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Abstract
Although the interaction between gp130 and the ErbB family has frequently been shown in cancer cells, the mechanism of this interaction remains unclear and controversial. In the present study, we found that specific tyrphostin inhibitors of ErbB2 (AG825 and AG879), but not ErbB1 inhibitor (AG1478), suppressed IL-6-induced tyrosine phosphorylation of STAT3 in schwannoma cells. However, biochemical evidence for transactivation of ErbB2 by IL-6 was not observed. Additionally, the inhibition of ErbB2 expression, with either a specific RNAi or transfection of an ErbB2 mutant lacking the intracellular domain did not inhibit the IL-6-induced tyrosine phosphorylation of STAT3. Thus, it seems that tyrphostins, which are known as specific inhibitors of the ErbB2 kinase, may have non-specific suppressive effects on the IL-6/STAT3 pathway.
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Fig. 1.
Specific tyrphostin ErbB2 inhibitors block IL-6-induced tyrosine phosphorylation of STAT3. (A, B) The effects of an ErbB1 inhibitor (AG1478) or ErbB2 inhibitors (AG825, AG879) on IL-6- induced tyrosine phosphorylation of STAT3 in RT4 cells were analyzed. ErbB2 inhibitors dose-dependently reduced tyrosine phosphorylation of STAT3 induced by IL-6. (C) STAT3 DNA binding activity was analyzed using a STAT3-interacting oligonucleotide conjugated to agarose beads, after 20 min of IL-6 treatment. STAT3 was immunoprecipitated and analyzed via immunoblotting, with antibodies against pSTAT3 or STAT3. (D) An ErbB1/ErbB2 dual inhibitor, GW583340, did not block IL-6-induced tyrosine phosphorylation of STAT3.
Fig. 2.
Effects of ErbB1 and ErbB2 inhibitors on IL-6-induced tyrosine phosphorylation of STAT3 in primary Schwann cells. (A) The selective inhibition of IL-6-induced (20 min) tyrosine phosphorylation of STAT3 by AG825 was demonstrated in primary Schwann cells. (B) Dose-dependent curves showing the effect of AG825 on RT4 (▪) cells and primary Schwann cells (•). The intensity of IL-6- stimulated pSTAT3 band in the absence of AG825 was set as 100% of relative intensity. The curve shows that primary Schwann cells were more sensitive to AG825 than RT4 cells.
Fig. 3.
IL-6 does not transactivate ErbB2 in Schwann cells. (A) Western blot analysis with anti-phospho-ErbB2 antibodies showed that IL-6 did not induce tyrosine phosphorylation of ErbB2 at tyrosines 877, 1221 or 1222, whereas NRG induced strong tyrosine phosphorylation of ErbB2 at tyrosine 877 and mild phosphorylation at tyrosines 1221 and 1222. (B) Cell lysates were subjected to immunoprecipitations using either an anti-gp130 antibody or an anti-ErbB2 antibody, and then immunoblotting was performed using an anti-phosphotyrosine antibody (4G10, pY). IL-6 did not induce tyrosine phosphorylation of ErbB2, while it did stimulate the tyrosine phosphorylation of gp130. (C) Cell lysates were subjected to immunoprecipitations using either an anti-gp130 antibody or an anti-IL-6R antibody, and then immunoblotting was performed using an antibody to ErbB2 to demonstrate interactions between gp130 and ErbB2 or between IL-6R and ErbB2. IL-6 induced an interaction between IL-6R and gp130, but did not induce an interaction between ErbB2 and gp130 or IL-6R.
Fig. 4.
Specific ablation of ErbB2 function does not inhibit IL-6-induced tyrosine phosphorylation of STAT3. (A) Cells were transfected with control siRNA or ErbB2 siRNA, and IL-6-induced tyrosine phosphorylation of STAT3 was analyzed using Western blot analysis. The suppression of ErbB2 expression with a specific RNAi did not affect the IL-6-induced tyrosine phosphorylation of STAT3. (B) Cells were transfected with an ErbB2 mutant lacking the intra-cellular domain (ErbB2TR) or empty vector. Two days after transfection, cells were incubated with IL-6 for 20 min, fixed and double-immunostained with an anti-Myc antibody and an anti- pSTAT3 antibody. (C) Quantitative analysis was performed by calculating the percentage of pSTAT3-positive nuclei among Myc- positive cells. Means±S.E from three independent experiments are shown.
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