NFAT5 Contributes to Osmolality-Induced MCP-1 Expression in Mesothelial Cells
Figure 2
Expression and activation of NFAT5 in Met5A cells. 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, NaCl, or mannitol as indicated. (a) Cells were incubated for 24 h and subsequently processed for immunoblotting as described in Section 2. To demonstrate comparable protein loading, the blots were also probed for actin. A representative blot from 3 independent experiments is shown. (b) Relative NFAT5 protein abundance was quantified by densitometric analysis of immunoblots and normalized to that of actin to correct for differences in protein loading. Means ± SEM for ; * versus isosmotic control. (c) Cells were incubated for 16 h. Thereafter, RNA was extracted and the abundance of MCP-1 mRNA transcript 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; * versus isosmotic control. (d) Cells were incubated for 1 h, and subsequently cytoplasmic and nuclear extracts prepared and processed for immunoblotting as described in Section 2. To demonstrate purity of extracts and comparable protein loading, the blots were also probed for histone H1 and actin. A representative blot from 4 independent experiments is shown. (e) Relative NFAT5 nuclear versus cytoplasmic abundance was quantified by densitometric analysis of immunoblots. Means ± SEM for ; * versus isosmotic control. (f) Activity of the transactivation domain of NFAT5 during osmotic stress. Met5A cells were cotransfected with a vector encoding the fusion protein GAL4dbd-TonEBP-TAD (amino acids 548-1541 of NFAT5 fused to the yeast GAL4 DNA binding domain) together with the reporter vector pFR-SEAP. Cells were kept in isosmotic medium (300 mosm/kg H2O) or were exposed to hyperosmotic medium (400 mosm/kg H2O). After 48 h, SEAP activity was measured as described in Section 2. Means ± SEM for ; * versus isosmotic control. (g) Met5A cells were transiently transfected with a reporter construct in which the SEAP gene is under control of two TonE sites. Cells were kept in isosmotic (300 mosm/kg H2O) medium or were exposed to hyperosmotic medium, with osmolalities between 325 and 550 mosm/kg H2O as indicated. After 24 h, SEAP activity was measured as described in Section 2. Means ± SEM for ; * versus isosmotic control.
