Macrophage Migration Inhibitory Factor Regulates AKT Signaling in Hypoxic Culture to Modulate Senescence of Human Mesenchymal Stem Cells

Abstract

Hypoxic culture has been shown to delay premature senescence occurring during in vitro culture. Human mesenchymal stem cells (hMSCs) cultured under hypoxia have been reported to maintain their stemness properties and delay senescence compared to the cells cultured under normoxia. However, the molecular mechanism by which hypoxia regulates premature senescence has not been fully revealed. In this study, hMSCs were cultured under the conditions of 21% (normoxia) and 1% O2 (hypoxia) tension and analyzed for cell growth, expression of MSC surface markers, multilineage differentiation, and cellular senescence. Our results showed that more cells retained MSC surface markers in hypoxic culture than those in normoxic culture, and hypoxia was able to enhance multilineage differentiation of hMSCs. The hypoxic condition also delayed cellular senescence of hMSCs, increased activation of AKT signaling, and upregulated both intra- and extracellular levels of macrophage migration inhibitory factor (MIF) compared to the normoxic condition. Inhibition of AKT activity in hypoxic culture increased the number of cells with positive staining for senescence-associated β-galactosidase activity, upregulated expression levels of senescence-associated markers p16 and p21 mRNA transcripts, and decreased expression levels of potency-associated markers, including NANOG, OCT3/4, and SOX2. On the other hand, upregulated intra- and extracellular levels of MIF by stable MIF overexpression in normoxic culture increased the activation of AKT while decreasing mRNA expression of senescence-associated markers and increasing expression of potency-associated markers. Taken together, our findings suggest that hMSCs in hypoxic culture produce endogenous MIF to activate AKT signaling to delay the progression of cellular senescence.

Role of macrophage migration inhibitory factor in age-related lung disease

“…MIF inhibits the expression of cellular senescence genes (106,152). Cellular senescence is a hallmark of aging and is characterized by cell cycle arrest, either from repetitive cell division or from accumulated damage from oxidative stress (27,116).…”

“…Cellular senescence occurs in aging-related lung disease such as idiopathic pulmonary fibrosis, lung cancer, and COPD (22,97). MIF inhibits the expression of cyclin-dependent kinase inhibitors including p21 and p16 (84,106,109,123,152). The pathological manifestations and clinical consequences of MIF's role in cellular senescence have not been thoroughly defined.…”

Effects of Human Fibroblast-Derived Extracellular Matrix on Mesenchymal Stem Cells

“…The procedure of cell culture is required for MSC expansion but unavoidably introduces factors that may change cells in culture. To tackle this issue, research strategies such as use of low oxygen [14,15] and addition of growth factors [16,17] or extracellular matrix (ECM) proteins [18] have been applied to improve culture conditions.…”

Stromal-dependent tumor promotion by MIF family members

“…These findings highlight the potential importance of MSC-derived cytokines and chemokines in malignant disease progression. Intriguingly, MIF is consistently one of the highest expressed cytokines/chemokines found in human bone marrow-, cord blood- and placental-derived MSCs [70] and hypoxia induces MIF expression and secretion beyond its already high steady state levels [71]. Hypoxia-induced MIF reportedly provides similar evasion from cell senescence in MSCs [71] as that which is observed in fibroblasts [56] although it utilizes an Akt-dependent pro-survival pathway to accomplish this as opposed to an inhibitory effect on tumor suppressor p53 [56].…”

“…Beyond that, it is tempting to speculate that MIF – and/or D-DT – may provide some functional contribution(s) to MSC differentiation processes in normal and/or malignant disease processes. Given that MIF has been found to contribute to differentiation processes in other cell types [74–76] and, in fact, regulates the expression of MSC lineage specifying transcription factors Oct3/4 and Sox2 in MSCs [71], it is not unlikely that MIF family members may participate in MSC differentiation. It should be noted that MIF participates in both epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) [77, 78] – two differentiation-like processes that serve to coordinate metastatic dissemination and distal secondary tumor growth, respectively [79].…”