Identification and characterization of ARID1A-interacting proteins in renal tubular cells and their molecular regulation of angiogenesis.

BACKGROUND: Defects and deficiency of AT-rich interactive domain-containing protein 1A (ARID1A) encoded by a tumor suppressor gene ARID1A have recently been suggested to get involved in angiogenesis, a crucial process in carcinogenesis. However, molecular mechanisms of ARID1A deficiency to induce angiogenesis in kidney cancer remain underinvestigated. METHODS: We performed large-scale identification of ARID1A protein interactors in renal tubular epithelial cells (RTECs) using immunoprecipitation (IP) followed by nanoLC-ESI-LTQ-Orbitrap tandem mass spectrometry (MS/MS). Their roles in angiogenesis were investigated using various assays. RESULTS: A total of 74 ARID1A-interacting proteins were identified. Protein-protein interactions analysis revealed that these identified proteins interacted directly or indirectly with ARID1A. Among them, the direct interaction between ARID1A and ß-actin was validated by IP and reciprocal IP followed by Western blotting. Small interfering RNA (siRNA) was used for single and double knockdowns of ARID1A and ACTB. Semi-quantitative RT-PCR demonstrated that deficiency of ARID1A, but not ACTB, significantly affected expression of angiogenesis-related genes in RTECs (VEGF and FGF2 were increased, whereas PDGF and EGF were decreased). However, the knockdowns did not affect TGFB1 and FGF1 levels. The quantitative mRNA expression data of VEGF and TGFB1 were consistent with the secreted levels of their protein products as measured by ELISA. Only secreted products derived from ARID1A-deficient RTECs significantly increased endothelial cells (ECs) migration and tube formation. Some of the other carcinogenic features could also be confirmed in the ARID1A-deficient RTECs, including increased cell migration and chemoresistance. Double knockdowns of both ARID1A and ACTB did not enhance the effects of single ARID1A knockdown in all assays. CONCLUSIONS: We report herein a large dataset of the ARID1A-interacting proteins in RTECs using an IP-MS/MS approach and confirm the direct interaction between ARID1A and ß-actin. However, the role of ARID1A deficiency in angiogenesis is independent of ß-actin.

Epigallocatechin-3-gallate plays more predominant roles than caffeine for inducing actin-crosslinking, ubiquitin/proteasome activity and glycolysis, and suppressing angiogenesis features of human endothelial cells.

A recent expression proteomics study has reported changes in cellular proteome (set of proteins) of human endothelial cells (ECs) induced by caffeine and epigallocatechin-3-gallate (EGCG), the most abundant bioactive compounds in coffee and green tea, respectively. Although both common and differential changes were highlighted by bioinformatics prediction, no experimental validation was performed. Herein, we reanalyzed these proteome datasets and performed protein-protein interactions network analysis followed by functional investigations using various assays to address the relevance of such proteome changes in human ECs functions. Protein-protein interactions network analysis revealed actin-crosslink formation, ubiquitin-proteasome activity and glycolysis as the three main networks among those significantly altered proteins induced by caffeine and EGCG. The experimental data showed predominant increases of actin-crosslink formation, ubiquitin-proteasome activity, and glycolysis (as reflected by increased F-actin and ß-actin, declined ubiquitinated proteins and increased intracellular ATP, respectively) in the EGCG-treated cells. Investigations on angiogenesis features revealed that EGCG predominantly reduced ECs proliferation, migration/invasion, endothelial tube formation (as determined by numbers of nodes/junctions and meshes), barrier function (as determined by levels of VE-cadherin, zonula occludens-1 (ZO-1) and transendothelial resistance (TER)), and angiopoietin-2 secretion. However, both caffeine and EGCG had no effects on matrix metalloproteinase-2 (MMP-2) secretion. These data indicate that EGCG exhibits more potent effects on human ECs functions to induce actin-crosslink, ubiquitin-proteasome activity and glycolysis, and to suppress angiogenesis processes that commonly occur in various diseases, particularly cancers.

High glucose induces phosphorylation and oxidation of mitochondrial proteins in renal tubular cells: A proteomics approach.

Mitochondrial dysfunction has been thought to play roles in the pathogenesis of diabetic nephropathy (DN). However, precise mechanisms underlying mitochondrial dysfunction in DN remained unclear. Herein, mitochondria were isolated from renal tubular cells after exposure to normal glucose (5.5 mM glucose), high glucose (25 mM glucose), or osmotic control (5.5 mM glucose + 19.5 mM mannitol) for 96 h. Comparative proteomic analysis revealed six differentially expressed proteins among groups that were subsequently identified by tandem mass spectrometry (nanoLC-ESI-ETD MS/MS) and confirmed by Western blotting. Several various types of post-translational modifications (PTMs) were identified in all of these identified proteins. Interestingly, phosphorylation and oxidation were most abundant in mitochondrial proteins whose levels were exclusively increased in high glucose condition. The high glucose-induced increases in phosphorylation and oxidation of mitochondrial proteins were successfully confirmed by various assays including MS/MS analyses. Moreover, high glucose also increased levels of phosphorylated ezrin, intracellular ATP and ROS, all of which could be abolished by a p38 MAPK inhibitor (SB239063), implicating a role of p38 MAPK-mediated phosphorylation in high glucose-induced mitochondrial dysfunction. These data indicate that phosphorylation and oxidation of mitochondrial proteins are, at least in part, involved in mitochondrial dysfunction in renal tubular cells during DN.