Inhibition of the EGFR prevents TNF-α-induced ERK activation. A and B, LLC-PK₁ (A) or MDCK (B) cells were grown to confluence and serum-depleted for 3 h before the treatments. The cells were pretreated with 100 nm AG1478 for 30 min in serum-free DMEM followed by the addition of 10 ng/ml TNF-α for 2 or 10 min (A) or 10 min (B) or 100 ng/ml EGF for 10 min as indicated. Where used, the inhibitor was present throughout the TNF-α or EGF treatment. At the end of the treatments the cells were lysed, and pERK and ERK were detected using Western blotting. C, MDCK cells were transfected with NR siRNA or an siRNA against canine EGFR. Forty-eight hours after transfection the cells were serum-depleted and then treated with 10 ng/ml TNF-α (5 min) or 100 ng/ml EGF (10 min). ERK and pERK were detected as above. The graphs below the blots summarize densitometric analysis of the pERK blots (see “Experimental Procedures”). Data presented are the mean ± S.E. of n = 3 experiments. D and E, LLC-PK₁ cells were transfected with human EGFR. 48 h later the cells were serum-depleted and treated with 10 ng/ml TNF-α (15 min) or 10 ng/ml EGF (5 min). At the end of the treatment cells were lysed and immunoprecipitated (IP) using EGFR (D) or phosphotyrosine (PY) (E) antibody as described under “Experimental Procedures.” The immunoprecipitated proteins and samples of the total cell lysates were analyzed by Western blotting (WB) using Tyr(P) and EGFR antibodies as indicated. In D the transfected EGFR in the total cell lysates was also visualized. The EGFR antibody in the total cell lysates and precipitates from untransfected cells yielded only a marginal signal (not shown).

Inhibition of EGFR prevents TNF-α-induced GEF-H1 and RhoA activation. A, confluent LLC-PK₁ cells were serum-depleted, then pretreated with 100 nm AG1478 (AG, 30 min) in serum-free DMEM followed by the addition of 10 ng/ml TNF-α for 10 min. Active GEFs were precipitated using GST-RhoA(G17A). GEF-H1 in the precipitates and total cell lysates (active and total, respectively) was detected by Western blotting. B, MDCK cells were transfected with NR siRNA or EGFR-specific siRNA. Forty-eight hours after transfection cells were treated with 10 ng/ml TNF-α (10 min), and active RhoA was captured using Rhotekin GST-RBD. RhoA in the precipitates (active) and total cell lysates was detected by Western blotting. The total cell lysates were redeveloped using an EGFR antibody. The blots were quantified by densitometry (see “Experimental Procedures”). The graphs below the blots show the mean ± S.E. from n = 3 independent experiments.

EGF activates the ERK/GEF-H1/RhoA pathway. A–D, LLC-PK₁ cells were serum-depleted for 3 h. In C, 48 h before the experiment, cells were transfected using NR or GEF-H1-specific siRNA. The cells were exposed to 10 ng/ml EGF for the indicated times (A) or 1 min (B) or 10 min (D) or 2 min (C). In D, cells were treated with 10 μm PD98059 before and during stimulation with EGF. At the end of the treatments cells were lysed, and the amount of active RhoA (A, C, and D) or GEF-H1 (B) was detected and analyzed as described earlier. The graphs below the blots show data from the densitometric analysis (mean ± S.E. n = 3). E, LLC-PK₁ cells were serum-depleted and treated using 10 ng/ml TNF-α (10 min) or 100 ng/ml EGF (10 min) or 10 ng/ml HGF (30 min). Cells were lysed, and active GEFs were precipitated using RhoA(G17A) as in B. Vav2 in the precipitates or cell lysates (active and total, respectively) were detected by Western blotting. The total cell lysates were redeveloped to detect pERK and ERK levels.

The TACE inhibitor TAPI-1 prevents TNF-α-induced ERK and RhoA activation. LLC-PK₁ cells were treated for 15 min with 10 μm TAPI-1 before the addition of TNF-α or EGF (10 ng/ml, 10 min). pERK (A) or active RhoA (B) levels were detected and quantified as described earlier. In A the maximal effect (EGF-induced pERK) was taken as 100%, as the control (untreated) values in some experiments were too close to the background.

Src kinase mediates TNF-α-induced ERK, GEF-H1, and RhoA activation. A, D, and E, LLC-PK₁ cells were pretreated with 10 μm Src family inhibitor PP2 (15 min) followed by 10 ng/ml TNF-α for 5 min. The inhibitor was present during the TNF-α treatment. Levels of pERK (A), RhoA (D), or GEF-H1 (E) were determined as described earlier. B and C, LLC-PK₁ cells were transfected with HA-tagged ERK2 with or without dominant negative Src kinase (B) or active Src kinase (C). Forty-eight hours after transfection the cells were treated with TNF-α (10 ng/ml, 10 min) as indicated. In C, cells were pretreated for 1 h with TAPI-1 as indicated before the addition of TNF-α. At the end of the treatment the cells were lysed, and HA-ERK2 was immunoprecipitated (IP). Phospho-ERK was detected in the precipitates. The blots were stripped and redeveloped using an HA-antibody to detect precipitated HA-ERK2. In B, the transfected HA-ERK2 and avian dominant negative Src were also detected in the total cell lysates. Please note that the avian Src antibody does not react with mammalian Src. In the experiments shown on C neither the transfected HA-ERK nor the avian Src was detectable in the total cell lysates (not shown). No signal was visible when untransfected cell lysates are used (non). The graph in B shows densitometric analysis done as described under “Experimental Procedure” (mean ± S.E. n = 3).

A and B, TNF-α-induced Src activation is upstream of the EGFR and TACE. LLC-PK₁ cells were treated with 100 nm AG1478 (A) or 10 μm TAPI-1 (B) before the addition of 10 ng/ml TNF-α for the indicated times. The cell lysates were probed using a phospho-SFK antibody. This antibody detects all Src family kinases phosphorylated at Tyr-416 (corresponding to activated SFK). The graph summarizes densitometric quantification (mean ± S.E. of n = 3 experiments). Actin was used to verify equal loading, as the total SFK antibody did not react with Src kinases in LLC-PK₁ cells. C, EGF-induced ERK activation is independent of Src kinases. LLC-PK₁ and MDCK cells were treated with PP2 and EGF as indicated, and pERK was detected as described earlier. D–F, TNF-α-induced EGFR activation requires Src and TACE. LLC-PK₁ cells were transfected with EGFR. Cells were treated as indicated. EGFR was immunoprecipitated (IP), and phosphotyrosine (PY) and EGFR were detected as in Fig. 1. The graph in F shows densitometric analysis of three independent experiments, performed as described under “Experimental Procedures.” In each experiment the normalized phosphorylation was expressed as -fold increase from the corresponding untreated sample or the sample treated with TAPI-1 or PP2 alone. The blots shown in D and E are from the same experiment run on the same gel (D) or on separate gels (E). WB, Western blot.

A, TNF-α activation of NFκB is independent of Src and EGFR. LLC-PK₁ cells grown on coverslips were pretreated with 100 nm AG1478 or PP2 for 30 min followed by 10 ng/ml TNF-α for 30 min. The cells were fixed and permeabilized, and p65 NFκB was visualized by immunostaining with anti-p65 and CY3-coupled secondary antibody. The nuclei were counterstained using DAPI. The pictures show p65 (red) and DAPI (purple) staining of the same field as well as the merged image. B and C, TNF-α enhances proliferation through the EGFR. MDCK cells were plated in 96-well plates, serum-depleted overnight, and then exposed to 10 ng/ml TNF-α or 100 ng/ml EGF for 24 h. DNA synthesis was assessed using a BrdU incorporation assay as described under “Experimental Procedures.” In each experiment the BrdU signal in the control cells were taken as 1, and-fold changes were calculated. B shows data from n = 4 independent experiments performed in triplicate. C shows data of a typical experiment performed in 6 parallel determinations. In C, the asterisk indicates that the column is significant versus control (p < 0.05). D–F, shown is the effect of TNF-α on epithelial cell growth measured using ECIS. Changes in the capacitance (D) and impedance (E) were followed using ECIS as described under “Experimental Procedures.” Curves represent the averages of the duplicate measurements. D and E show capacitance and resistance data of the same measurement, respectively. All curves were normalized to the first obtained point. The red dashed lines on D and E indicate the 2 and 8 h time points, and the blue dashed lines indicate the 0.25 capacitance point. F summarizes data from capacitance measurements. In each measurement the time required for a 75% drop in the capacitance (0.25 value on the normalized capacitance curve) was determined and expressed as -fold change compared with the control in the same experiment taken as 1. The graph shows data from n = 8 curves obtained in 4 independent experiments (mean ± S.E., n = 8). The asterisk indicates p < 0.01 versus control.