Abstract 10888: Microrna-143 is Critical Regulator of Cell Cycle Activity in Stem Cells with Co-expression of Akt and Angiopoietin-1 Transgenes via Transcriptional Regulation of Cyclin D1
Background: MicroRNAs (miRs) transcriptionally regulate various stem cell functions including survival, differentiation and proliferation. We report that simultaneous expression of Akt and angiopoietin-1 in MSCs (AAMSCs) supported their cell cycle progression in comparison with their adenoviral (Ad) null vector transduced counterparts (NullMSCs; controls) with a critical role for microRNA-143 (miR-143) downstream of FOXO1 transcription factor.
Methods and Results: MSCs from young male rats were successfully transduced with Ad vectors encoding for Akt and angiopoietin-1 transgenes for their simultaneous overexpression (>5-fold gene level and >3-fold Akt and angiopoietin-1 protein expression in AAMSCs vsNullMSCs). AAMSCs showed higher phosphorylation of FOXO1 which activated ERK5, a distinct mitogen induced MAPK pathway that drove transcriptional activation of Cyclin D1 and Cdk4. These molecular events increased mitogenesis in AAMSCs by more than 8% higher S-phase cell population which was confirmed by analysis of BrdU uptake by the cells (15%) and immunohistology for Ki67 (11%) in AAMSCs using NullMSCs as controls. microRNA array supported by real time PCR data showed significantly higher expression of miR-143 in AAMSCs (4.73-fold vs NullMSCs). Luciferase assay indicated a dependent relationship between miR-143 and ERK5 in MSCs. Pretreatment of cells with FOXO1 specific siRNA abrogated miR-143. On the contrary, inhibition of miR-143 did not change FOXO1 phosphorylation. However, miR-143 inhibition significantly abolished phosphorylation of ERK5 and abrogated Cyclin D1 with concomitant arrest of the cells in G0/G1 phase and reduced the number of cells entering cell cycle as determined by flow cytometry. During in vivo studies, male GFP expressing AAMSCs transplanted into the wild type female infarcted rat hearts showed significantly higher number of Ki67 expressing cells (p<0.05 vs NullMSCs) 7 days after engraftment (n= 4 animals/ group) which was substantiated by sry-gene analysis by real time PCR (n= 4 animals/group).
Conclusions: Simultaneous overexpression of Akt and angiopoietin-1 in MSCs activates cell cycle progression through upregulation of miR-143 and stimulation of signaling downstreams of FOXO1 and ERK5.
- © 2011 by American Heart Association, Inc.