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2001 Abstract: 1792 TGFb Activation of cAMP Responsive Element-Binding Protein (CREB) is Dependent on PKA and Smads in Pancreatic Acinar cells

2001 Digestive Disease Week

# 1792 TGFb Activation of cAMP Responsive Element-Binding Protein (CREB) is Dependent on PKA and Smads in Pancreatic Acinar cells
Lizhi Zhang, Kathleen vanLeeuwen, Craig D. Logsdon, Diane M. Simeone, Ann Arbor, MI

Background: TGFb is an important regulator of growth and differentiation in the pancreas, yet the transcription factors that mediate the physiological responses elicited by TGFb are poorly understood. cAMP responsive element-binding protein (CREB) is critical for a variety of cellular processes, including proliferation, differentiation and adaptive response. It promotes cellular gene expression following its phosphorylation at Ser133. We hypothesized that TGFb activates CREB in pancreatic acinar cells and sought to identify the signaling mechanisms involved.

Methods: Studies were performed in isolated mouse pancreatic acini. CREB activation was examined using electrophoretic mobility shift assays (EMSAs) and Western blotting with an anti-phospho-CREB antibody. The role of protein kinase A (PKA) was assessed with an in vitro kinase assay using biotinylated PKA peptide substrate (Kemptite). The ability of TGFb to elevate intracellular cAMP levels was evaluated using an enzyme immunoassay. Adenovirus-mediated gene transfer of dominant-negative Smad2 (dnSmad2) and dominant-negative Smad4 (dnSmad4) was performed to study the role of Smads in TGFb-mediated activation of CREB.

Results: TGFb (100 pM) activated CREB in pancreatic acinar cells, as demonstrated by both EMSAs and Western blotting using an anti-phospho-CREB antibody. TGFb induced a significant increase in CREB activation at 1 hour, with maximal results at 2 hours (fold increase 2.01 ± 0.23 vs. control, p<0.01). TGFb also induced a rapid increase in PKA activity, maximal at 15 minutes (fold increase 2.17 ± 0.17 vs. control, p<0.01). Pretreatment of acini with the specific PKA inhibitor PKI (1 mM) inhibited both TGFb-mediated activation of PKA and CREB. Interestingly, intracellular cAMP levels did not increase in response to TGFb, but were significantly increased by forskolin. Adenoviral-mediated gene transfer of both dnSmad2 and dnSmad4 blocked TGFb s ability to phosphorylate CREB.

Conclusions: These results demonstrate that TGFb activated the transcription factor CREB in pancreatic acini via cAMP-independent activation of PKA. CREB activation by TGFb was also dependent on signaling by Smad2/Smad4. These studies suggest novel signaling crosstalk between PKA and Smads in the TGFb signaling pathway in pancreatic acinar cells.

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