Proteomics Analysis Identifies Nuclear Protein Effectors Of Pkc-δ Signaling Under High Glucose Stress In HUVEC

Background and Objective: Disturbances of endothelial cell homeostasis are well-recognized as primary cellular events in the development of diabetic vascular complication. Although previous studies have demonstrated that high ambient glucose would lengthen cell proliferation, disturb cell cycle, increase DNA damage, and slightly accelerated cell death, the precise molecular mechanism by which diabetes leads to endothelial cell injury remains to be fully understood. Recent a large body of evidences have strongly implicated activation of protein kinase C (PKC) in the pathogenesis of diabetic vascular disease. However, a global characterization of signal mechanism of PKC isoform involved is still missing.Among the various PKC isoforms,βandδisoforms appear to be activated preferentially in the vasculatures of diabetic animals and in vitro. Although experimental animal and clinical trail have suggested that more than PKC-βactivation is involved in the vascular pathology of diabetic retinopathy, the present knowledge about the role of PKC-δis very fragmentary, and results are inconclusive. Studies have been demonstrated that chronic exposure of cells (such as endothelial cells, mesangial cells, or smooth muscle cells) to high glucose increases diacylglycerol-activated PKC-δ. Moreover, recent findings implicate a role of PKC-δin promoting apoptosis in pericyte exposed to high glucose through two pathways involving ROS induction of NF-κB activity and deactivation of PDGF receptorβ, in ovine fetal pulmonary artery endothelial cells via ERK or Akt pathway by regulating NO generation and eNOs expression, and in beta-cells exposed to fatty acid through a mechanism that involves inhibition of FOXO1 activation. The multiplicity of situations in which PKC-δacts suggest that PKC-δaffects diverse initial or terminal event in apoptotic pathway.However, all these approaches as mentioned above only focus on one known molecule or signal pathway. In fact, cell signal cascade is not point-to-point linear paradigm, but complicated and cross-talking. Not like the previous research, we first detected nucleus translocation of PKC-δin HUVEC exposed to high glucose (25 mmol/L) for 6 days. As we know, nucleus contains the genetic information and is the place of gene expression, therefore it is important to indentify nuclear proteins for an understanding of genome regulation and function as well as for providing clues about the molecular function of novel proteins. We also detected a significant increased cell apoptosis and cell-cycle arrest by constitutively active PKC-δusing an adenoviral vector. So we hypothesized, PKC-δappeared to be an upstream element, triggering a serious of signaling events that recruitment of many downstream effectors involved in the regulation of cell behavior. Thus identification of the molecules that participate in PKC-δsignaling may provide important insights into the molecular events that lead to the apoptosis and cycle arrest.Functional proteomics is emerging technology to study molecular mechanisms of cell function. It is ideal tool for unbiased and large-scale clarifying quantitative protein change in physiological and diseased conditions. This tool offer us a new way to discover novel proteins or proteins that seldom studied before, but they are really associated with changes of cell structures and functions. Now proteomic analysis is widely utilized on many fields of clinic or basic medical research, however, it is rare performed in diabetes, especially in studying PKC-δ-induced apoptosis in HUVEC, this is a creative way to further study the downstream effectors of PKC-δsignaling in its pro-apoptosis function.Methods:1. Recombinant adenovirus infected HUVEC and groupingA recombinant adenoviral vector was constructed to express PKC-δand packaged, Ad5-Null was used as empty vector to infect HUVEC as control. There are totally 5 groups: the normal glucose control(NC, D-glucose 5.6mmol/L), high glucose group(HG, D-glucose 25mmol/L), empty vector control(EC, cells transfected with Ad5-Null and cultured in medium containing 25mmol/L glucose), PKC-δoverexpression group(PO, cells transfected with Ad5-PKCδand cultured in medium containing 25mmol/L glucose) and PKC-δinhibition group(PI, cells transfected with Ad5-PKCδand cultured in medium containing 25mmol/L glucose plus 10umol/L Rottlerin).2. Immunofluorescence Labeling of PKC-δand Confocal MicroscopyCells cultured on glass coverslips were incubated successively with The PKC-δpolyclonal antibodies and the FITC-conjugated goat anti-rabbit IgG in the dark. Fluorescence intensity of PKC-δwas detected by Confocal Microscopy.3. Cell-cycle assay and apoptosis assayAfter incubation in culture medium for 6 days, Cell-cycle was analyzed by flow cytometry and apoptosis ratio was tested by Annexin-FITC cell apoptosis kit followed with flow cytometry.4. Two-Dimensional electrophoresis and Mass Spectrometry analysisNuclear protein extraction was gain from the following four groups: Cells of normal control group(NC), high glucose group(HG), PKCδoverexpression group(PO) also the empty vector control (EC). Protein sample for each group was processed by IEF in a stepwise fashion at 250V for 2h, 500V for 1h, 1000V for 1h rapidly, 10000V for 6 h, lastly, focused for a total of 70000Vh. Protein spots were dispensed on the SDS-PAGE gel by the following second-dimension electrophoresis and analyzed by PDQuest software. Differential expression proteins were digested into mass of peptides by trypsin.5. Bioinformatics analysisInput the data of peptide mass fingerprint to the MASCOT database and search for the probalble proteins according to their mass, PI, percentage of Sequence coverage.Results:1. The fluorescence intensity per cell was increased (1.6 folds) in cytoplasma and the cytosol-to-nucleus fluorescence ratio was decreased in the presence of high glucose(25mmol/L). When PKC-δoverexpressed, the fluorescence intensity per cell after adenovirus transfection was significantly increased up to 1.8-fold comparing with non-trasfected high glucose control(P<0.05) and the cytosol-to-nucleus fluorescence ratio of PKC-δwas decreased markedly (1.49±0.17 vs 2.31±0.33 , p<0.05).2.Approximately (4.1±0.67)% of the cells underwent early apoptosis in response to 5.6mmol/L glucose exposure for 6 days, whereas approximately (22±2.7)% of the cells were early apoptotic in present of 25mmol/L glucose(p<0.05). Treatment of the cells with high glucose exposure also arrested the cells in G0/G1 phase of the cell cycle obviously(p<0.05), the percentage of G0/G1 phase for cells in 25mmol/L glucose is approximately 1.3-fold up over that of cells in 5.6mmol/L glucose. PKC-δoverexpression lead to further cell cycle arrest and increased ratio of apoptosis induced by high glucose (p<0.05). Whereas, rottlerin treatment(10umol/L) significantly affect the basal level of cell apoptosis in 25mmol/L glucose, reducing cell apoptosis by approximately 80% and recovering the arrested G0/G1 phase to the normal.3.There were a total of close to 600 protein spots in each gel, after the intra- and inter-group cross-match, 51 spots significantly altered and were identified as high glucose-induced and PKCδ-associated proteins. All the 51 spots gave satisfactory MALDI-MS spectra.4. Finally, we classified all the 37 proteins identified by bioinformatics analysis according to their known function involving in (1) cell cycle and apoptosis regulation, (2) tumor suppression, (3) transcription, (4)stress, (5)signal transduction in nucleus and so on. Among them, SGK1, CCND3, CDKN2A, hLin-9, RAPL and GRP78 participated in various regulating pathway for apoptosis and cell cycle.Conclusion:Our data elucidate that PKC-δis clearly an important mediator of cell apoptosis and cycle arrest pathway with diverse downstream effectors such as SGK1, CCND3, CDKN2A, hLin-9, RAPL and GRP78 in HUVEC under high glucose stress. Cell apoptosis occurs early as an initial event in diabetic vascular complication, and the mechanism of this cell death is not fully known. Therefore, this finding provide new insights into the mechanism of diabetes associated blood vessel damage, showing that specific inhibitors for PKC-δor its key downstream effector may have potential for therapeutic agents for the prevention of human diabetic macro-vascular and micro-vascular complications.