Figure 4
HKLM activates the MAP kinase signaling pathway. THP-1 cells were treated with HKLM for different time points and cell lysates were prepared as described in methods. Samples were run on denaturing gels. Phosphorylated MEK1/2, ERK1/2, JNK, p-38 and c-Jun are depicted in the upper panels and total respective proteins are shown in the lower panels. As shown, HKLM treatment increases the phosphorylation of MEK1/2, ERK1/2, JNK, p38 and c-Jun in a time-dependent manner (A). THP-1 cells were pretreated with MEK-ERK inhibitors (U0126: 10 μM for 2 hr); PD98059: 10 μM for 1 hr) or JNK inhibitor (SP600125: 20 μM for 30 min) or p38 inhibitor (SB203580: 10 μM for 1 hr) and then treated with HKLM (3x107/ml) for 24 hr. Cells and supernatants were collected. Cells were used for the isolation of total RNA and MMP-9 mRNA was assessed by real-time RT-PCR (B). Secreted levels of MMP-9 protein were determined in supernatants by ELISA (C). The results obtained from three independent experiments are shown. The data are presented as mean ± SE. An asterisk (*) represents P-value of <0.05.