The Effects Of MAPKs On The Expression Of IL-12 In NOD Mice And Macrophage Development In C57 Mice

BACKGROUNDThe MAP kinase cascade is one of the most ancient and evolutionarily conserved signaling pathways, which is also important for many processes in immune responses. There are three major groups of MAP Kinases in mammalian cells—the extracellular signal-regulated protein kinases (ERK), the c-Jun NH2-terminal kinase (JNK)/stress-avtivated protein kinases (SAPK), and the p38 MAP kinases. These MAP kinases are activated by dual phosphorylation at the tripeptide motif Thr-Xaa-Tyr. The sequence of this tripeptide motif is different in each group of MAP kinases: ERK(Thr-Glu-Tyr), JNK(Thr-Pro-Tyr), and p38(Thr-Gly-Tyr). The dual phosphorylation of Thr and Tyr is mediated by a conserved protein kinase cascade. The ERK MAP kinases are avtivated by the MAP kinase kinases(MKK) MEK1 and MEK2; the JNK pathway is activated by MKK4 and MKK7; and the p38MAP kinases are activated by MKK3, MKK4, and MKK6. These MAP kinase kinases are activated, in turn, by several different MAP kinase kinase kinases(MKKK). Different stimuli can lead to the activation of these MKKK. The MAP kinase signal transduction pathway activates downstream substrates by the conserved three grades cascades responses. MAPK is a hinge in the signal transduction pathway. Activated MAPKs regulate cellular proliferation, differentiation, transformation, and apoptosis by phosphorylating nuclear transcription factors, scaffold proteins and enzymes. MAPKs have intimate relations with inflammatory, tumor and other diseases.OBJECTIVES1: To explore the relations of MAPKs activation and the expression of IL-12 in NOD mice.2: To explore the effects of cAMP on M-CSF-induced MAPKs activation and on macrophage development.METHODS1: The relationship between MAPKs signal pathway and IL-12's abnormal expression was investigated in macrophages from NOD mice with type1 diabetes mellitus (T1DM). The exact molecular mechanism of MAPKs'abnormal activation was further explored in T1DM. Bone marrow-derived murine macrophages (BMMs) were got from NOD mice and C57 mice(control), respectively, stained with Giemsa, then compared with different morphology under microscope. The level of IL-12 was measured with ELISA. The level was measured after pretreated with MAPKs'inhibitors again. Finally, MAPKs'activation was detected in different stimulating factors with Western blot. Through comparing the different activation of MAPKs in NOD mice and C57 mice, new ideas were provided for the treatment of T1DM.2: Observation under microscope of the morphology of bone marrow-derived murine macrophages; Western blot analysis of the effects of M-CSF on the activation of MAPKs; Western blot analysis of the effects of cAMP on M-CSF-induced MAPKs activation; FACS analysis of the effect of cAMP on F4/80 expression during macrophage development.RESULTS1: The morphology of NOD BMMs was different from that of C57 BMMs with Giemsa staining; ELISA analysis showed that the expression of IL-12 was increase in NOD. With the inhibitor of p38 MAPK, the expression was decrease; Western blot analysis displayed that the abnormal activation of p38 MAPK maybe was related with the level of MAPKKK in NOD BMMs.2: Macrophage morphology showed the successful establishment of the model of macrophage development; Western blot analysis displayed that M-CSF activated ERK,JNK and p38 in both mature and immature macrophages, cAMP inhibited M-CSF-induced ERK,JNK and p38 activation in a time-dependent manner; FACS analysis revealed that macrophage development was impaired upon cAMP pretreatment. CONCLUSIONSp38 MAP Kinase abnormal avtivation induces to produce an overabundance of IL-12 in NOD mice. The matter maybe is related with abnormal avtivation in the level of MAPKKK; cAMP modulates macrophage development by suppressing M-CSF-induced MAPKs activation.