Regulation of transforming growth factor beta-induced responses by protein kinase A in pancreatic acinar cells

Abstract

TGF-beta is an important regulator of growth and differentiation in the pancreas and has been implicated in pancreatic tumorigenesis. We have recently demonstrated that TGF-beta can activate protein kinase A (PKA) in mink lung epithelial cells (Zhang L, Duan C, Binkley C, Li G, Uhler M, Logsdon C, Simeone D. Mol Cell Biol 24: 2169-2180, 2004). In this study, we sought to determine whether TGF-beta activates PKA in pancreatic acinar cells, the mechanism by which PKA is activated, and PKA's role in TGF-beta-mediated growth regulatory responses. TGF-beta rapidly activated PKA in pancreatic acini while having no effect on intracellular cAMP levels. Coimmunoprecipitation experiments demonstrated a physical interaction between a Smad3/Smad4 complex and the regulatory subunits of PKA. TGF-beta also induced activation of the PKA-dependent transcription factor CREB. Both the specific PKA inhibitor H89 and PKI peptide significantly blocked TGF-beta's ability to activate PKA and CREB. TGF-beta-mediated growth inhibition and TGF-beta-induced p21 and SnoN expression in pancreatic acinar cells were blocked by H89 and PKI peptide. This study demonstrates that this novel cross talk between TGF-beta and PKA signaling pathways may play an important role in regulating TGF-beta signaling in the pancreas.

Fig. 1.

TGF-β activates PKA in pancreatic acinar cells. Isolated pancreatic acini were treated with TGF-β (100 pM) for the indicated time periods (A) or at the indicated doses for 15 min (B). In vitro kinase assays for PKA activity were performed using a biotinylated PKA peptide substrate (Kemptide, Promega). A specific PKA inhibitor H89 (3 μM) was also used to pretreat cells for 30 min. Acinar cells were treated with 10 μM forskolin for indicated times, and PKA assays were performed (C). Results are expressed as fold increase of control from 3 separate experiments (*P < 0.01 vs. control).

Fig. 2.

TGF-β does not have sensitizing effect on caerulein (CAE)-induced trypsin activity, expressed as ng/mg protein, from isolated acini preparations incubated with caerulein at a submaximal dose (100 pM) or at supramaximal dose (100 nM), or with TGF-β at 100 pM, as indicated in methods. Control acini were incubated with the caerulein and the TGF-β vehicle (*P < 0.001 vs. untreated controls).

Fig. 3.

TGF-β-mediated activation of PKA and CREB is dependent on Smad4 in pancreatic acinar cells. PKA and CREB activities following TGF-β stimulation were measured in isolated mouse pancreatic acinar cells treated with PKI peptide (10 μM) or H89 (3 μM) for 30 min prior to TGF-β treatment (100 pM for 30 min). Western blot analysis of cell lysates (A), ELISA assay for phosphorylated CREB from nuclear extracts (B), and PKA activity assay (C) were performed. PKA activity and CREB activation following TGF-β stimulation were also measured in mouse pancreatic acinar cells that were infected with either AddnSmad4 virus or AdGFP control virus for 48 h. Cell lysates were then subjected to PKA activity assays and Western blot analysis (D and E). Results are representative of 4 separate experiments (*P < 0.01 vs. control).

Fig. 4.

cAMP levels are not increased with TGF-β treatment. In the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX, 100 μM), cAMP levels were measured in acini after treatment with TGF-β (100 pM) or forskolin (10 μM) by using a Biotrak enzyme immunoassay assay kit. Results are from 3 separate experiments (*P < 0.01 vs. control).

Fig. 5.

TGF-β induces the interaction of an activated Smad3/Smad4 complex with PKA regulatory subunits. Isolated acini were treated with 100 pM TGF-β for the indicated time periods. Cell lysate (500 μg) was used for immunoprecipitation (IP) with 1 μg of anti-PKA regulatory subunit Iβ and anti-PKA catalytic subunit α antibodies. Samples were subsequently immunoblotted with anti-Smad2, anti-Smad3 and anti-Smad4 antibodies. Cell lysates (50 μg) were run at the same time as positive controls. WB, Western blot. Data are representative of 2 separate experiments, which provided the same results.

Fig. 6.

TGF-β-induced SnoN expression is mediated by the activation of PKA. Mouse acinar cells were preincubated either with PKI peptide (10 μM) or H89 (3 μM) for 30 min and TGF-β (100 pM) for 3 h. The cell lysates were examined for SnoN expression by Western blotting analysis. The membrane was also probed with β-actin antibody for loading control.

Fig. 7.

TGF-β-induced p21 expression and growth inhibition is mediated by the activation of PKA. TGF-β-induced p21 expression can be blocked by PKI peptide and H89 (A). Acinar cells were pretreated with PKI peptide (10 μM) or H89 (3 μM) for 30 min. TGF-β (100 pM) was added for 16 h, and 30 μg of protein was used to perform Western blotting (top). The same membrane was stripped and reprobed with anti-β-actin antibody to demonstrate equal loading (bottom). Experiments were repeated 3 times with essentially the same results. TGF-β-mediated growth inhibition was determined by [³H]thymidine incorporation assays (B). Acinar cells were treated with TGF-β (100 pM) for 60 h. Some cells were also treated with 10 μM PKI peptide or 3 μM H89. Results are expressed as a percentage of control from 4 separate experiments (*P < 0.01 vs. control).