NFAT5 Contributes to Osmolality-Induced MCP-1 Expression in Mesothelial Cells
Figure 4
Role of NF-κB in osmolality-induced MCP-1 expression. (a) Activation of NF-κB by osmolality. Met5A cells were transiently transfected with a reporter construct in which the SEAP gene is under control of κB sites. Cells were kept in isosmotic (300 mosm/kg H2O) medium or were exposed to hyperosmotic medium (400 mosm/kg H2O). Medium osmolality was elevated by addition of glucose or NaCl. After 24 h, SEAP activity was measured as described in Section 2. Means ± SEM for ; # versus isosmotic control. (b) Phosphorylation of the p65 subunit by osmolality. Met5A cells were kept in isosmotic medium (300 mosm/kg H2O) or were exposed to hyperosmotic medium (400 mosm/kg H2O). Medium osmolality was elevated by addition of glucose or NaCl. After 16 h, cells were processed for immunoblotting as described in Section 2. Abundance of phosphorylated p65 or whole p65 was tested using specific antibodies. A representative blot of three independent experiments is shown. (c) MCP-1 secretion. Met5A cells were preincubated for 1 h with the NF-κB inhibitor Bay 11-7082 (5 μM) or with vehicle DMSO only. Cells were kept in isosmotic (gray column; 300 mosm/kg H2O) medium or were exposed to hyperosmotic medium (black column; 400 mosm/kg H2O). Medium osmolality was elevated by addition of glucose or NaCl. After 24 h, medium samples were collected and the concentration of MCP-1 in the cell culture supernatant was determined by ELISA as described in Section 2. Means ± SEM for per point; *. (d) MCP-1 transcription. Met5A cells were preincubated for 1 h with the NF-κB inhibitor Bay 11-7082 (5 μM) or with vehicle DMSO only. Cells were kept in isosmotic (gray column; 300 mosm/kg H2O) medium or were exposed to hyperosmotic medium (black column; 400 mosm/kg H2O). Medium osmolality was elevated by addition of glucose or NaCl. After 24 h, RNA was extracted from the cells and the abundance of MCP-1 mRNA transcript was determined by qRT-PCR as described in Section 2. Relative MCP-1 mRNA abundance was normalized to that of β-actin to correct for differences in RNA input. Means ± SEM for per point; *.
