The Role Of Notch-p18Axis In Hematopoietic Stem Cells

Hematopoietic stem cells (HSC) have been routinely and successfully used in the clinic in diverse applications that include congenital deficiencies and hematological malignancies. However, the mechanisms that control the self-renewal and differentiation of HSCs remain to be further investigated. Although it is known that Notch pathway is one of the critical signaling pathways that regulate the balance between self-renewal and differentiation of stem cells, the signal transduction of the Notch pathway in HSCs are less well understood. Our preliminary data showed significant downregulation of p18, a CKI (cyclin dependent kinase inhibitor) after ectopic expression of Notchl in murine LKS cells. In this research, we used DAPT-an inhibitor of Notch pathway, and3#compound-an inhibitor of p18to investigate the direct link between p18downregulation and elevated Notch signaling in HSCs. The results indicated that(1) DAPT blocks Notch signaling pathway in HSCs, resulting in the exhaustion of HSCs. After the treatment of DAPT, the expression of the cell cycle related genes CDK1, CDK2, CDK4, CDK6, p27and apoptosis related genes Bcl-2, Bcl-xl, Bax, p53,Puma in LKS cells increased, the expression of p18, p21and Bim in LKS cells decreased. HSCs at Go phase decreased and HSCs at G1phase increased. The number of apoptotic HSCs increased. The expansion capacity of HSCs decreased. It is concluded that DAPT not only enhances the exhaustion of HSCs, but also enhance the apoptosis of HSCs.(2) The p18inhibitor has no effects on cell cycle and apoptosis of HSCs. We examined the cell-cycle status and apoptosis of HSCs after the treatment of p18inhibitor. The fraction of viable cells in each phase of cell cycle remained unchanged after treatment of p18inhibitor. We also stained for the apoptotic marker, annexin V, in the cells cultured with p18inhibitor, and found no statistical difference in the apoptotic fractions between control and compound p18inhibitor groups. This suggests that cell-cycle regulation and apoptosis may not be the main reason for the increase of self-renewal of HSCs treated with p18inhibitor. (3) p18maybe an important downstream mediator in the Notch signaling pathway. A co-culture system in which enriched HSCs are cultured onto the murine stromal cell lines MS5-Deltal that have been engineered to express Notch ligands was used to gauge the effect of DAPT and p18inhibitor on HSCs. The expansion of HSCs increased after treatment with p18inhibitor and decreased after treatment with DAPT respectively, interestingly, the expansion of HSCs after treatment with p18inhibitor and DAPT was less than that with p18inhibitor alone, but more than that with DAPT alone.HES1was downregulated and p18was upregulated after treatment with DAPT. p18inhibitor did not significantly affect the expression of the genes involved in Notch signaling. LKS cells treated by DAPT and p18inhibitor had the same effect on the gene expression with that treated only by DAPT. The expression of cell cycle related genes p21, CDK1, CDK4increased after treatment with DAPT. it is concluded that p18maybe a important protein in Notch signaling pathway to affect the function of HSCs.