P028. Protein kinase D1 mediates CREB activation and increased SOCS3 expression induced by PGE2 in colon epithelial cells
Y. Chen1, J. Xiong1, W. Xu2, X. Huang1, Y. Wang1, S. Liu1, F. Deng2, 1Southern Medical University, Department of Gastroenterology, Nanfang Hospital, Guangzhou, China, 2Southern Medical University, Department of Cell Biology, School of Basic Medical Sciences, Guangzhou, China
Ulcerative colitis (UC) is a chronic disease associated with long periods of quiescent disease followed by fulminant exacerbation. Imminent relapse in UC is associated with high mucosal expression of suppressor of cytokine signaling 3 (SOCS3). Hence, knowing mechanisms underlying mucosal SOCS3 expression may provide important clues as to rational therapy for ulcerative colitis. However, the molecular mechanisms that regulate SOCS3 expression and signal transduction pathway remain poorly understood. Here we report that protein kinase D (PKD), a newly described serine/threonie kinase that has been implicated in inflammatory response, contributes to CREB signaling and increased SOCS3 expression in the colonic epithelial cells, activation of PKD1 induced by pro-inflammatory cytokine prostaglandin E2 activates CREB phosphorylation and up-regulates SOCS3 expression.
The colon epithelial cell line Caco-2 & HT-29 and the intestine cell line IEC-6 were stimulated with interleukin (IL)-6 or prostaglandin E(2) (PGE2) to allow correlations between SOCS3 expression with signal transducer and activator of transcription 3 (STAT3), and adenosine 3′,5′-cyclic monophosphate (cAMP) signaling, respectively, and the phosphorylation of PKD and downstream signaling moleculars were detected by Western blot. We also transfected the plasmids and the small interference RNA of PKD1 into Caco-2 & HT-29 to evaluate its effect on related molecular phosphorylation and SOCS3 expression with PGE2 treatment. Immunofluorescence and Co-immunoprecipitation were made to prove the relevance between PKD1 and cAMP response element-binding protein (CREB).
Overexpression of wild type or constitutive active mutant of PKD1 increased SOCS3 expression, whereas overexpression of dominant negative of PKD1 significantly reduced SOCS3 expression in the colonic epithelial cells. Furthermore, Knockdown of PKD1 by siRNA transfection or specific PKD inhibitor KB-NB142–70 treatment remarkably inhibited CREB (ser133) phosphorylation and SOCS3 expression induced by PGE2 but not IL-6 in the colon epithelial cell line. There is no effect of siRNA transfection on PKA and STAT3 phosphorylation with or without PGE2 treatment, In addition, ser744/748 phosphorylation of PKD1 and interaction of PKD1 with CREB were also enhanced with PGE2 treatment, and PKD1 activation induced by PGE2 promoted phosphorylated CREB translocation into nucleus and subsequently upregulated SOCS3 expression.
Our data suggest that PKD1 may contribute to inflammatory response of ulcerative colitis through CREB phosphorylation and increased SOCS3 expression, which may be a potential target for anti ulcerative colitis.