Figure 3

pERK is necessary for TGFβ-induced cell cycle arrest in benign pancreas duct cells. (a) pRB expression was evaluated via immunohistochemistry, showing increased staining in the exocrine tissue of Tgfbr2^DN and Tgfbr1^+/− mice compared with wild-type (WT) controls. Dashed lines surround islets. (b–d) Pancreas tissue from wild-type (WT) mice was dual-stained for PCNA and pERK, and both PCNA⁺ and pERK⁺PCNA⁺ cells quantified per 20x field. We subsequently dual-stained the small intestine of wild type (WT), Tgfbr2^DN, and Tgfbr1^+/− mice for PCNA and pERK, affirming the pERK deficiency in Tgfbr2^DN and Tgfbr1^+/− groups, though there was no change in PCNA staining. The white arrows indicate nuclei that are dual positive for pERK and PCNA. (e) benign human pancreatic ductal epithelial (HPDE) cells were starved of growth supplements, and incubated with 5–10 ng/ml exogenous TGFβ1, and pERK examined after 30 min. (f) Thirty minutes following incubation with TGFβ1, pERK was isolated by immunoprecipitation and interaction with TGFBR1 assessed by western blotting. (g) pERK was isolated by immunoprecipitation 24 h following incubation with TGFβ1, and interaction with SMAD4 and p21 measured by western blotting. (h) inhibition of pERK via U0126 prevented TGFβ1-induced nuclear translocation of p21 in HPDE cells. (i) HPDE cells were pre-incubated with U0126 prior to TGFβ-treatment. In the absence of pERK, despite the compensatory upregulation of ERK1 (small upper band in the ERK doublet), TGFβ1 failed to induce downstream SMAD2 phosphorylation or p21 upregulation, as well as repression of CDK2. (j) HPDE cells were again pre-incubated with U0126 prior to TGFβ-treatment, and the interaction between p21 and CDK2 was assessed by immunoprecipitation after 24 h. (*P<0.05. N=4 mice per group unless otherwise specified).