Impact of hyperglycemia on phospholipase C and phospholipase D expression in neutrophil-like HL-60 cells
Date
2011
DOI
Authors
Muller, Enrique
Version
OA Version
Citation
Abstract
Diabetes mellitus is a metabolic disease characterized by functional abnormalities of polymorphonuclear neutrophils (PMN) and inflammation. The aim of this study was to define the impact of diabetic control on the expression of phosphoinositide-specific phospholipase C (PLC) and phosphatidylcholine-specific phospholipase D (PC-PLD) in neutrophils.Testing diabetic conditions requires prolonged incubation periods to expose cells to hyperglycemia and advanced glycation endproducts (AGE). This is not possible for neutrophils because they are terminally differentiated cells with a lifespan of 24 to 48 hours. Therefore, superoxide (O2[-]) production, a measurement of basic cytotoxic PMN function and expression levels of target molecules and expression of PLC and PLD were evaluated in human pro-myelocytic leukemic cells (HL-60) which can be cultured and differentiated into neutrophil-like cells.
The cells were cultured in diabetic conditions by adding 25 mM glucose to replicate hyperglycemia and S100B RAGE ligand to simulate the effects of AGE. Superoxide release assays were carried out after corroborating the differentiation of HL-60 cells into neutrophil like cells; an increase of 20% in mean optical density values during 5 minutes was seen on the diabetic samples versus the control. This increase in O2[-] production is consistent with previous reports and most likely due to the priming of the neutrophils because of the diabetic conditions [1].
Expression of PLC and PLD isoforms were measured at protein level. PLC[Beta]1 demonstrated a slight decrease in expression levels. However, PLC[Beta]2 1evels were cleary shown to be elevated over one and a half times the control values in the diabetic groups. PLC[Beta] levels increase cytosolic Ca++ influx thus resulting in increased PKC phosphorylation [2]. Both PLCy1 and PLCy2 were elevated in diabetes, PLCy1 showed a more dramatic increase than PLCy2. PLD catalyzes the hydrolysis of the terminal diester bond of glycerophospholipids, resulting in the formation of phosphatidic acid, which can be converted by diacylglycerol (DAG) kinase into DAG and then phosphorylate protein kinase C (PKC). PA can also activate p47[phox] and subsequently NADPH oxidase directly, resulting in O2[-] production [3, 4]. PLD1 has been shown to regulate motility and adhesion of phagocytes in response to fMLP and IL-8 [5, 6]. The role of PLD2 is not well understood at this time, but it is theorized that it is involved in cell migration as well [6]. The results of this project show a statistically significant increase in expression levels of PC-PLD1 with a decrease in PC-PLD2 levels of almost 50%. Our data shows that diabetes causes an up-regulation of most isoforms and variants of PLC and PLD, which is consistent with elevated O2[-] generation.
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Thesis (MSD) --Boston University, Henry M. Goldman School of Dental Medicine, 2011 (Department of Periodontology and Oral Biology).
Includes bibliography: leaves 80-96.
Thesis (MSD) --Boston University, Henry M. Goldman School of Dental Medicine, 2011 (Department of Periodontology and Oral Biology).
Includes bibliography: leaves 80-96.
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This work is protected by copyright. Downloading is restricted to the BU community. If you are the author of this work and would like to make it publicly available, please contact open-help@bu.edu.