Abstract 51: Nucleostemin Induced by Pim-1 Kinase Is Critical to Maintain Pluripotency and Enhance Regenerative Potential of Cardiac Progenitor Cells

Abstract

Myocardial regeneration and repair in response to injury are governed in part by cell survival, proliferation and pluripotency. Proliferation and survival in cardiac progenitor cells (CPCs) are mediated by Pim-1, a serine threonine kinase, and nucleostemin (NS), a nucleolar stress sensor protein. The role of NS in regulating CPC pluripotency and the molecular mechanism of NS induction and action is largely unknown. The hypothesis of the study is that NS, induced by Pim-1 mediated stabilization of transcription factor c-Myc is critical to maintain CPC pluripotency and inhibits senescence. NS and c-Myc protein levels are increased in cultured CPCs overexpressing Pim-1 (3.1 and 5.5 fold, p<0.01) while knockdown of Pim-1 using sh-RNA decreases c-Myc and NS expression (-60%, -54%, p<0.05), similar to effects mediated by a Pim-1 kinase inhibitor (p<0.01), indicating that Pim-1 regulates both c-Myc and NS. c-Myc is necessary and sufficient for NS regulation, as indicated by the increase (3.1 fold, p<0.01) and decrease (-60.2%, p<0.01) in NS expression upon lentiviral mediated over-expression or knockdown of c-Myc, respectively. Regulation of NS promoter by c-Myc is evident from loss of GFP expression and fluorescence following knock down of c-Myc in CPCs isolated from transgenic mice expressing eGFP driven by the NS promoter. The role of NS in regulating CPC pluripotency is determined by silencing NS. Change in morphology (flat, round cells vs spindle shape control CPCs), decreased expression of stem cell marker c-Kit (-55%, p<0.05), up-regulation of cell cycle inhibitors p53 and p16 (4.2, 3.8 fold, p<0.01) and decreased proliferation (p<0.05) result from loss of NS in CPCs, suggestive of increased senescence and loss of pluripotency. NS-mediated regulation of CPC senescence is p53 dependent, as silencing p53 reverses CPC morphology and pluripotency lost by NS depletion. In conclusion, NS which is induced downstream of Pim-1 kinase maintains pluripotency and enhances regenerative potential in CPCs. These findings are consistent with cumulative evidence that Pim-1 induced cardiac regeneration is mediated in part by NS, providing an additional mechanistic basis for benefits of genetic engineering with Pim-1 to enhance NS expression in cardiac stem cells.