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.

Roles of heat-shock protein 90 and its four domains (N, LR, M and C) in calcium oxalate stone-forming processes.

Human heat-shock protein 90 (HSP90) has four functional domains, including NH2-terminal (N), charged linker region (LR), middle (M) and COOH-terminal (C) domains. In kidney stone disease (or nephrolithiasis/urolithiasis), HSP90 serves as a receptor for calcium oxalate monohydrate (COM), which is the most common crystal to form kidney stones. Nevertheless, roles of HSP90 and its four domains in kidney stone formation remained unclear and under-investigated. We thus examined and compared their effects on COM crystals during physical (crystallization, growth and aggregation) and biological (crystal-cell adhesion and crystal invasion through extracellular matrix (ECM)) pathogenic processes of kidney stone formation. The analyses revealed that full-length (FL) HSP90 obviously increased COM crystal size and abundance during crystallization and markedly promoted crystal growth, aggregation, adhesion onto renal cells and ECM invasion. Comparing among four individual domains, N and C domains exhibited the strongest promoting effects, whereas LR domain had the weakest promoting effects on COM crystals. In summary, our findings indicate that FL-HSP90 and its four domains (N, LR, M and C) promote COM crystallization, crystal growth, aggregation, adhesion onto renal cells and invasion through the ECM, all of which are the important physical and biological pathogenic processes of kidney stone formation.

Systematic comparisons of various markers for mast cell activation in RBL-2H3 cells.

Mast cell activation plays a key role in various allergic diseases and anaphylaxis. Several methods/techniques can be used for detection of mast cell activation. However, there was no previous systematic evaluation to compare the efficacy of each method/technique. The present study thus systematically compared various markers for mast cell activation induced by IgE cross-linking. The widely used RBL-2H3 mast cells were sensitized with anti-DNP (dinitrophenyl) IgE overnight and activated with DNP-BSA (bovine serum albumin) for up to 4 h. The untreated cells and those with anti-DNP IgE sensitization but without DNP-BSA activation served as the controls. Intracellular calcium level gradually increased to ~2-fold at 1 h, reached its peak (~5-fold) at 2 h, and returned to the basal level at 3-h post-activation. The increases in cellular tryptase level (by Western blotting) (~0.3- to 0.4-fold) and average cell size (~2.5-fold) and decrease of nucleus/cytoplasm ratio (~0.4- to 0.5-fold) were marginal at all time-points. By contrast, ß-hexosaminidase release and CD63 expression (by both flow cytometry and immunofluorescence detection/localization), secreted tryptase level (by Western blotting), and tryptase expression (by immunofluorescence detection/localization) stably and obviously increased (~10-fold as compared with the untreated control and sensitized-only cells or detectable only after activation). Based on these data, the stably obvious increases (by ≥ 10-fold) in ß-hexosaminidase release, CD63 expression (by both flow cytometry and immunofluorescence staining), secreted tryptase level (by Western blotting), and tryptase expression (by immunofluorescence staining) are recommended as the markers of choice for the in vitro study of mast cell activation using RBL-2H3 cells.

Defining physicochemical properties of urinary proteins that determine their inhibitory activities against calcium oxalate kidney stone formation.

We have recently reported a set of urinary proteins that inhibited calcium oxalate (CaOx) stone development. However, physicochemical properties that determine their inhibitory activities remained unknown. Herein, human urinary proteins were chromatographically fractionated into 15 fractions and subjected to various CaOx crystal assays and identification by nanoLC-ESI-Qq-TOF MS/MS. Their physicochemical properties and crystal inhibitory activities were subjected to Pearson correlation analysis. The data showed that almost all urinary protein fractions had crystal inhibitory activities. Up to 128 proteins were identified from each fraction. Crystallization inhibitory activity correlated with percentages of Ca2+-binding proteins, stable proteins, polar amino acids, alpha helix, beta turn, and random coil, but inversely correlated with number of Ox2--binding motifs/protein and percentage of unstable proteins. Crystal aggregation inhibitory activity correlated with percentage of stable proteins but inversely correlated with percentage of unstable proteins. Crystal adhesion inhibitory activity correlated with percentage of stable proteins and GRAVY, but inversely correlated with pI, instability index and percentages of unstable proteins and positively charged amino acids. However, there was no correlation between crystal growth inhibitory activity and any physicochemical properties. In summary, some physicochemical properties of urinary proteins can determine and may be able to predict their CaOx stone inhibitory activities.

Comparative analyses of various IgE-mediated and non-IgE-mediated inducers of mast cell degranulation for in vitro study.

In vitro investigations of mast cell (MC) degranulation are essential for studying many diseases, particularly allergy and urticaria. Many MC-degranulation inducers are currently available. However, there is no previous systematic comparative analysis of these available inducers in term of their efficacies to induce MC degranulation. Herein, we performed systematic comparisons of efficacies of five well-known and commonly used MC-degranulation inducers. RBL-2H3 cells were sensitized with 50 ng/ml anti-DNP IgE or biotinylated IgE followed by stimulation with 100 ng/ml DNP-BSA or streptavidin, respectively. For non-IgE-mediated inducers, the cells were treated with 5 µg/ml substance P, compound 48/80, or A23187. At 15-, 30-, 45- and 60-min post-induction, several common MC-degranulation markers (including intracellular [Ca2+], ß-hexosaminidase release, tryptase expression by immunofluorescence staining, cellular tryptase level by immunoblotting, secretory tryptase level by immunoblotting, CD63 expression by immunofluorescence staining, and CD63 expression by flow cytometry) were evaluated. The data showed that all these markers significantly increased after activation by all inducers. Among them, A23187 provided the greatest degrees of increases in intracellular [Ca2+] and ß-hexosaminidase release at all time-points and upregulation of CD63 at one time-point. These data indicate that all these IgE-mediated (anti-DNP IgE/DNP-BSA and biotinylated IgE/streptavidin) and non-IgE-mediated (substance P, compound 48/80, and A23187) inducers effectively induce MC degranulation, while A23187 seems to be the most effective inducer for MC degranulation.

Large-scale identification of calcium oxalate stone inhibitory proteins in normal human urine.

Recent evidence has shown that proteins in normal human urine can inhibit calcium oxalate (CaOx) kidney stone formation. Herein, we performed fast protein liquid chromatography (FPLC) to fractionate normal human urinary proteins using anion-exchange (DEAE) and size-exclusion (Superdex 200) materials. FPLC fractions (F1-F15) were examined by CaOx crystallization, growth, aggregation and crystal-cell adhesion assays. The fractions with potent inhibitory activities against CaOx crystals were then subjected to mass spectrometric protein identification. The data revealed that 13 of 15 fractions showed inhibitory activities in at least one crystal assay. Integrating CaOx inhibitory scores demonstrated that F6, F7 and F8 had the most potent inhibitory activities. NanoLC-ESI-Qq-TOF MS/MS identified 105, 93 and 53 proteins in F6, F7 and F8, respectively. Among them, 60 were found in at least two fractions and/or listed among known inhibitors with solid experimental evidence in the StoneMod database (https://www.stonemod.org). Interestingly, 10 of these 60 potential inhibitors have been reported with lower urinary levels in CaOx stone formers compared with healthy (non-stone) individuals, strengthening their roles as potent CaOx stone inhibitors. Our study provides the largest dataset of potential CaOx stone inhibitory proteins that will be useful for further elucidations of stone-forming mechanisms and ultimately for therapeutic/preventive applications.

Epigenetic regulation of epithelial-mesenchymal transition during cancer development.

Epithelial-mesenchymal transition (EMT) plays essential roles in promoting malignant transformation of epithelial cells, leading to cancer progression and metastasis. During EMT-induced cancer development, a wide variety of genes are dramatically modified, especially down-regulation of epithelial-related genes and up-regulation of mesenchymal-related genes. Expression of other EMT-related genes is also modified during the carcinogenic process. Especially, epigenetic modifications are observed in the EMT-related genes, indicating their involvement in cancer development. Mechanically, epigenetic modifications of histone, DNA, mRNA and non-coding RNA stably change the EMT-related gene expression at transcription and translation levels. Herein, we summarize current knowledge on epigenetic regulatory mechanisms observed in EMT process relate to cancer development in humans. The better understanding of epigenetic regulation of EMT during cancer development may lead to improvement of drug design and preventive strategies in cancer therapy.

Bioinformatics and computational analyses of kidney stone modulatory proteins lead to solid experimental evidence and therapeutic potential.

In recent biomedical research, bioinformatics and computational analyses have played essential roles for examining experimental findings and database information. Several bioinformatic tools have been developed and made publicly available for analyzing protein sequence, structure, functional motif/domain, and interactions network. Such properties are very helpful to define biochemical and functional roles of the protein(s) of interest. During the past few decades, bioinformatics and computational biotechnology have been widely applied to kidney stone research. This review summarizes commonly used tools and evidence of bioinformatics and computational biotechnology applied to kidney stone disease (KSD) with special emphasis on analyses of the stone modulatory proteins that play critical roles in kidney stone formation. Such analyses lead to solid experimental evidence to demonstrate mechanisms underlying their stone modulatory activities. The findings obtained from such analyses may also lead to better understanding of KSD pathogenesis and to further development of new therapeutic and preventive strategies.

Comparative analysis of markers for H2O2-induced senescence in renal tubular cells.

To address what marker(s) is/are most suitable for determining renal cell senescence, cell area, granularity, cycle shift/arrest, SA-ß-Gal, SIRT1 and p16 were evaluated after inducing senescence in HK-2 cells with 0.2-0.8 mM H2O2. Only cell area and granularity concentration-dependently increased at all time-points, whereas SA-ß-Gal, SIRT1 and p16 showed significant coefficient of determination (R2) at two time-points. Cell granularity had significant correlation coefficient (R) with other six, whereas SA-ß-Gal had significant R with five, and cell area, SIRT1 and p16 had significant R with four others. Comparing to SA-ß-Gal, other markers had significantly lower fold-changes only at 72-h with 0.8 mM H2O2, whereas p16 provided greater fold-changes at 48-h with 0.4 and 0.8 mM H2O2. Therefore, cell area, granularity, SA-ß-Gal and p16 may serve as the most suitable markers for determining H2O2-induced senescence in HK-2 renal cells, whereas other markers can be also used but with inferior quantitative precision.

ARID1A knockdown enhances carcinogenesis features and aggressiveness of Caco-2 colon cancer cells: An in vitro cellular mechanism study.

Loss of ARID1A, a tumor suppressor gene, is associated with the higher grade of colorectal cancer (CRC). However, molecular and cellular mechanisms underlying the progression and aggressiveness of CRC induced by the loss of ARID1A remain poorly understood. Herein, we evaluated cellular mechanisms underlying the effects of ARID1A knockdown on the carcinogenesis features and aggressiveness of CRC cells. A human CRC cell line (Caco-2) was transfected with small interfering RNA (siRNA) specific to ARID1A (siARID1A) or scrambled (non-specific) siRNA (siControl). Cell death, proliferation, senescence, chemoresistance and invasion were then evaluated. In addition, formation of polyploid giant cancer cells (PGCCs), self-aggregation (multicellular spheroid) and secretion of an angiogenic factor, vascular endothelial growth factor (VEGF), were examined. The results showed that ARID1A knockdown led to significant decreases in cell death and senescence. On the other hand, ARID1A knockdown enhanced cell proliferation, chemoresistance and invasion. The siARID1A-transfected cells also had greater number of PGCCs and larger spheroid size and secreted greater level of VEGF compared with the siControl-transfected cells. These data, at least in part, explain the cellular mechanisms of ARID1A deficiency in carcinogenesis and aggressiveness features of CRC.

Systematic analysis of modulating activities of native human urinary Tamm-Horsfall protein on calcium oxalate crystallization, growth, aggregation, crystal-cell adhesion and invasion through extracellular matrix.

Functions of Tamm-Horsfall protein (THP), the most abundant human urinary protein, have been studied for decades. However, its precise roles in kidney stone formation remain controversial. In this study, we aimed to clarify the roles of native human urinary THP in calcium oxalate monohydrate (COM) kidney stone formation. THP was purified from the human urine by adsorption method using diatomaceous earth (DE). Its effects on stone formation processes, including COM crystallization, crystal growth, aggregation, crystal-cell adhesion and invasion through extracellular matrix (ECM), were examined. SDS-PAGE and Western blotting confirmed that DE adsorption yielded 84.9% purity of the native THP isolated from the human urine. Systematic analyses revealed that THP (at 0.4-40 µg/ml) concentration-dependently reduced COM crystal size but did not affect the crystal mass during initial crystallization. At later steps, THP concentration-dependently inhibited COM crystal growth and aggregation, and prevented crystal-cell adhesion only at 40 µg/ml. However, THP did not affect crystal invasion through the ECM. Sequence analysis revealed two large calcium-binding domains (residues 65-107 and 108-149) and three small oxalate-binding domains (residues 199-207, 361-368 and 601-609) in human THP. Immunofluorescence study confirmed the binding of THP to COM crystals. Analyses for calcium-affinity and/or oxalate-affinity demonstrated that THP exerted a high affinity with only calcium, not oxalate. Functional validation revealed that saturation of THP with calcium, not with oxalate, could abolish the inhibitory effects of THP on COM crystal growth, aggregation and crystal-cell adhesion. These data highlight the inhibitory roles of the native human urinary THP in COM crystal growth, aggregation and crystal-cell adhesion, which are the important processes for kidney stone formation. Such inhibitory effects of THP are most likely mediated via its high affinity with calcium ions.

Dual modulatory effects of diosmin on calcium oxalate kidney stone formation processes: Crystallization, growth, aggregation, crystal-cell adhesion, internalization into renal tubular cells, and invasion through extracellular matrix.

Diosmin is a natural flavone glycoside (bioflavonoid) found in fruits and plants with several pharmacological activities. It has been widely used as a dietary supplement or therapeutic agent in various diseases/disorders. Although recommended, evidence of its protective mechanisms against kidney stone disease (nephrolithiasis/urolithiasis), especially calcium oxalate (CaOx) monohydrate (COM) that is the most common type, remained unclear. In this study, we thus systematically evaluated the effects of diosmin (at 2.5-160 nM) on various stages of kidney stone formation processes, including COM crystallization, crystal growth, aggregation, crystal-cell adhesion, internalization into renal tubular cells and invasion through extracellular matrix (ECM). The results showed that diosmin had dose-dependent modulatory effects on all the mentioned COM kidney stone processes. Diosmin significantly increased COM crystal number and mass during crystallization, but reduced crystal size and growth. While diosmin promoted crystal aggregation, it inhibited crystal-cell adhesion and internalization into renal tubular cells. Finally, diosmin promoted crystal invasion through the ECM. Our data provide evidence demonstrating both inhibiting and promoting effects of diosmin on COM kidney stone formation processes. Based on these dual modulatory activities of diosmin, its anti-urolithiasis role is doubtful and cautions should be made for its use in kidney stone disease.

ARID1A knockdown in human endothelial cells directly induces angiogenesis by regulating angiopoietin-2 secretion and endothelial cell activity.

AT-rich interactive domain 1A (ARID1A) is a novel tumor suppressor gene found in several human cells and its loss/defect is commonly observed in many cancers. However, its roles in angiogenesis, which is one of the hallmarks for tumor progression, remained unclear. Herein, we demonstrated the direct effects of ARID1A knockdown in human endothelial cells by lentivirus-based short-hairpin RNA (shRNA) (shARID1A) on angiogenesis. Functional assays revealed that shARID1A significantly enhanced cell proliferation and migration/invasion and endothelial tube formation compared with the control cells transfected with scramble shRNA (shControl). Additionally, the shARID1A-transfected cells had significantly increased podosome formation and secretion of angiopoietin-2 (ANG2), a key angiogenic factor. Moreover, neutralization of ANG2 with monoclonal anti-ANG2 antibody strongly reduced cell proliferation and migration/invasion and endothelial tube formation in the shARID1A-transfected cells. These findings indicate that down-regulation of ARID1A in human endothelial cells directly induces angiogenesis by regulating angiopoietin-2 secretion and endothelial cell activity.

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.

Effects of secretome derived from macrophages exposed to calcium oxalate crystals on renal fibroblast activation.

The association between kidney stone disease and renal fibrosis has been widely explored in recent years but its underlying mechanisms remain far from complete understanding. Using label-free quantitative proteomics (nanoLC-ESI-LTQ-Orbitrap MS/MS), this study identified 23 significantly altered secreted proteins from calcium oxalate monohydrate (COM)-exposed macrophages (COM-MP) compared with control macrophages (Ctrl-MP) secretome. Functional annotation and protein-protein interactions network analysis revealed that these altered secreted proteins were involved mainly in inflammatory response and fibroblast activation. BHK-21 renal fibroblasts treated with COM-MP secretome had more spindle-shaped morphology with greater spindle index. Immunofluorescence study and gelatin zymography revealed increased levels of fibroblast activation markers (α-smooth muscle actin and F-actin) and fibrotic factors (fibronectin and matrix metalloproteinase-9 and -2) in the COM-MP secretome-treated fibroblasts. Our findings indicate that proteins secreted from macrophages exposed to COM crystals induce renal fibroblast activation and may play important roles in renal fibrogenesis in kidney stone disease.

Caffeine inhibits hypoxia-induced renal fibroblast activation by antioxidant mechanism.

Caffeine has been demonstrated to possess anti-fibrotic activity against liver fibrosis. However, its role in renal fibrosis remained unclear. This study investigated the effects of caffeine on renal fibroblast activation induced by hypoxia (one of the inducers for renal fibrosis). BHK-21 fibroblasts were cultured under normoxia or hypoxia with or without caffeine treatment. Hypoxia increased levels of fibronectin, α-smooth muscle actin, actin stress fibers, intracellular reactive oxygen species (ROS), and oxidized proteins. However, caffeine successfully preserved all these activated fibroblast markers to their basal levels. Cellular catalase activity was dropped under hypoxic condition but could be reactivated by caffeine. Hif1a gene and stress-responsive Nrf2 signaling molecule were elevated/activated by hypoxia, but only Nrf2 could be partially recovered by caffeine. These data suggest that caffeine exhibits anti-fibrotic effect against hypoxia-induced renal fibroblast activation through its antioxidant property to eliminate intracellular ROS, at least in part, via downstream catalase and Nrf2 mechanisms.

Phytohormone priming elevates the accumulation of defense-related gene transcripts and enhances bacterial blight disease resistance in cassava.

Cassava bacterial blight (CBB) disease caused by Xanthomonas axonopodis pv. manihotis (Xam) is a severe disease in cassava worldwide. In addition to causing significant cassava yield loss, CBB disease has not been extensively studied, especially in terms of CBB resistance genes. The present research demonstrated the molecular mechanisms underlining the defense response during Xam infection in two cassava cultivars exhibiting different degrees of disease resistance, Huay Bong60 (HB60) and Hanatee (HN). Based on gene expression analysis, ten of twelve putative defense-related genes including, leucine-rich repeat receptor-like kinases (LRR-RLKs), resistance (R), WRKY and pathogenesis-related (PR) genes, were differentially expressed between these two cassava cultivars during Xam infection. The up-regulation of defense-related genes observed in HB60 may be the mechanism required for the reduction of disease severity in the resistant cultivar. Interestingly, priming with salicylic acid (SA) or methyl jasmonate (MeJA) for 24 h before Xam inoculation could enhance the defense response in both cassava cultivars. The disease severity was decreased 10% in the resistant cultivar (HB60) and was remarkably reduced 21% in the susceptible cultivar (HN) by SA/MeJA priming. Priming with Xam inoculation modulated cassava4.1_013417, cassava4.1_030866 and cassava4.1_020555 (highest similarity to MeWRKY59, MePR1 and AtPDF2.2, respectively) expression and led to enhanced resistance of the susceptible cultivar in the second infection. The putative cis-regulatory elements were predicted in an upstream region of these three defense-related genes. The different gene expression levels in these genes between the two cultivars were due to the differences in cis-regulatory elements in their promoter regions. Taken together, our study strongly suggested that the induction of defense-related genes correlated with defense resistance against Xam infection, and exogenous application of SA or MeJA could elevate the defense response in both cultivars of cassava. This finding should pave the way for management to reduce yield loss from disease and genetic improvement in cassava.