cAMP Agonist Forskolin Disrupts Mitochondrial Metabolism and Induces Senescence in Human Mesenchymal Cells

Abstract Backgroud: Adult-derived mesenchymal stem cells (MSCs) can be used in therapies for the treatment of various diseases. However, MSCs derived from aging tissues or long-term MSC cultures could have diminished therapeutic effects compared with MSCs derived from younger tissues, but the underlying mechanism has not been completely established. Dysfunction of energy metabolism is one of the main mechanisms underlying cell senescence. Although cyclic adenosine monophosphate (cAMP) is known to inhibit cell division and proliferation in vitro, its impact on MSC senescence has not been described. Methods: In this study, we used forskolin, an adenylate cyclase agonist and cAMP inducer, to disrupt metabolism in human adipose-derived MSCs and investigate the effects of metabolic dysfunction on MSC senescence. cAMP and ATP levels were identified by its ELISA kits. The identification of MSCs senescence was assessed by MTS assay, cell cycle tests, β-galactosidase staining, and genes evaluation through qRT-PCR and Western blotting. Functions of the forskolin treated-MSCs were evaluated by oil Red O staining and alkaline phosphatase staining, as well as mRNA tests and wound healing assay. Mitochondrial morphology and function tests were performed by transmission electron microscopy and oxygen consumption analysis, mitochondrial superoxide assay, quantification of nitric oxide.Results: Treatment of human MSCs with forskolin resulted in senescence phenotypes, including reduced proliferation, cell-cycle arrest, and enhanced expression of the cell aging markers P16 and P21. Furthermore, the senescent MSCs exhibited increased adipogenesis capacity and decreased osteogenesis capacity as well as a senescence-associated secretory phenotype characterized by increased expression of several inflammatory factors. Forskolin-associated MSC senescence was mainly caused by oxidative stress–induced disruption of mitochondrial metabolism, and the senescent MSCs had high levels of reactive oxygen species and reduced sirtuin gene expression. Lastly, we found that cAMP inhibitor SQ22536 protects MSCs from forskolin-induced senescence and senescence-related-inflammatory-phenotype (SASP). Conclusions: Our results indicate that forskolin can cause senescence of human MSCs through oxidative stress–induced mitochondrial metabolic dysfunction.