IPP-1 controls Akt/CREB phosphorylation extension in A(2a) adenosine receptor signaling cascade in MIN6 pancreatic beta-cell line

Abstract

Signaling through A(2a) adenosine receptor specifically prevent pancreatic beta-cells (PBCs) loses under diabetogenic conditions. However, signaling mediators of this receptor in PBCs remained unidentified. Thus, we aimed to investigate the possible involvement of PKA/Akt/IPP-1/CREB pathway in MIN6 beta-cells. In addition, we investigated IPP-1 role in A(2a) receptor signaling pathway. The expression of A(2a) receptor in MIN6 cell line was evaluated by RT-PCR and its functionality confirmed by quantification of cAMP in response to the CGS 21680, an A(2a) receptor agonist. MTT and Brdu assays were used to evaluate cell viability and proliferation, respectively. PKA activity and insulin release were evaluated using ELISA methods. P-Akt/Akt, p-IPP-1/IPP-1, and p-CREB/CREB levels were assessed using western blotting. IPP-1 knock down assessments was performed using specific siRNA. Our result revealed that MIN6 cells express A(2a) receptor which actively increased cAMP levels (with EC50 = 2.41 mu M) and PKA activity. Activation of this receptor increased cell viability, proliferation and insulin release. Moreover, we mentioned A(2a) receptor stimulation increased p-Akt, p-IPP-1, and p-CREB levels in dose (max at 10 mu M of CGS 21680) and time (max at 30 min after CGS 21680 treatment) dependent manner. Interestingly, herein, we found in IPP-1 knocked down cells, A(2a) receptor failed to activate Akt and CREB. Altogether, we mentioned that in MIN6 cells A(2a) receptor increase cell viability, proliferation and insulin release through PKA/Akt/IPP-1/CREB signaling pathway. In addition, we conclude A(2a) receptor signaling through this pathway is dependent to activation of IPP-1.