P498A new role of AMP-activated protein kinase in regulating proliferation of mesenchymal stem cells

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Abstract

Purpose

It is widely accepted that cardiac regeneration needs differentiated cells to restore an optimal function. So, the use of mesenchymal stem cells (MSCs) predifferentiated in cardiomyocytes is a promising approach. It has been shown that a decrease in stem cell proliferation should favour their differentiation. The AMP-activated protein kinase (AMPK) has been largely described as a sensor of cell energy state. But AMPK also plays roles in others cell functions such as mitochondrial biogenesis, cell proliferation or cytoskeleton organisation. We firstly characterized AMPK pattern in MSCs and then, we tested the hypothesis that AMPK can promote MSC differentiation by blocking proliferation.

Methods

MSCs isolated from WT or AMPKalpha1 KO adult mouse bone marrow were stimulated with the specific AMPK activator, A-769662. The phosphorylation state of signaling elements was measured by immunoblot. Adenine nucleotides were measured by high performance liquid chromatography. Cell viability was quantified by FACS after Annexin V and Propidium Iodide staining, cell proliferation was quantified by EdU incorporation and gene expression was analysed by real-time PCR.

Results

Immunoblotting showed that A-769662 is able to stimulate AMPK signaling pathway in WT but not in KO MSCs. Chronic exposure of MSCs to hypoxia did not cause changes in AMP/ATP ratio and failed to activate AMPK. This hypoxic tolerance of MSCs could be explained by their particular glycolytic metabolism. On the other hand, the A-769662-mediated AMPK stimulation inhibited cell proliferation in WT (51 ± 6 % of inhibition, p < 0.05) but not in KO. Moreover, the absence of AMPK in KO induced a 2-fold increase of basal MSC proliferation. A-769662 also reduced the percentage of WT MSCs in S phase (6.0 ± 1.1 versus 11.2 ± 0.5 % for control, p < 0.05) whereas did not affect cell viability (92.3 ± 1.6 vs 91.8 ± 2.2 % for control). Real-time PCR and immonublotting demonstrated that none of the well-known AMPK targets, p21 and p70S6K, was involved in the A-769662-induced cell cycle arrest.

Conclusions

In conclusion, MSCs resist to hypoxia independently of AMPK. However, our results showed that a pharmacological activation of AMPK promotes a principal prerequisite for cell differentiation, the arrest of MSC proliferation, without affecting cell viability. The AMPK targets involved in this effect remains to be discovered.

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