Macrophage migration inhibitory factor confers resistance to senescence through CD74-dependent AMPK-FOXO3a signaling in mesenchymal stem cells

Introduction: Mesenchymal stem cells (MSCs)-based therapies have had positive outcomes in animal models of cardiovascular diseases. However, the number and function of MSCs decline with age, reducing their ability to contribute to endogenous injury repair. The potential of stem cells to restore damaged tissue in older individuals can be improved by specific pretreatment aimed at delaying senescence and improving their regenerative properties. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that modulates age-related signaling pathways, and hence is a good candidate for rejuvenative function. Methods: Bone marrow-derived mesenchymal stem cells (BM-MSCs) were isolated from young (6-month-old) or aged (24-month-old) male donor rats. Cell proliferation was measured using the CCK8 cell proliferation assay; secretion of VEGF, bFGF, HGF, and IGF was assessed by RT-qPCR and ELISA. Apoptosis was induced by hypoxia and serum deprivation (hypoxia/SD) for up to 6 hr, and examined by flow cytometry. Expression levels of AMP-activated protein kinase (AMPK) and forkhead box class O 3a (FOXO3a) were detected by Western blotting. CD74 expression was assayed using RT-qPCR, Western blotting, and immunofluorescence. Results: In this study, we found that MSCs isolated from the bone marrow of aged rats displayed reduced proliferative capacity, impaired ability to mediate paracrine signaling, and lower resistance to hypoxia/serum deprivation-induced apoptosis, when compared to younger MSCs. Interestingly, pretreatment of aged MSCs with MIF enhanced their growth, paracrine function and survival. We detected enhanced secretion of VEGF, bFGF, HGF, and IGF from MIF-treated MSCs using ELISA. Finally, we show that hypoxia/serum deprivation-induced apoptosis is inhibited in aged MSCs following MIF exposure. Next, we found that the mechanism underlying the rejuvenating function of MIF involves increased CD74-dependent phosphorylation of AMPK and FOXO3a. Furthermore, this effect was abolished when CD74, AMPK, or FOXO3a expression was silenced using small-interfering RNAs(siRNA). Conclusions: MIF can rejuvenate MSCs from a state of age-induced senescence by interacting with CD74 and subsequently activating AMPK-FOXO3a signaling pathways. Pretreatment of MSCs with MIF may have important therapeutic implications in restoration or rejuvenation of endogenous bone marrow-MSCs in aged individuals.