Figure 1.
Cell type specific activation of Erk.
(A) AKR-2B and NIH 3T3 fibroblast were treated with TGF-β (2 ng/ml) for times ranging from 0 to 4 h. Cell lysates were probed with antibodies specific to phospho-Erk (P-Erk). Blots were then stripped and reprobed for total Erk as a loading control. Typical results are shown representing four independent time course experiments. (B) Graph of densitometric analysis of western blots from time courses from 0–3 h (n = 4) of phospho-Erk. Shown is the mean fold increase of Phospho-Erk relative to total Erk for each time point with the 0 time set as 1. Statistically significant change from 0 time is noted as, (*) P<0.05 and (**) P<0.01. (C) Mv1Lu and NMuMG epithelial cell lines were treated with TGF-β (2 ng/ml) for times ranging from 0 to 3 h. Cell lysates were probed with antibodies specific to phospho-Erk (P-Erk). Blots were then stripped and reprobed for total Erk as a loading control. Triplicate blots were performed on three independent time course experiments with typical results shown.
Figure 2.
Erk is activated in fibroblasts via the PI3K/c-Raf/MEK pathway.
(A) AKR-2B fibroblasts were treated with the PI3K inhibitor LY294002 or MEK inhibitor U0126 (10 µM) 30 minutes prior to addition of TGF-β (2 ng/ml) for 2 h. Cell lysates were probed with an antibody specific to phospho-Erk, blots were then stripped and reprobed for total Erk as a loading control. (B) AKR-2B fibroblasts were treated with TGF-β (2 ng/ml) for the indicated times. Cells were also treated with LY294002 (10 µM) 30 minutes before TGF-β was added for 2 h. Blots were probed using an antibody specific to phospho-c-Raf (Ser338), and total Erk as a loading control. The loading control blot was obtained using the lower molecular mass portion of the same blot. (C) AKR-2B fibroblasts were treated with LY294002, U0126 or Ras inhibitor FPTII (10 µM or 20 µM, respecitively) 30 minutes prior to addition of TGF-β (2 ng/ml) for 2 h. Cell lysates were probed with antibodies specific to phospho-Erk or phospho-Akt (S473) with the blots stripped and reprobed for the corresponding total protein as a loading control. Comparisons of the relative intensity of bands of Phopho-Erk relative to total Erk loading control was expressed as fold increase relative to untreated control (set at 1). Statistical analysis was used to determine if treatments were not significant (NS), or significant at P<0.05 (*) or P<0.01 (**). Analysis was performed on four independent blots and the mean values (±SEM) shown.
Figure 3.
Erk activation requires activation of Pak2 and c-Raf.
(A) AKR-2B fibroblasts were infected at an MOI of 1∶125 with adenovirus containing either dominant-negative PAK2 (Ad-EGFPdnPAK2) or Ad-EGFP as a negative control. Cells were treated with TGF-β (2 ng/ml) for 2 h prior to lysis. Cell lysates were then probed for phospho-Erk, and phospho-c-Raf (Ser338). The phospho-Erk1/2, and the stripped/reprobed total Erk loading control blot was obtained using the lower molecular mass portion of the same blot as that for P-cRaf. The same cell lysates were analyzed on a separate blot for PAK2 to confirm expression. (B) Cell lysates were analyzed for phospho-Erk and total Erk levels, in MEF cells that contained the Pak2 gene with flanking flox sites and a MEF cell line derived from this parental line in which Cre recombinase had been used to excise the Pak2 gene, following treatment with or without TGF-β for 2 h.
Figure 4.
TGF-β directs Erk phosphorylation of Smad2 linker region.
(A) Western blots from AKR-2B fibroblast cell lysates treated with TGF-β (2 ng/ml) for the indicated time periods with or without U0126 (10 µM) for 120 min. Blots were probed for smad2 phosphorylated within the linker region at S245, 250 and 255, striped and reprobed for total smad2 to demonstrate each loading. (B) Receptor mediated phosphorylation of smad2 was also determined in the same samples using antibodies specific to C-terminal Ser 465/467. The blots were stripped and reprobed for total smad2 to demonstrate similar loading of all samples. Representative western-blots are shown with each time course performed in triplicate with consistent results. (C) Western blot and relative quantification of smad2 linker region phosphorylation in AKR-2B fibroblasts treated for 30 min. with or without TGF-β (2 ng/ml) or EGF (50 ng/ml). P-Erk blots are also shown to indicate Erk activation. Intensity of P-smad2 linker region band was determined and expressed graphically as fold increase (using total Erk band intensity as the loading control) relative to untreated control values for each experiment. The mean values of three independent experiments are shown (±SEM). Letters above each column indicate the different statistically significant (P>0.05) groupings. (D) Representative western blots showing phosphorylation of smad2 linker region of AKR-2B fibroblasts treated for 120 min. with or without TGF-β (2 ng/ml) and/or inhibitors U0126 or LY364947. Blots were stripped and reprobed for total smad 2 as a loading control.
Figure 5.
Nuclear Smad levels are controlled by the proteasome and activated Erk.
(A) AKR-2B fibroblasts were treated for 3 h with TGF-β (2 ng/ml) with or without MG132 (10 µM), 30 minutes prior to TGF-β addition. Nuclear and cytoplasmic fractions were isolated and probed for smad2 linker region phosphorylation (245/250/255), or receptor phosphorylation (465/467). Linker phosphorylation blots were stripped and reprobed for total smad2 as a loading control, while receptor phosphorylated blots were stripped and reprobed for GAPDH to monitor the presence of cytoplasmic protein in the nuclear fraction. (B) Photomicrographs of NIH 3T3 fibroblasts treated with TGF-β (2 ng/ml) for 3 h with or without MG132 (10 µM) added 30 minutes prior to TGF-β treatment. Cells were incubated with phospho-smad2 (S245/250/255) linker antibody and specific immune complexes detected using Rhodamine X conjugated secondary antibody. (C) Cell lysates from AKR-2B fibroblasts pulsed for 10 minutes with TGF-β (2 ng/ml) with or without U0126 (10 µM) were probed for receptor phosphorylated smad2 and smad3. Blots were stripped and reprobed for β-actin as a loading control. (D) The density of phospho-smad2 and 3 bands for each time point relative to its β-actin control were determined. The mean values for each time point (n = 3 for smad 2, n = 4 for smad 3) are displayed with the solid line representing the curve for TGF-β+U0126 and the dotted line representing TGF-β treatment.
Figure 6.
Erk activity is integral for TGF-β signaling and inducing growth in fibroblasts.
(A) Fold change in expression levels of two smad regulated genes, PAI-1 and smad7, relative to untreated controls levels are shown from AKR-2B fibroblasts treated for 10 minutes with or without TGF-β (2 ng/ml), with or without U0126 (10 µM) for 3 h. The mean values (±SEM) of six independent experiments are shown with statistical evaluation indicating differences between groups as (NS) not significant, (*) P<0.05, (***) P<0.001. (B) Thymidine incorporation was determined in serum deprived NIH 3T3 cells treated with TGF-β (5 ng/ml) with or without infection with Ad-dnPAK2 or Ad-EGFP (MOI = 125∶1) or U0126 (10 µM) prior to treatment. The effect of treatment is expressed as a fold change in thymidine incorporation relative to untreated cells (control = 1). The mean (±SEM) of triplicate assays is shown with statistical evaluation indicating differences between groups as previously describe. (C) Schematic representation of TGF-β signaling pathways in fibroblasts, indicating the proposed interactions and their subcellular locations. Arrows represent a functional link between the subsequent proteins, not necessarily a direct interaction.