Figure 6.

Involvement of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). (a) Catestatins induce the phosphorylation of ERK and JNK. LAD2 cells (1 × 10⁶ cells) were stimulated with 5 μm wild-type catestatin (WT), Gly364Ser (G364S), Pro370Leu (P370L), Arg374Gln (R374Q), and scrambled catestatin (sCst), or diluent 0·01% acetic acid (Ctrl, control) for 5 min, and the levels of phosphorylated ERK (p-ERK), JNK (p-JNK), and p38 (p-p38), and unphosphorylated ERK, JNK, and p38 in the cellular lysates were determined by Western blot analysis. Upper panel: representative of three separate experiments with similar results. Lower panel: Bands were quantified by densitometry using the software program Image Gauge (LAS-4000plus) to allow correction for protein loading. Data represent the ratio of the intensity of each phosphorylated protein (p-ERK, p-JNK or p-p38) divided by the amount of the respective unphosphorylated protein (ERK, JNK or p38). Values are the mean ± SD of three independent experiments. *P < 0·05 as compared between stimulated and untreated cells (Ctrl, control). (b) Inhibitory effects of ERK and JNK inhibitors. LAD2 cells (1 × 10⁶ cells) were pre-treated with 10 μm U0126, 20 μm SP600125 or 0·1% DMSO for 2 hr, and the cells were then exposed to 10 μm wild-type catestatin (WT), Gly364Ser (G364S), Pro370Leu (P370L), Arg374Gln (R374Q) or diluent 0·01% acetic acid (Ctrl, control) for 6 hr. The concentrations of granulocyte–macrophage colony-stimulating factor (GM-CSF), monocyte chemoattractant protein-1 (MCP-1/CCL2), macrophage inflammatory protein-1α (MIP-1α/CCL3) and MIP-1β/CCL4 in the culture supernatants were measured by an ELISA. Values are the mean ± SD of four separate experiments and were compared between untreated cells (Ctrl, control) and stimulated cells without inhibitor (w/o inhibitor), **P < 0·01, #P < 0·001, and between cells with the presence and absence of ERK inhibitor. *P < 0·05, **P < 0·01, #P < 0·001.