Subcellular Proteomic Approach For Identifying Signaling Effectors Of PKC-β₂ Under High Glucose In HUVEC

Background:With constantly increase of the global prevalence of diabetes, diabetes mellitus has become a frequent and severe public health problem, its unique vascular complications are the major cause of blindness, disability and death. Recently, endothelial dysfunction is well-recognized as a primary contributor in the development of diabetic vascular complication. A large quantity of evidences have showed that activation of protein kinase C (PKC) plays a pivotal role in the pathogenesis of diabetic vascular disease. Among the various PKC isoforms, especially, PKC-β₂ subtype predominantly expressed in endothelial cells, which is the key target cells in the vascular lesions. Further more, high glucose-induced activation of PKC-β₂ plays a core effect in the important pathophysiological chain of diabetes vascular disease. However, previous studies only focus on one known molecule or signal pathway, a global characterization of signal mechanism of PKC-β₂ isoform involved remains incompletely understood.. To date, little is known about the crosstalk events at the subcellular level regulating by PKC isoform. The work here demonstrates the novel downstream effectors of PKC-β₂-signaling at the subcellular level under high glucose stress in HUVEC. Purpose and Methods:To analyse potential novel effectors that participate in the PKC-β₂ signaling in subcellular compartment of endothelial cells, we have constructed a model system of constitutively active PKC-β₂ in human umbilical vein endothelial cells(HUVECs) exposed to high ambient glucose. For 2-DE analysis, HUVECs were treated into 4 groups: the normal glucose control (NG, D-glucose 5.6mmol/L); high glucose group (HG, D-glucose 25mmol/L); empty vector control (EV, cells transfected with Ad5-Null and cultured in medium containing 25mmol/L glucose); PKC-β₂ overexpression group (PO, cells transfected with Ad5-PKC-β₂ and cultured in medium containing 25mmol/L glucose). The classical subcellular fractionation methods have been used to extract the nuclear and cytoplasm fraction from HUVECs. Changes in protein patterns were identified using a proteomic approach consisting of two-dimensional (2-DE) gel electrophoresis and mass spectrometry(MALDI-TOF MS). And the altered expression of representative proteins was validated by Western blotting. Furthermore, a Human Protein-Protein Interaction (PPI) network of potential connections on PKC-β₂-associated proteins that we found in the proteomics study was obtained by using Biological General Repository for Interaction Datasets (BioGRID 3.1).Result:(1)In this study, we got two-dimensional electrophoresis gel patterns from nuclear and cytoplasmic fraction protein of HUVECs respectively. From nuclear fraction protein, series of two-dimensional electrophoresis have allowed us to detect a total of 800 spots approximately. After the intra- and inter-group cross-match, a total of 38 spots significantly altered (±over 1.5-fold, p<0.05) and were identified as high glucose-induced and PKC-β₂-associated nuclear proteins. From cytoplasm fraction, a total of close to 600 spots was detected in 2-DE mapping. And 28 spots associated with PKC-β₂ significant alteration(±over 1.5-fold, p<0.05). (2)Subsequent identification of 50 altered peptides is made by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry(MALDI-TOF MS) and bioinformatics analysis. Most of the identified proteins involved inâ‘ biosynthesis and metabolism;â‘¡protein kinase family-associated;â‘¢cell cycle, apoptosis and proliferations;â‘£transcription factors-associated, or others. On the basis of our results, diverse novel downstream effectors of PKC-β₂ signaling were identified: mitogen-activated protein kinase 3(MAPK3), Protein DBF4 homolog B(DBF4B), Cell division cycle 7(CDC7), Peroxisome proliferator-activated receptor delta (PPARδ), NF-kappa-B inhibitor-interacting Ras-like protein (NKIRAS1), they respectively involved in glucose and lipid metabolism crosstalk, inflammatory response, cell proliferation, the alteration of cell cycle. (3)Among the identified proteins, two proteins were further confirmed using Western blot analyses. We validated a decrease in the nuclear localization of PPARδ, and a decrease in their cytosolic localization of NKIRAS1 in response to PKC-β₂ sustained activation. (4)In addition, among the 50 different proteins identified by MS, 14 proteins had successfully connected by Protein-Protein Interaction (PPI) network.Conclusion:This is the first study using subcellular and functional proteomics to analyze key proteins alteration at subcellular level in endothelial cells before and after PKC-β₂ isoform activation, via analyzing protein profile changes. We found that PKC-β₂ may be involved in high glucose-induced glucose and lipid crosstalk by regulating PPARδ. And NKIRAS1 may play a important role of a potential PKC-β₂-IκB-NF-κB signaling pathway in HUVEC under high glucose. This finding provide direct, system-wide insights into the mechanism of diabetes associated blood vessel damage and it will enable a better understanding of the molecular mechanisms underlying PKC-β₂-induced crosstalk events at the subcellular level.