Nur77 and Nurr1, Two Novel Players In MSC Migration and Immune Function.

Abstract 3854

Mesenchymal stromal cells (MSC) have a promising potential for cellular therapies, based on their immunomodulatory functions and their differentiation capacity. However, it remains unclear how MSC exert their functions and especially homing towards effector sites is poorly understood. An imperative for future therapies is to understand the process of MSC migration. We have previously shown that only 10–20% of culture-expanded MSC is capable to migrate in vitro, but these migratory cells can not be discriminated by markers. To identify genes specific for migratory MSC, we performed a gene expression study. Using a Transwell system (12mm pore size), migrating and non-migrating fetal bone marrow MSC (FBMSC) were physically separated and RNA was extracted. MSC that were exposed to a SDF-1 gradient (0.4mm pore size) and cultured FBMSC were included as controls. Only 9 genes were differentially expressed between migratory and non-migratory MSC. This set did not include cell surface markers that enable selection for a migratory subset. The nuclear orphan receptor family members Nurr1 and Nur77 were 2-fold upregulated in migratory MSC. These results were confirmed by RQ-PCR. Nurr1 and Nur77 are members of the NR4A nuclear orphan receptor family and were first described as early response transcription factors upon growth factor stimulation. We observed that the chemokine SDF-1 and the growth factor PDGF-BB increased the expression of Nur77 and Nurr1 in MSC. To investigate the role of Nur77 and Nurr1 in migration, FBMSC were transduced with lentivirus to achieve overexpression. Both overexpression of Nur77 and Nurr1 increased specific migration towards SDF-1 compared to Mock transduced FBMSC (n=4) (Nur77 mean 297% ± 186 relative to Mock, p≤ 0.014; Nurr1 mean 227% ± 79, p≤ 0.014). We previously described that cell cycle influences MSC migration, with S- and G2/M-phase of the cell cycle negatively influencing migration. In MSC cultures overexpressing Nur77 or Nurr1, the percentage of cells in S-phase of the cell cyle was significantly reduced (Nur77 15.2±7.8%, p≤0.007; Nurr1 16.4±9.1%, p≤0.039) compared to Mock-transduced cells (24.0±8.9%). Possibly, part of the Nur77 and Nurr1 effect is regulated by an effect on cell cycle. Together these data suggest that Nur77 and Nurr1 are involved in migration of MSC, which offers perspectives to modulate migration of MSC. Because these nuclear orphan receptors were not implied in MSC biology to date, we study their immunomodulatory effect. NR4A genes modulate immune responses in a variety of cells by influencing cytokine production. The immunomodulatory effect of MSC also seems to be based on secretion of a wide variety of cytokines, chemokines and growth factors. We therefore investigated whether Nur77 and Nurr1 play a role in cytokine and growth factor production in MSC. Compared to Mock transduced MSC, overexpression of Nur77 and Nurr1 in MSC resulted in increased basal levels of IL-6 protein (Nur77 3.4±2.4 fold, p≤0.007; Nurr1 3.8±3.1 ns) and IL-8 protein (Nur77 5.9±3.3 fold, p≤0.046; Nurr1 4.7±2.2, p≤0.031) and HGF mRNA expression (Nur77 5.4±0.8 fold, p≤0.021; Nurr1 3.4±1.18, p≤0.026). This was further enhanced upon stimulation with TNFα for 24hrs relative to the unstimulated Mock (IL-6 Mock 22.9±19.5 fold; Nur77 35.1±21.9 fold; Nurr1 35.8±24.1 fold; IL-8 Mock 58±37 fold; Nur77 160±141 fold; Nurr1 262±181 fold). HGF mRNA levels were slightly elevated by stimulation with IFNγ for 24hrs (Mock 1.8±1.6 fold; Nur77 7.0±3.8 fold; Nurr1 7.1±1.7 fold; ns). Expression levels of IDO1 and TGF-ß, also involved in immune modulation by MSC, were not influenced by Nur77 or Nurr1. In conclusion, we identified two novel genes involved in MSC migration. Intriguingly these genes are also involved in cytokine production, suggesting that the fraction with the best migratory capacity is able to modulate the immune response at its effector site distinct from the bulk of culture expanded MSC that lack migratory capacity. Modulation of Nur77 and Nurr1 expression in MSC could therefore offer perspectives to ameliorate cellular therapy.