RING Finger Protein10Regulates Cell Cycle Exit And Neuronal Differentiation Of Stem Cells

Rnf10is a member of the RING finger protein family. Recently, a number ofRING finger proteins were reported to be involved in neuronal differentiation,development and proliferation. Rnf10contain C3HC4type RING finger domain thatis known to involve in protein-protein interaction, DNA binding and metal ionbinding, however specific function of Rnf10is still under investigation. Previouslypublished data showed that Rnf10can bind to promoter of myelin-associatedglycoprotein (MAG) gene and contribute to myelin production of Schwann cells(peripheral nerve cells). Rnf10also have protein binding ability and bind withtranscription factor Meox2between histidine/glutamine rich domain and thehomeodomain. Together with Meox2it can enhance the transcription of p21WAF1promoter in NIHT3cell line. To determine Rnf10importance in central nervoussystem we examined its protein expression in different brain regions and in cerebellarneurons and glial cells. Rnf10is extensively expressed in brain regions, neurons andglial cells. In glia Rnf10express in whole cell but in neurons it is restricted to cellbody only and does not express in specialized structures. This differential expressionshowed that Rnf10may have generalized as well as specific function. Moreover, toinvestigate Rnf10role in terminal differentiation we observed its expression inundifferentiated and differentiated P19cells. The mRNA levels and proteinexpression of Rnf10increase significantly upon the retinoic acid-induced neuronaldifferentiation of P19cells. Knockdown of Rnf10by RNA interference significantlyimpaired neuronal differentiation of P19cells by attenuating the expression ofneuronal markers. Cell cycle profiling revealed that Rnf10-depleted cells were unableto establish cell cycle arrest at G2phase after RA treatment. In agreement with flowcytometry analysis, increased cell proliferation was observed after RA induction inRnf10knockdown cells as determined by a BrdU incorporation assay. Furthermore,we also determined the mRNA levels and protein expression of p21, p27and p57during RA induced neuronal differentiation. Expression of p21and p27was increasedupon differentiation signal while p57expression remained unchanged. However,Rnf10knockdown only resulted in a reduction of p21expression, while p27and p57expression remained the same, indicating that Rnf10regulate cell cycle exit throughthe p21pathway. In order to establish the fact that reduce expression of p21cyclin kinase inhibitor also effect neuronal differentiation of RA induced P19cells weknockdown p21protein. Similar to Rnf10inhibition, p21knockdown also results inimpaired neuronal differentiation of P19cells. Ectopic p21expression partiallyrescued the effect of Rnf10depletion on the neuronal differentiation of P19cells.Collectively, these results showed that increase in Rnf10expression upon RAinduction is necessary for the positive regulation of cyclin kinase inhibitor p21expression, which leads to cell cycle arrest and is critical for neuronal differentiation.In addition to Rnf10study, we have also worked on chemical preconditioningof neural stem cells to propose a potential approach for cell transplantation in stroke.A substantial loss of transplanted neural stem cells is a major limitation to celltransplantation therapy of stroke. In this study, we provided in vitro evidence thatdoxycycline preconditioning of neural stem cells have resulted in decreased cell deathand increased cell viability after oxygen-glucose deprivation-reoxygenationconditions that best mimics cerebral ischemia-reperfusion injury. Resistance tooxidative stress is one of the mechanisms of doxycycline-induced cytoprotection inneural stem cells as it significantly reduced the superoxide anion production.Moreover, doxycycline preconditioning also induced the expression of Nrf2which isa basic transcription factor for a series of antioxidative and cytoprotective genes.Collectively, we suggested that doxcycline preconditioning of neural stem cells is apotential strategy to improve effectiveness of cell transplantation therapy.