Figure 9.

Upregulation of claudin 1 by TPA was significantly inhibited by the PKC inhibitor GF109203 and mediated by the ERK pathway in MCF7 cells but not in MDA-MB231 cells. (A) MCF7 cells were treated with 100 nM TPA or vehicle control (DMSO) for 18 hours. Pretreatment with the PKC α/β/μ inhibitor (Go6976, 1 μM) or the PKC δ inhibitor (Rottlerin, 5 μM) was initiated 1 hour prior to TPA treatment as indicated and continued for 19 hours. A Western blot of a representative experiment is shown, with β-actin as a loading control. In three experimental replicates, there was a consistent trend (not statistically significant, ANOVA, P=.148) towards reduced claudin 1 upregulation by TPA treatment in the presence of the inhibitors. (B) Cells were treated with 100 nM TPA or vehicle control (DMSO) for 18 hours. Pretreatment with the PKC inhibitor (GF109203x, 3 μM), the ERK inhibitor (U0126, 10 μM), or the JNK inhibitor II (20 μM) was initiated 1 hour prior to TPA treatment as indicated and continued for 19 hours. Western blots of a representative experiment are shown. Upregulation of claudin 1 in MCF7 cells was blocked by both the PKC and ERK inhibitors, but not the JNK inhibitor. (C) The relative protein levels of claudin 1 (panel B) were determined by densitometry (mean ± SE for n=3 experimental replicates). ANOVA P<.05; *P<.05 Dunnett’s multiple comparison test. (D) The upregulation of claudin 1 by TPA in the MDA-MB231 cells was not blocked by any of the inhibitors used for pre-treatment.