Ubiquitin specific peptidase 22 regulates the transcription activity of mitogen-activated protein kinase kinase 6 gene / 中南大学学报(医学版)

To clone human mitogen-activated protein kinase kinase 6 (MKK6) gene promoter and explore its transcription activity by ubiquitin specific peptidase 22 (USP22).
 Methods: MKK6 gene promoter was amplified by PCR and two bases mutation within USP22 binding site was subsequently introduced. The wild type and mutant MKK6 promoter were inserted into the luciferase report vector pGL3-Basic, respectively. Recombinant plasmids were co-transfected with plasmid pRL-TK into HeLa cells, and the luciferase activities were measured by dual luciferase reporter system. Furthermore, the direct interaction between USP22 and MKK6 promoter was detected by chromatin immunoprecipitation (ChIP) assay. Finally, the MKK6 transcription activity was measured after knockdown of USP22.
 Results: The recombinant luciferase report vectors containing wild or mutant type of MKK6 promoter were successfully constructed. Mutation of USP22 binding site resulted in decrease of MKK6 promoter-driven luciferase activity in HeLa cells (P<0.05). USP22 could interact directly with MKK6 promoter. Down-regulation of USP22 led to the decreased MKK6 mRNA expression (P<0.05).
 Conclusion: USP22 could regulate the transcription activity of MKK6 gene in HeLa cells.

Regulation of P38 and MKK6 on HMGB1 expression in alveolar macrophages induced by cyclic mechanical stretch / 生理学报

The aim of the present study was to investigate the role of mitogen-activated protein kinase kinase 6 (MKK6)-P38 signaling pathway in cyclic mechanical stretch-induced high mobility group box 1 protein (HMGB1) expression in alveolar macrophages. In the study, Sprague-Dawley rats were anesthetized and then sacrificed by bloodletting. The lungs were lavaged six times with prechilled PBS. Alveolar macrophages were isolated from lavage samples. Recombinant plasmids were transfected into alveolar macrophages with liposome DOTAP. Alveolar macrophages transfected with P38(AF)/pGFP and MKK6b(E)/pGFP plasmids were taken as treated groups, while the groups that transfected with pcDNA3 plasmid and pGFP plasmid served as blank transfection group and control group, respectively. All the groups were then cultured in 6-well Bioflex cell culture plates and exposed to cyclic mechanical stretch at 20% elongation using Flexercell 4000T cell stretching unit. The results showed that the transfection of MKK6b(E) led to a marked increases in P38 kinase activity compared with control group. In contrast, the transfection of P38(AF) significantly inhibited P38 kinase activity. Compared with control group, HMGB1 protein and mRNA expression in MKK6b(E) transfected cells increased markedly, while HMGB1 expression in P38(AF) transfected cells decreased markedly. These results suggest that MKK6-P38 MAPK signaling pathway regulates the expression of HMGB1 induced by cyclic mechanical stretch in alveolar macrophages.

Exposure to power-frequency magnetic fields can induce activation of P38 mitogen-activated protein kinase / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study the effects of 50 Hz power-frequency magnetic fields on signal transduction pathway of P38 mitogen-activated protein kinase (P38 MAPK), and explore the cellular signal transduction mechanism of the biological effects induced by power-frequency magnetic fields.</p><p><b>METHODS</b>Chinese hamster lung (CHL) cell line was exposed to power-frequency magnetic fields with two intensities(0.1 and 0.4 mT) for different exposure durations. The cytoplasmic protein was extracted. The phosphorylated(activated) and non-phosphorylated P38 MAPK and MKK3/MKK6 were measured by Western blotting analysis with their specific corresponding antibodies.</p><p><b>RESULTS</b>Power-frequency magnetic fields at 0.4 mT for 10 min could transitorily induce the activation of P38 MAPK and after 15 min the phosphorylation of P38 MAPK restored to control level, while 0.1 mT power-frequency magnetic fields could not induce the activation of P38 MAPK within 24 h. However, both 0.1 mT and 0.4 mT power-frequency magnetic fields could not phosphorylate(activate) the MKK3/MKK6, which is a general upstream kinase of P38 MAPK.</p><p><b>CONCLUSION</b>Power-frequency magnetic fields could transitorily activate the P38 MAPK, but not MKK3/MKK6. The activation mechanism of P38 MAPK needs to be further identified.</p>