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Subcellular proteomic approach for identifying the signaling effectors of protein kinase C-?? under high glucose conditions in human umbilical vein endothelial cells.

ABSTRACT: The high glucose?induced activation of protein kinase C??2 (PKC??2) has an essential role in the pathophysiology of diabetes?associated vascular disease. In the present study, human umbilical vein endothelial cells (HUVECs) were cultured in high and normal glucose conditions prior to being infected with a recombinant adenovirus to induce the overexpression of PKC??2. The activity of PKC??2 was also decreased using a selective PKC??2 inhibitor. A series of two?dimensional electrophoresis images detected ~800 spots in the nuclei, and ~600 spots in the cytosol. Following intra? and inter?group cross?matching, 38 significantly altered spots were identified as high glucose?induced and PKC??2?associated nuclear proteins. In addition to the observation that the regulation of key proteins involved in the nuclear factor (NF)??B signaling cascade occurred in the cytosol, various transcription factors, including peroxisome proliferator?activated receptor ? (PPAR??), were also altered in the nuclei. A human protein?protein interaction network of potential connections of PKC??2?associated proteins was constructed in the proteomics investigation using Biological General Repository for Interaction Datasets. The results indicated that PKC??2 may be involved in high glucose?induced glucose and lipid crosstalk by regulating PPAR??. In addition, NF??B inhibitor?interacting Ras?like protein 1 may be important in the PKC??2?NF??B inhibitor?NF??B signaling pathway in HUVECs under high?glucose conditions.

SUBMITTER: Zhang M

PROVIDER: S-EPMC4626174 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Subcellular proteomic approach for identifying the signaling effectors of protein kinase C-β₂ under high glucose conditions in human umbilical vein endothelial cells.

Zhang Min M Sun Fang F Chen Fangfang F Zhou Bo B Duan Yaqian Y Su Hong H Lin Xuebo X

Molecular medicine reports 20150930 5

The high glucose‑induced activation of protein kinase C‑β2 (PKC‑β2) has an essential role in the pathophysiology of diabetes‑associated vascular disease. In the present study, human umbilical vein endothelial cells (HUVECs) were cultured in high and normal glucose conditions prior to being infected with a recombinant adenovirus to induce the overexpression of PKC‑β2. The activity of PKC‑β2 was also decreased using a selective PKC‑β2 inhibitor. A series of two‑dimensional electrophoresis images d ...[more]

PMID: 26459836

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Project description:BackgroundThe balance between endothelial cell survival and apoptosis during stress is an important cellular process for vessel integrity and vascular homeostasis, and it is also pivotal in angiogenesis during the development of many vascular diseases. However, the underlying molecular mechanisms remain largely unknown. Although both transcription and alternative splicing are important in regulating gene expression in endothelial cells under stress, the regulatory mechanisms underlying this state and their interactions have not yet been studied on a genome-wide basis.ResultsHuman umbilical vein endothelial cells (HUVECs) were treated with cobalt chloride (CoCl2) both to mimic hypoxia and to induce cell apoptosis and alternative splicing responses. Cell apoptosis rate analysis indicated that HUVECs exposed to 300 microM CoCl2 for 24 hrs were initially counterbalancing apoptosis with cell survival. We therefore used the Affymetrix exon array system to determine genome-wide transcript- and exon-level differential expression. Other than 1583 differentially expressed transcripts, 342 alternatively spliced exons were detected and classified by different splicing types. Sixteen alternatively spliced exons were validated by RT-PCR. Furthermore, direct evidence for the ongoing balance between HUVEC survival and apoptosis was provided by Gene Ontology (GO) and protein function, as well as protein domain and pathway enrichment analyses of the differentially expressed transcripts. Importantly, a novel molecular module, in which the heat shock protein (HSP) families play a significant role, was found to be activated under mimicked hypoxia conditions. In addition, 46% of the transcripts containing stress-modulated exons were differentially expressed, indicating the possibility of combinatorial regulation of transcription and splicing.ConclusionThe exon array system effectively profiles gene expression and splicing on the genome-wide scale. Based on this approach, our data suggest that transcription and splicing not only regulate gene expression, but also carry out combinational regulation of the balance between survival and apoptosis of HUVECs under mimicked hypoxia conditions. Since cell survival following the apoptotic challenge is pivotal in angiogenesis during the development of many vascular diseases, our results may advance the knowledge of multilevel gene regulation in endothelial cells under physiological and pathological conditions.

Project description:Human umbilical vein endothelial cells (HUVECs) are a widely-used model system to study pathological and physiological processes associated with the cardiovascular system. An understanding of genes and proteins that are expressed in any cell type is a fundamental need which facilitates studies of molecular changes in disease states and response to various stimuli. In this study, we employed next generation sequencing and mass spectrometry to profile the transcriptome and proteome of primary HUVECs. Analysis of 145 million paired-end reads from next generation sequencing confirmed expression of 12,186 protein-coding genes (FPKM>0.1), 439 novel long non-coding RNAs and revealed 6,089 novel isoforms that were not annotated in GENCODE. A comparison of the transcripts against human gene expression data for 53 tissues catalogued by the Genotype-Tissue Expression (GTEx) project revealed a number of HUVEC-specific genes. Proteomics analysis identified 6,477 proteins including confirmation of N-termini for 1,091 proteins and isoforms for 149 proteins for which transcriptomic evidence was observed. Alternate translational start sites for seven proteins and alternate splicing in five proteins were also identified. A database search to specifically identify other post-translational modifications provided evidence for a number of modification sites on 117 proteins which included ubiquitylation, lysine acetylation and mono, di- and tri-methylation events. Based on the data from this study and a survey of other databases, we provide evidence for 11 “missing proteins,” which are proteins for which there was insufficient or no protein level evidence. Peptides supporting missing protein and novel events were validated by comparison of MS/MS fragmentation patterns with synthetic peptides. By creating a custom database of proteins containing sample-specific single amino acid variants (SAAV), we also identified 245 variant peptides derived from 207 expressed proteins. Overall, we believe that the integrated approach employed in this study is widely applicable to study any primary cell type for deeper molecular characterization.

Dataset Information

Subcellular proteomic approach for identifying the signaling effectors of protein kinase C-?? under high glucose conditions in human umbilical vein endothelial cells.

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Subcellular proteomic approach for identifying the signaling effectors of protein kinase C-β₂ under high glucose conditions in human umbilical vein endothelial cells.

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