MicroRNA-143 is a critical regulator of cell cycle activity in stem cells with co-overexpression of Akt and angiopoietin-1 via transcriptional regulation of Erk5/cyclin D1 signaling

We report that simultaneous expression of Akt and angiopoietin-1 (Ang-1) transgenes supported mitogenesis in stem cells with a critical role for microRNA-143 (miR-143) downstream of FoxO1 transcription factor. Mesenchymal stem cells (MSC) from young male rats were transduced with Ad-vectors encoding for Akt ((MSC)-M-Akt) and Ang-1 (Ang-1MSC) transgenes for their individual or simultaneous overexpression ((MSC)-M-AA; >5-fold gene level and >4-fold Akt and Ang-1 protein expression in (MSC)-M-AA vs. Ad-Empty transduced MSC; (MSC)-M-Emp). AAMSC had higher phosphorylation of FoxO1, which activated Erk5, a distinct mitogen-induced MAPK that drove transcriptional activation of cyclin D1 and Cdk4. Flow cytometry showed >10% higher S-phase cell population that was confirmed by BrdU assay (15%) and immunohistology for Ki67 (11%) in (MSC)-M-AA using (MSC)-M-Emp as controls. miR array supported by real-time PCR showed induction of miR-143 in (MSC)-M-AA (4.73-fold vs. (MSC)-M-Emp). Luciferase assay indicated a dependent relationship between miR-143 and Erk5 in (MSC)-M-AA. FoxO1-specific siRNA upregulated miR-143, whereas inhibition of miR-143 did not change FoxO1 activation. However, miR-143 inhibition repressed phosphorylation of Erk5 and abrogated cyclin D1 with concomitant reduction in cells entering cell cycle. During in vivo studies, male GFP+ (MSC)-M-AA transplanted into wild-type female infarcted rat hearts showed significantly higher numbers of Ki67-expressing cells (p < 0.05 vs. (MSC)-M-Emp) 7 d after engraftment (n = 4 animals/group). In conclusion, co-overexpression of Akt and Ang-1 in MSC activated cell cycle progression by upregulation of miR-143 and stimulation of FoxO1 and Erk5 signaling.