Upregulation of RelB in the miR-122 knockout mice contributes to increased levels of proinflammatory chemokines/cytokines in the liver and macrophages.

OBJECTIVE: MicroRNA-122 (miR-122) is the most abundant miRNA in the liver and it plays an important role in regulating liver metabolism and tumor formation. Previous studies also reveal an anti-inflammatory function of miR-122; however, relatively little is known about the mechanisms by which miR-122 suppresses inflammation. This study aims to search the effect of miR-122 on proinflammatory chemokines/cytokines production in mice. METHODS: Quantitative real-time PCR, Western blot analysis, and ELISA were performed to examine gene expression. TargetScan, miRanda, and microT v3.0 were used to search for possible miR-122 target sites in the 3'-untranslated regions (3'-UTR) of candidate genes. Luciferase reporter assay and site-directed mutagenesis were applied to verify miR-122 target sequences. LPS was applied to peritoneal macrophages and mice to evaluate inflammatory response. RESULTS: The expression of proinflammatory chemokines, including Ccl2, Ccl4, Ccl20, Cxcl2, and Cxcl10, and Relb in the livers of miR-122 knockout (KO) mice was increased. We identified Relb as a direct miR-122 target. Overexpressing RelB in the mouse liver increased the expression of Ccl2, Ccl4, Ccl20, Cxcl2, and Cxcl10. Peritoneal macrophages from miR-122 KO mice had a higher level of RelB, and they showed a stronger NF-κB activation and more TNF-α and IL-6 secretion after LPS stimulation. Overexpression of RelB in a macrophage cell line augmented LPS-induced TNF-α and IL-6 production. miR-122 KO mice showed a greatly increased mortality rate and generated a stronger and lasting inflammatory response to LPS. CONCLUSIONS: Deletion of miR-122 caused an upregulation of proinflammatory chemokines and RelB in the liver. Increased RelB may contribute to increases in these chemokine in the liver. Intriguingly, deletion of miR-122 also enhanced the sensitivity of macrophages and mice to LPS. Our results reveal that reducing RelB expression is a new mechanism by which miR-122 regulates inflammation.

Fibronectin and pellet suspension culture promote differentiation of human mesenchymal stem cells into insulin producing cells.

Multipotential mesenchymal stem cells (MSCs) isolated from bone marrow can differentiate into multiple mesenchymal tissues and exhibit a neuronal phenotype under appropriate induction conditions. Methods promoting neural differentiation have been adapted to derive insulin producing cells (IPCs) from embryonic stem cells, but it remains unclear whether neuronal cell-based differentiation method will be able to derive IPCs from MSCs. Using a four-stage differentiation protocol which contains neuronal differentiation factor and IPC-conversion reagent-nicotinamide, the potential of human MSCs to differentiate into IPCs was evaluated by means of reverse transcription-polymerase chain reaction, immunostaining, and functional analysis. MSCs in monolayer spontaneously expressed genes for islet transcription factors, Nkx6.1 and Ngn3, but did not express insulin after treatment in this protocol. Pellet suspension culture and the addition of fibronectin enhanced pancreatic differentiation with increase in insulin and Glut2 gene expression. Switching of cells to high-glucose culture further increased immunostaining for proinsulin and insulin. IPCs secreted insulin in response to elevated glucose concentration, which was regulated by reagents that increase cyclic AMP production or modify calcium influx. Our data suggest that MSCs in the monolayer do not undergo IPC differentiation and pellet suspension culture with fibronectin promotes IPCs derived from MSCs.

Sodium butyrate activates ERK to regulate differentiation of mesenchymal stem cells.

Histone deacetylase inhibitors such as sodium butyrate are known to regulate the differentiation of a variety of cells. Mesenchymal stem cells (MSCs) differentiate into osteoblasts and adipocytes under transcriptional control of Runx2 and PPARgamma2, respectively. How these two transcription factors are regulated by sodium butyrate in order to specify the alternate cell fates remains a pivotal question. Sodium butyrate stimulated osteogenic differentiation and increased expression of Runx2 and genes regulated by Runx2 when cells were induced to undergo osteogenic differentiation. Sodium butyrate suppressed the adipogenic differentiation and decreased the expression of PPARgamma2 and LPL when MSCs were treated under conditions that promote adipogenic differentiation. Sodium butyrate also decreased the ratio of RANKL/OPG gene expression by MSCs. Analysis of MSCs induced in the presence of sodium butyrate revealed an immediate increase in ERK phosphorylation by sodium butyrate. The MEK-specific inhibitor, PD98059 but not p38- or JNK-specific inhibitor and the transfection with dominant negative ERK expressing plasmids blocked the sodium butyrate-induced regulation of MSC differentiation and increase in the RANKL/OPG ratio. Our results suggest that sodium butyrate modulates MSC differentiation and the RANKL/OPG ratio via activating ERK, and could be applied for in vivo bone growth using MSCs.