Rab11a drives adhesion molecules to the surface of endometrial epithelial cells

Abstract

Is Rab11a GTPase, a regulator of intracellular trafficking, of significance in endometrial functions? Rab11a is an important component of the cascades involved in equipping the endometrial epithelium (EE) with 'adhesiveness' and 'cohesiveness'. 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. 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. 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. shRNA-mediated attenuation of Rab11a expression led to a significant (P < 0.01) decline in the surface localization of αVβ3 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 αVβ3 integrin and maintenance of E-cadherin levels at the surface of EE cells. N/A. 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. 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. 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.