The Role of AKAP6 in mouse ESCs - Knock-down of AKAP6 promotes mESCs apoptosis, not differentiation.

Abstract

Background Researches focusing on Embryonic stem cells have the infinite possibility of cell based therapy for cure disease. Thus, it is important to know the regulating mechanism of cell signaling molecules in stem cells. Herein, we report the novel aspects of apoptosis in mouse embryonic stem cells, which was regulated by AKAP protein expression.

Methods and results mouse Embryonic Stem Cells (mESCs) were differentiated into Embryoid bodies by hanging drop method and their RNA harvested at 1, 4, 7 day. EB represented more higher AKAP6 expression than undifferentiated ESCs. Using shAKAP6 plasmids, transient transfection experiments and making of stable knock-down cell lines were performed. To elucidate AKAP6 effect on mESC differentiation, stable AKAP6 knock-down cell lines were formed and then, knock-down cells were differentiated into EBs. Expression level of three germ lineage markers was checked by real-time PCR. However, stemness markers and three germ layer markers didn't show any reasonable changes, although AKAP6 expression was resonably decreased in shAKAP6 cells. Form these results, we concluded that AKAP6 didn't affect mESC differentiation. But, When culturing of stable knock-down cell lines, we observed morphological differences between control and shAKAP6 cells. Staining actin filaments for clarifying cell structural differences showed disrupted actin arrangement in AKAP6 knock-down cells. After, we performed transient knock-down of AKAP6 and observed that frequent membrane ruffling occurred in AKAP6 knock-down mESCs. Membrane ruffling is widely known as migration indicator and/or apoptosis indicator. Migration signaling molecules were detected. When AKAP6 was suppressed, migratory proteins were decreased. Specially, FAK, which is generally known as anti-apoptotic factor, was decreased in AKAP6 knock-down mESCs. With these reasons, we assumed that AKAP6 knock-down induces apoptosis in mESCs. To confirm apoptotic characters, we performed Annexin V/PI FACS analysis and we detected cleaved caspase 3 expression. In AKAP6 knock-down mESCs, Annexin V/PI double positive populations were higher than control cells and also, increased cleaved caspase 3 expression was shown by immunofluorescence and westernblot analysis.

Conclusion We demonstrated the effect of AKAP6 in mouse Embryonic Stem Cells. Particularly, we explained that knock-down of AKAP6 was not a differentiation factor. Our findings proposed that knock-down of AKAP6 was close to a potential apoptotic factor. These results suggest a novel therapeutic effects of stem cells in apoptosis related disease.