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* Department of Periodontology and Oral Biology, Goldman School of Dental Medicine, Boston University, Boston, MA 02118;
Department of Anesthesiology, Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women’s Hospital and Harvard University Medical School, Boston, MA 02118; and
Section of Endocrinology, Diabetes and Nutrition, Department of Medicine, School of Medicine, Boston University, Boston, MA 02118
The protein kinase C (PKC) family of intracellular enzymes plays a crucial role in signal transduction for a variety of cellular responses of mononuclear phagocytes including phagocytosis, oxidative burst, and secretion. Alterations in the activation pathways of PKC in a variety of cell types have been implicated in the pathogenesis of the complications of diabetes. In this study, we investigated the consequences of PKC activation by evaluating endogenous phosphorylation of PKC substrates with a phosphospecific PKC substrate Ab (pPKC(s)). Phosphorylation of a 40-kDa protein was significantly increased in mononuclear phagocytes from diabetics. Phosphorylation of this protein is downstream of PKC activation and its phosphorylated form was found to be associated with the membrane. Mass spectrometry analysis, immunoprecipitation, and immunoblotting experiments revealed that this 40-kDa protein is pleckstrin. We then investigated the phosphorylation and translocation of pleckstrin in response to the activation of receptor for advanced glycation end products (RAGE). The results suggest that pleckstrin is involved in RAGE signaling and advanced glycation end product (AGE)-elicited mononuclear phagocyte dysfunction. Suppression of pleckstrin expression with RNA interference silencing revealed that phosphorylation of pleckstrin is an important intermediate in the secretion and activation pathways of proinflammatory cytokines (TNF-
and IL-1
) induced by RAGE activation. In summary, this study demonstrates that phosphorylation of pleckstrin is up-regulated in diabetic mononuclear phagocytes. The phosphorylation is in part due to the activation of PKC through RAGE binding, and pleckstrin is a critical molecule for proinflammatory cytokine secretion in response to elevated AGE in diabetes.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by U.S. Public Health Service Grant DE15566.
2 Deceased during the preparation of this manuscript.
3 Address correspondence and reprint requests to Dr. Thomas E. Van Dyke, Department of Periodontology and Oral Biology, Goldman School of Dental Medicine, Boston University, 100 East Newton Street G-107, Boston, MA 02118. E-mail address: tvandyke{at}bu.edu
4 Abbreviations used in this paper: PKC, protein kinase C; RAGE, receptor for advanced glycation end products; AGE, advanced glycation end products; p-PKC(s), phospho-(Ser) PKC substrate Ab; BCA, bicinchoninic acid; MS, mass spectrometry; Xcorr, cross-correlation score; IP, immunoprecipitation; siRNA, small-interfering RNA; DM, diabetes mellitus; cPKC, conventional PKC; PH, pleckstrin homology.
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  V. Tchaikovski, S. Olieslagers, F.-D. Bohmer, and J. Waltenberger Diabetes Mellitus Activates Signal Transduction Pathways Resulting in Vascular Endothelial Growth Factor Resistance of Human Monocytes Circulation, July 14, 2009; 120(2): 150 - 159. [Abstract] [Full Text] [PDF]
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