Mitogen-activated protein kinase kinase 6 mediates mechanical stretch-induced high-mobility group box 1 protein expression in pulmonary alveolar epithelial cells

Recent studies have demonstrated that high-mobility group box 1 protein (HMGB1) plays an important role in the development of ventilator-induced lung injury. However, the molecular mechanisms that are involved in this process are poorly understood. The aim of this study was to explore the role of mitogen-activated protein kinase kinase 6 (MKK6) in the HMGB1 expression in pulmonary alveolar epithelial cells induced by mechanical stretch. Pulmonary alveolar epithelial cells (A549 cell line) were divided into five groups based on adenoviral infection, including control group, empty adenovirus vector group, wild-type MKK6 group, constitutively active mutant MKK6(E) group, and dominant-negative mutant MKK6(A) group. Each group was then subdivided into stretched and unstretched groups. The expression of transfected genes was detected by fluorescence microscopy and western blotting. MKK6 activity was measured using a kinase activity assay. The expression levels of HMGB1 mRNA and protein were measured by reverse transcription polymerase chain reaction and western blotting, respectively. Cytokines were investigated using the LiquiChip system. Mechanical stretch significantly enhanced MKK6 activity and HMGB1 protein expression in A549 cells. Transfection with adenoviral MKK6(E) also led to a statistically significant increase in HMGB1 expression level, whereas the introduction of MKK6(A) interfered with stretch-induced HMGB1 expression. We also found that the level of HMGB1 expression was positively correlated with cytokine abundance. Mechanical stretch can induce HMGB1 and cytokine expression in A549 cells by activating MKK6.