inhibitory factor or saline had no effect on pregnant rats. Further investigations revealed morphological changes in the endometrium, an increase in the expression of luminal epithelial NFκB and significantly (P < 0.05) higher expression levels of endometrial RAGE, TNF-α and IL-6 in HMGB1-treated rats, compared with untreated pseudo-pregnant rats. LIMITATIONS, REASONS FOR CAUTION: The mechanisms, contributing to a decline in the cellular and extracellular levels of HMGB1 during the receptive phase, remain to be ascertained. WIDER IMPLICATIONS OF THE FINDINGS: An excess of HMGB1 in the UF may be associated with infertility in women.

Endometrial polyps associated with endometrial hyperplasia in an obese bonnet monkey (Macaca radiata): a case report.

BACKGROUND: A 10-year-old, female bonnet monkey (Macaca radiata) showed abnormal menstrual cycle length with heavy menstrual bleeding for 6-8 days. METHODS: Uterine ultrasound and histological examinations of endometrium by endometrial biopsy. RESULTS: An ultrasound examination of the uterine cavity showed presence of an enlarged polypoid mass. Further endometrial histology confirmed the presence of simple endometrial hyperplasia. CONCLUSIONS: We report for the first time that endometrial polyp is associated with endometrial hyperplasia in obese bonnet monkey.

Uterine secretome and its modulation in rat (Rattus norvegicus).

The present study identifies uterine fluid (UF) proteins that display differential abundance during the embryo-permissive phase in nonconception and conception cycles in rats. UF samples were collected from nonpregnant rats in the proestrous (n=17) and metestrous (n=18) phases and also from pregnant (n=17) and pseudopregnant (n=17) rats on day 4 post coitus. UF protein profile in the metestrous phase was compared with that in the proestrous phase. Similarly, UF protein profile of the pregnant rats was compared with that of the pseudopregnant rats. Two-dimensional PAGE, followed by densitometric analysis of the paired protein spots, revealed differential abundance of 44 proteins in the metestrous phase, compared with that in the proestrous phase. Of these, 29 proteins were identified by matrix-assisted laser desorption/ionization time-of-flight or liquid chromatography-tandem mass spectrometry. Functional groups such as proteases, protease inhibitors, and oxidoreductases were enriched in differentially abundant proteins. Total protease activity in UF was found to be significantly (P<0.05; t-test) higher in the proestrous phase, compared with that in the metestrous phase. Furthermore, 41 UF proteins were found to be differentially abundant in pregnant rats, compared with pseudopregnant rats. Of these, 11 proteins could be identified. Immunoblotting analysis confirmed significantly higher (P<0.05; t-test) abundance of ß-actin, Rho-specific guanine nucleotide dissociation inhibitor alpha (Rho-GDIα), and peroxiredoxin-2 and -6 in the metestrous phase, compared with that in the proestrous phase. Compared with pseudopregnant rats, pregnant rats had significantly higher (P<0.05; t-test) levels of UF ß-actin and Rho-GDIα. Furthermore, these proteins could be detected in the culture supernatants of endometrial epithelial cell lines, thereby providing an evidence of their secretion from endometrial epithelial cells. Data obtained from the study expand our knowledge on the uterine milieu that favours embryo implantation.