RESUMO
Thymosin ß4 (Tß4), a G-actin-sequestering peptide, improves neurological outcome in rat models of neurological injury. Tissue inflammation results from neurological injury, and regulation of the inflammatory response is vital for neurological recovery. The innate immune response system, which includes the Toll-like receptor (TLR) proinflammatory signaling pathway, regulates tissue injury. We hypothesized that Tß4 regulates the TLR proinflammatory signaling pathway. Because oligodendrogenesis plays an important role in neurological recovery, we employed an in vitro primary rat embryonic cell model of oligodendrocyte progenitor cells (OPCs) and a mouse N20.1 OPC cell line to measure the effects of Tß4 on the TLR pathway. Cells were grown in the presence of Tß4, ranging from 25 to 100 ng/ml (RegeneRx Biopharmaceuticals Inc., Rockville, MD), for 4 days. Quantitative real-time PCR data demonstrated that Tß4 treatment increased expression of microRNA-146a (miR-146a), a negative regulator the TLR signaling pathway, in these two cell models. Western blot analysis showed that Tß4 treatment suppressed expression of IL-1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6), two proinflammatory cytokines of the TLR signaling pathway. Transfection of miR-146a into both primary rat embryonic OPCs and mouse N20.1 OPCs treated with Tß4 demonstrated an amplification of myelin basic protein (MBP) expression and differentiation of OPC into mature MBP-expressing oligodendrocytes. Transfection of anti-miR-146a nucleotides reversed the inhibitory effect of Tß4 on IRAK1 and TRAF6 and decreased expression of MBP. These data suggest that Tß4 suppresses the TLR proinflammatory pathway by up-regulating miR-146a.
Assuntos
Diferenciação Celular , MicroRNAs/metabolismo , Oligodendroglia/metabolismo , Transdução de Sinais , Timosina/biossíntese , Receptores Toll-Like/metabolismo , Regulação para Cima , Animais , Citocinas/genética , Citocinas/metabolismo , Células Hep G2 , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Camundongos , MicroRNAs/genética , Proteína Básica da Mielina/genética , Proteína Básica da Mielina/metabolismo , Oligodendroglia/patologia , Ratos , Ratos Wistar , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Timosina/genética , Receptores Toll-Like/genéticaRESUMO
Thymosin beta 4 (Tß4), a G-actin sequestering peptide, increases oligodendrogenesis and improves functional outcome in models of neurological injury. The molecular mechanisms of Tß4 mediated oligodendrogenesis are unclear. The p38 mitogen-activated protein kinase (p38MAPK) regulates oligodendrocyte (OL) differentiation and myelin gene expression in other models. Therefore, we investigated p38MAPK signaling pathways. We used primary rat neural progenitor cells (NPCs) and a mouse oligodendrocyte progenitor cell (OPC) line (N20.1 cells) to investigate the molecular mechanisms of Tß4-enhanced oligodendrogenesis. NPCs were isolated from rat subventricular zone (SVZ) of the lateral ventricles (n = 12). Primary NPCs and N20.1 cells were grown in the presence of 0, 25, and 50 ng/mL of Tß4 (RegeneRx Biopharmaceuticals Inc, Rockville, MD) for 14 days. Quantitative real-time PCR and Western blot data showed significant induction of both expression and phosphorylation of p38MAPK with simultaneous inhibition of phosphorylation of extracellular signal regulated kinase (ERK1), c-Jun N-terminal kinase 1 (JNK1), leading to reduction of phosphorylation of c-Jun, a potent negative regulator of transcription of myelin genes. These effects were reversed with transfection of Tß4siRNA. Our data indicate that Tß4 treatment induces OL differentiation by inducing p38MAPK with parallel inactivation of ERK1 and JNK1, thus preventing the accumulation of phosphorylated c-Jun.