Expression of Ras related proteins in ES cell-derived AVP secreting cells
A. Kiyota1, Y. Sugimura1, H. Takagi1, K. Fukuoka1, H. Nagasaki2 & Y. Oiso1
Rab proteins (Rab GTPase) have been implicated in the trafficking and exocytosis of secretary vesicle and play a central role in the secretion of hormones or neurotransmitters. However, the involvement of Rabs and Rab effectors in the regulation of AVP secretion is still unknown. Embryonic stem (ES) cells are pluripotent cells that differentiate into all cell types. Recently, it has been reported that when dissociated ES cells are quickly reaggregeted in low cell-adhesion culture wells and cultured as floating aggregates in growth fator free chemically defined medium (SFEBq/gfCDM culture), ES cells differentiate into rostral hypothalamic progenitor cells including vasopressin (AVP) secreting cells. The aim of this study is to examine the involvement of Rab proteins in SFEBq/gfCDM cultured ES-cell aggregates (SgESa). To examine the expression of Rabs in SgESa, whole cell lysates from SgESa cultured for day 28 were subjected into SDS-PAGE and immunoblotting was performed. We confirmed that AVP was expressed in the SgESa and found that Rab3a, Rab27a, and Rabphilin3a were expressed in the SgESa. Next, AVP release upon high potassium and osmolality stimulation in SgESa on day 28 was analyzed. AVP concentrations in conditioned media were higher in high potassium- or osmolality- stimulated SgESa (incubated with high K+ artificial cerebrospinal fluid (aCSF) or high mannitol aCSF respectively) compared to control (incubated with aCSF). These results confirmed that ES cell-derived AVP secreting cells (ESAVP cells) were successfully induced. Immunocytochemical analysis of ESAVP cells using confocal laser scanning microscopy showed that rabphilin3a was mainly distributed in the subplasma membrane region, and partially colocalized with copeptin (precursor of AVP). After the high potassium and osmotic stimulation, the distribution of copeptin colocalized with rabphilin3a seemed to be enriched in plasma membrane region. Our results revealed that Rab proteins and Rab effectors may be involved in AVP release in ES-AVP cells.
Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.
Funding: This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.