Regulation of P38 and MKK6 kinase on heat-induced Raw264.7 cell apoptosis / 基础医学与临床

Objective To elucidate the role of P38 signaling pathway on heat-induced apoptosis in monocytic cell line Raw264.7.Methods Raw cells were transfected with constitutively active mutant MKK6b(E)and dominant negative mutant P38(AF),or the empty cloning vector pcDNA3 and apoptosis was detected by flow cytometric analysis.Results The ectopic expression of P38 mutant was confirmed by immunostaining with the antibody against the Flag-epitope tag.Expression of MKK6b(E)led to a marked increase in P38 kinase activity in transfected cells and induced a 4-fold increase in the number of apoptotic cells as compared to that in cultures of control transfected cells.Meanwhile the expression of MKK6b(E)increased the apoptotic rate of Raw cells induced by heat.In contrast,the dominant-negative mutant P38(AF)inhibited Raw cells apoptosis induced by heat.Conclusion The activation of the MKK6-P38 MAP kinase signaling pathway is required for heat-induced apoptosis in Raw264.7 cells.

Regulation of dopamine receptors on cocaine-induced MAPK signaling pathway and c-fos gene expression / 中国病理生理杂志

AIM: To investigate the role of D1 and D3 dopamine receptor on MAPK signal transduction and c-fos gene expression after acute cocaine treatment. METHODS: Activations of extracellular signal-regulated kinase (ERK), the c-Jun N-terminal kinase (JNK), p38 activation and expression of c-fos in wild type and D1 and D3 receptor mutant mice after acute cocaine treatment were checked by Western blotting. RESULTS: ERK activation and c-fos induction was enhanced in D3 mutant mice and abolished in D1 mutant mice by acute cocaine treatment, while p38 and JNK activation was not obviously modulated by the D1 and D3 receptors by acute cocaine treatment. Meanwhile, c-fos induction was inhibited when SL327, a specific MEK inhibitor, was injected before cocaine treatment. CONCLUSION: D1 and D3 receptors play opposite roles in the regulation of ERK activation and c-fos gene expression after acute cocaine treatment. The expression of c-fos gene depends on ERK signal pathway after acute cocaine treatment.

Effect of LPS on intracellular localization of p38 protein kinase in Raw264.7 cells / 中国病理生理杂志

AIM: To investigate the activation dynamics and the intracellular localization of p38 protein kinase in Raw264.7 cells after lipopolysaccharide (LPS) stimulation.METHODS: Protein kinase assay and immunogold electron microscope technique were used to check the activation dynamics and distribution of p38 MAPK in Raw264.7 cells before and after LPS stimulation. RESULTS: The kinase assay results showed that a marked increase in p38 activity was detected 15 min after LPS treatment, and reached maximal activity 30 min post stimulation, then dropped down and got closed to the pre-stimulated level 2 h later. The optimal LPS concentration for treatment was 100 ?g/L. The immunogold electron microscope data showed that p38 spread evenly in every part of the cytosol of the non-stimulated and EGF stimulated Raw264.7 cells, such as endoplasmic reticulum, mitochondria, lysosome, while the golden granules intensity in the cytosol area decreased and in the nuclear area increased significantly after LPS stimulation.CONCLUSION: p38 MAPK moves to the nuclei of Raw264.7 cells on account of stimulation by LPS.