IL-33 Is Involved in the Anti-Inflammatory Effects of Butyrate and Propionate on TNFα-Activated Endothelial Cells

Short-chain fatty acids (e.g., butyrate and propionate) are able to diminish endothelial cell activation. The aim of this study was to investigate whether intracellular IL-33 mediates the effects of butyrate and propionate on TNFα-induced IL-8 production and vascular cell adhesion molecule-1 (VCAM-1) expression. In addition, it was investigated whether regulating NF-κB and MAPK signaling pathways are involved. Intracellular IL-33 was measured in human endothelial cells (HUVECs) pre-incubated for 24 h with butyrate (0.1 mM or 5 mM), propionate (0.3 mM or 10 mM), or trichostatin A (TSA, 0.5 μM) prior to TNFα (1 ng/mL) stimulation (24 h). The effects of butyrate, propionate, and TSA on TNFα-induced IL-8, vascular cell adhesion molecule-1 (VCAM-1), NF-κB, and MAPK signaling pathways in normal HUVECs and IL-33 siRNA (siIL-33)-transfected HUVECs were compared to study the role of IL-33 in the protective effects of butyrate and propionate. Endogenous IL-33 was highly expressed in the perinuclear in HUVECs, which was significantly reduced by TNFα stimulation. The TNFα-induced reduction in IL-33 was prevented by pre-incubation with butyrate or propionate. Butyrate (0.1 mM), propionate (0.3 mM), and TSA inhibited the IL-8 production and activation of NF-κB. Interestingly, this effect was not observed in siIL-33-transfected HUVECs. The effects of butyrate (5 mM), propionate (10 mM), and TSA (0.5 μM) on VCAM-1 expression and activation of MAPK signaling pathways were not affected by siIL-33 transfection. In conclusion, we showed that the inhibitory effects of butyrate and propionate on TNFα-induced IL-8 production were mediated by the HDACs/IL-33/NF-κB pathway, while their effects on VCAM-1 expression might be associated with the HDACs/MAPK signaling pathway, independently of IL-33.     

Monitoring Human Milk β-Casein Phosphorylation and O-Glycosylation Over Lactation Reveals Distinct Differences between the Proteome and Endogenous Peptidome

Human milk is a vital biofluid containing a myriad of molecular components to ensure an infant’s best start at a healthy life. One key component of human milk is β-casein, a protein which is not only a structural constituent of casein micelles but also a source of bioactive, often antimicrobial, peptides contributing to milk’s endogenous peptidome. Importantly, post-translational modifications (PTMs) like phosphorylation and glycosylation typically affect the function of proteins and peptides; however, here our understanding of β-casein is critically limited. To uncover the scope of proteoforms and endogenous peptidoforms we utilized mass spectrometry (LC-MS/MS) to achieve in-depth longitudinal profiling of β-casein from human milk, studying two donors across 16 weeks of lactation. We not only observed changes in β-casein’s known protein and endogenous peptide phosphorylation, but also in previously unexplored O-glycosylation. This newly discovered PTM of β-casein may be important as it resides on known β-casein-derived antimicrobial peptide sequences.     

UVB-Stimulated TNFα Release from Human Melanocyte and Melanoma Cells Is Mediated by p38 MAPK

Ultraviolet (UV) radiation activates cell signaling pathways in melanocytes. As a result of altered signaling pathways and UV-induced cellular damage, melanocytes can undergo oncogenesis and develop into melanomas. In this study, we investigated the effect of UV-radiation on p38 MAPK (mitogen-activated protein kinase), JNK and NFκB pathways to determine which plays a major role in stimulating TNFα secretion in human HEM (melanocytes) and MM96L (melanoma) cells. MM96L cells exhibited 3.5-fold higher p38 activity than HEM cells at 5 min following UVA + B radiation and 1.6-fold higher JNK activity at 15–30 min following UVB+A radiation, while NFκB was minimally activated in both cells. Irradiated HEM cells had the greatest fold of TNFα secretion (UVB: 109-fold, UVA + B: 103-fold & UVB+A: 130-fold) when co-exposed to IL1α. The p38 inhibitor, SB202190, inhibited TNFα release by 93% from UVB-irradiated HEM cells. In the UVB-irradiated MM96L cells, both SB202190 and sulfasalazine (NFκB inhibitor) inhibited TNFα release by 52%. Although, anisomycin was a p38 MAPK activator, it inhibited TNFα release in UV-irradiated cells. This suggests that UV-mediated TNFα release may occur via different p38 pathway intermediates compared to those stimulated by anisomycin. As such, further studies into the functional role p38 MAPK plays in regulating TNFα release in UV-irradiated melanocyte-derived cells are warranted.     

14-3-3γ Regulates Lipopolysaccharide-Induced Inflammatory Responses and Lactation in Dairy Cow Mammary Epithelial Cells by Inhibiting NF-κB and MAPKs and Up-Regulating mTOR Signaling

As a protective factor for lipopolysaccharide (LPS)-induced injury, 14-3-3γ has been the subject of recent research. Nevertheless, whether 14-3-3γ can regulate lactation in dairy cow mammary epithelial cells (DCMECs) induced by LPS remains unknown. Here, the anti-inflammatory effect and lactation regulating ability of 14-3-3γ in LPS-induced DCMECs are investigated for the first time, and the molecular mechanisms responsible for their effects are explored. The results of qRT-PCR showed that 14-3-3γ overexpression significantly inhibited the mRNA expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) and inducible nitric oxide synthase (iNOS). Enzyme-linked immunosorbent assay (ELISA) analysis revealed that 14-3-3γ overexpression also suppressed the production of TNF-α and IL-6 in cell culture supernatants. Meanwhile, CASY-TT Analyser System showed that 14-3-3γ overexpression clearly increased the viability and proliferation of cells. The results of kit methods and western blot analysis showed that 14-3-3γ overexpression promoted the secretion of triglycerides and lactose and the synthesis of β-casein. Furthermore, the expression of genes relevant to nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPKs) and lactation-associated proteins were assessed by western blot, and the results suggested that 14-3-3γ overexpression inactivated the NF-κB and MAPK signaling pathways by down-regulating extracellular signal regulated protein kinase (ERK), p38 mitogen-activated protein kinase (p38MAPK) and inhibitor of NF-κB (IκB) phosphorylation levels, as well as by inhibiting NF-κB translocation. Meanwhile, 14-3-3γ overexpression enhanced the expression levels of β-casein, mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase 1 (S6K1), serine/threonine protein kinase Akt 1 (AKT1), sterol regulatory element binding protein 1 (SREBP1) and peroxisome proliferator-activated receptor gamma (PPARγ). These results suggest that 14-3-3γ was able to attenuate the LPS-induced inflammatory responses and promote proliferation and lactation in LPS-induced DCMECs by inhibiting the activation of the NF-κB and MAPK signaling pathways and up-regulating mTOR signaling pathways to protect against LPS-induced injury.     

Comparative Molecular Dynamics Simulations of Mitogen-Activated Protein Kinase-Activated Protein Kinase 5

The mitogen-activated protein kinase-activated protein kinase MK5 is a substrate of the mitogen-activated protein kinases p38, ERK3 and ERK4. Cell culture and animal studies have demonstrated that MK5 is involved in tumour suppression and promotion, embryogenesis, anxiety, cell motility and cell cycle regulation. In the present study, homology models of MK5 were used for molecular dynamics (MD) simulations of: (1) MK5 alone; (2) MK5 in complex with an inhibitor; and (3) MK5 in complex with the interaction partner p38α. The calculations showed that the inhibitor occupied the active site and disrupted the intramolecular network of amino acids. However, intramolecular interactions consistent with an inactive protein kinase fold were not formed. MD with p38α showed that not only the p38 docking region, but also amino acids in the activation segment, αH helix, P-loop, regulatory phosphorylation region and the C-terminal of MK5 may be involved in forming a very stable MK5-p38α complex, and that p38α binding decreases the residual fluctuation of the MK5 model. Electrostatic Potential Surface (EPS) calculations of MK5 and p38α showed that electrostatic interactions are important for recognition and binding.     

AKIP1, a Cardiac Hypertrophy Induced Protein that Stimulates Cardiomyocyte Growth via the Akt Pathway

Cardiac adaptation to unremitting physiological stress typically involves hypertrophic growth of cardiomyocytes, a compensatory response that often fails and causes heart disease. Gene array analysis identified AKIP1 (A Kinase Interacting Protein 1) as a hypertrophic gene and we therefore hypothesized a potential role in the hypertrophic response. We show for the first time that both AKIP1 mRNA and protein levels increased in hypertrophic cardiomyocytes under conditions of sustained cardiac stress, including pressure overload and after myocardial infarction and in vitro in phenylephrine (PE) stimulated neonatal rat ventricular cardiomyocytes (NRVCs). AKIP1 overexpression in NRVCs markedly stimulated hypertrophic growth responses, including significantly increased cell size, augmented cytoskeletal organization and protein synthesis. Although, AKIP1 was not essential for PE induced hypertrophy in NRVCs, it did potentiate neurohormonal induced protein synthesis. AKIP1 did, however, not induce expression of pathological marker genes like ANP and β-MHC. ERK and Akt kinase signaling pathways have been linked to hypertrophy and AKIP1 specifically induced phosphorylation of Akt. This phosphorylation of Akt was essential for activation of ribosomal rpS6 and translation elongation factor eEF2 and this readily explains the increased protein synthesis. Akt inhibition fully blocked AKIP1 induced hypertrophy, showing that this pathway is critically involved. In conclusion, our results show that AKIP1 is induced in hypertrophic hearts and can stimulate adaptive cardiomyocyte growth, which involves Akt signaling.     

Inflammatory Regulation by TNF-α-Activated Adipose-Derived Stem Cells in the Human Bladder Cancer Microenvironment

Mesenchymal stem cells (MSCs), such as adipose-derived stem cells (ADSCs), have the most impressive ability to reduce inflammation through paracrine growth factors and cytokines that participate in inflammation. Tumor necrosis factor (TNF)-α bioactivity is a prerequisite in several inflammatory and autoimmune disease models. This study investigated the effects of TNF-α stimulate on ADSCs in the tumor microenvironment. The RNAseq analysis and cytokines assay demonstrated that TNF-α stimulated ADSCs proliferation and pro-inflammatory genes that correlated to leukocytes differentiation were upregulated. We found that upregulation of TLR2 or PTGS2 toward to IRF7 gene-associated with immunomodulatory and antitumor pathway under TNF-α treatment. In TNF-α-treated ADSCs cultured with the bladder cancer (BC) cell medium, the results showed that apoptosis ratio and OCT-4 and TLR2 genes which maintained the self-renewal ability of stem cells were decreased. Furthermore, the cell survival regulation genes including TRAF1, NF-kB, and IRF7 were upregulated in TNF-α-treated ADSCs. Additionally, these genes have not been upregulated in BC cell medium. A parallel study showed that tumor progressing genes were downregulated in TNF-α-treated ADSCs. Hence, the study suggests that TNF-α enhances the immunomodulatory potential of ADSCs during tumorigenesis and provides insight into highly efficacious MSC-based therapeutic options for BC.     

miR-200c-3p Regulates Epitelial-to-Mesenchymal Transition in Epicardial Mesothelial Cells by Targeting Epicardial Follistatin-Related Protein 1

Recent findings suggest that epithelial to mesenchymal transition (EMT), a key step during heart development, is involved in cardiac tissue repair following myocardial infarction (MI). MicroRNAs (miRNAs) act as key regulators in EMT processes; however, the mechanisms by which miRNAs target epicardial EMT remain largely unknown. Here, by using an in vitro model of epicardial EMT, we investigated the role of miRNAs as regulators of this process and their potential targets. EMT was induced in murine epicardial-mesothelial cells (EMCs) through TGF β1 treatment for 48, 72, and 96 h as indicated by the expression of EMT-related genes by qRT-PCR, WB, and immunofluorescence. Further, enhanced expression of stemness genes was also detected. Among several EMT-related miRNAs, miR-200c-3p expression resulted as the most strongly suppressed. Interestingly, we also found a significant upregulation of Follistatin-related protein 1 (FSTL1), a miR-200c predicted target already identified as a potent cardiogenic factor produced by epicardial cells that promotes regeneration following MI. Dual-luciferase reporter assay demonstrated that miR-200c-3p directly targeted the 3′-untranslated region of FSTL1 in EMCs. Consistently, WB analysis showed that knockdown of miR-200c-3p significantly increased FSTL1 expression, whereas overexpression of miR-200c-3p counteracted TGF β1-mediated FSTL1 upregulation. Importantly, FSTL1 silencing maintained epithelial features in EMCs, despite EMT induction by TGF β1, and attenuated EMT-associated traits, including migration and stemness. In conclusion, epicardial FSTL1, an important cardiogenic factor in its secreted form, induces EMT, stemness, and migration of EMCs in a miR-200c-3p dependent pathway.     

DAG/PKCδ and IP3/Ca2+/CaMK IIβ Operate in Parallel to Each Other in PLCγ1-Driven Cell Proliferation and Migration of Human Gastric Adenocarcinoma Cells,through Akt/mTOR/S6 Pathway

Phosphoinositide specific phospholipase Cγ (PLCγ) activates diacylglycerol (DAG)/protein kinase C (PKC) and inositol 1,4,5-trisphosphate (IP3)/Ca²⁺/calmodulin-dependent protein kinase II (CaMK II) axes to regulate import events in some cancer cells, including gastric adenocarcinoma cells. However, whether DAG/PKCδ and IP3/Ca²⁺/CaMK IIβ axes are simultaneously involved in PLCγ1-driven cell proliferation and migration of human gastric adenocarcinoma cells and the underlying mechanism are not elucidated. Here, we investigated the role of DAG/PKCδ or CaMK IIβ in PLCγ1-driven cell proliferation and migration of human gastric adenocarcinoma cells, using the BGC-823 cell line. The results indicated that the inhibition of PKCδ and CaMK IIβ could block cell proliferation and migration of BGC-823 cells as well as the effect of inhibiting PLCγ1, including the decrease of cell viability, the increase of apoptotic index, the down-regulation of matrix metalloproteinase (MMP) 9 expression level, and the decrease of cell migration rate. Both DAG/PKCδ and CaMK IIβ triggered protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/S6 pathway to regulate protein synthesis. The data indicate that DAG/PKCδ and IP3/Ca²⁺/CaMK IIβ operate in parallel to each other in PLCγ1-driven cell proliferation and migration of human gastric adenocarcinoma cells through Akt/mTOR/S6 pathway, with important implication for validating PLCγ1 as a molecular biomarker in early gastric cancer diagnosis and disease surveillance.     

Bacterial Outer Membrane Protein OmpX Regulates β1 Integrin and Epidermal Growth Factor Receptor (EGFR) Involved in Invasion of M-HeLa Cells by Serratia proteamaculans

Opportunistic pathogen Serratia proteamaculans are able to penetrate the eukaryotic cells. The penetration rate can be regulated by bacterial surface protein OmpX. OmpX family proteins are able to bind to host cell surface to the epidermal growth factor receptor (EGFR) and the extracellular matrix protein fibronectin, whose receptors are in return the α5 β1 integrins. Here we elucidated the involvement of these host cell proteins in S. proteamaculans invasion. We have shown that, despite the absence of fibronectin contribution to S. proteamaculans invasion, β1 integrin was directly involved in invasion of M-HeLa cells. Herewith β1 integrin was not the only receptor that determines sensitivity of host cells to bacterial invasion. Signal transfer from EGFR was also involved in the penetration of these bacteria into M-HeLa cells. However, M-HeLa cells have not been characterized by large number of these receptors. It turned out that S. proteamaculans attachment to the host cell surface resulted in an increment of EGFR and β1 integrin genes expression. Such gene expression increment also caused Escherichia coli attachment, transformed with a plasmid encoding OmpX from S. proteamaculans. Thus, an OmpX binding to the host cell surface caused an increase in the EGFR and β1 integrin expression involved in S. proteamaculans invasion.     

Secretion of Human Protein C in Mouse Milk

To determine the production of recombinant human protein C (rec-hPC) in milk, we created two homozygous mice lines for the goat β-casein/hPC transgene. Females and males of both lines (#10 and #11) displayed normal growth, fertility, and lactated normally. The copy number of the transgene was about fivefold higher in #10 line as compared to #11 line. mRNA expression of the transgene was only detected in the mammary glands of both lines. Furthermore, mRNA expression was fourfold higher on day 7 than on day 1 during lactation. Northern blot analysis of mRNA expression in the #10 line of transgenic (Tg) mice indicated a strong expression of the transgene in the mammary glands after seven days of lactation. Comparison of rec-hPC protein level with that of mRNA in the mammary glands showed a very similar pattern. A 52-kDa band corresponding to the hPC protein was strongly detected in mammary glands of the #10 line during lactation. We also detected two bands of heavy chain and one weak band of light chain in the milk of the #10 and #11 lines. One single band at 52 kDa was detected from CHO cells transfected with hPC cDNA. hPC was mainly localized in the alveolar epithelial cell of the mammary glands. The protein is strongly expressed in the cytoplasm of the cultured mammary gland tissue. hPC protein produced in milk ranged from 2 to 28 ng/mL. These experiments indicated that rec-hPC can be produced at high levels in mice mammary glands.     

Transcriptional Modulation of the Hippo Signaling Pathway by Drugs Used to Treat Bipolar Disorder and Schizophrenia

Recent reports suggest a link between positive regulation of the Hippo pathway with bipolar disorder (BD), and the Hippo pathway is known to interact with multiple other signaling pathways previously associated with BD and other psychiatric disorders. In this study, neuronal-like NT2 cells were treated with amisulpride (10 µM), aripiprazole (0.1 µM), clozapine (10 µM), lamotrigine (50 µM), lithium (2.5 mM), quetiapine (50 µM), risperidone (0.1 µM), valproate (0.5 mM), or vehicle control for 24 h. Genome-wide mRNA expression was quantified and analyzed using gene set enrichment analysis (GSEA), with genes belonging to Hippo, Wnt, Notch, TGF- β, and Hedgehog retrieved from the KEGG database. Five of the eight drugs downregulated the genes of the Hippo pathway and modulated several genes involved in the interacting pathways. We speculate that the regulation of these genes, especially by aripiprazole, clozapine, and quetiapine, results in a reduction of MAPK and NFκB pro-inflammatory signaling through modulation of Hippo, Wnt, and TGF-β pathways. We also employed connectivity map analysis to identify compounds that act on these pathways in a similar manner to the known psychiatric drugs. Thirty-six compounds were identified. The presence of antidepressants and antipsychotics validates our approach and reveals possible new targets for drug repurposing.     

Combination of Aβ Secretion and Oxidative Stress in an Alzheimer-Like Cell Line Leads to the Over-Expression of the Nucleotide Excision Repair Proteins DDB2 and XPC

Repair of oxidative DNA damage, particularly Base Excision Repair (BER), impairment is often associated with Alzheimer’s disease pathology. Here, we aimed at investigating the complete Nucleotide Excision Repair (NER), a DNA repair pathway involved in the removal of bulky DNA adducts, status in an Alzheimer-like cell line. The level of DNA damage was quantified using mass spectrometry, NER gene expression was assessed by qPCR, and the NER protein activity was analysed through a modified version of the COMET assay. Interestingly, we found that in the presence of the Amyloid β peptide (Aβ), NER factors were upregulated at the mRNA level and that NER capacities were also specifically increased following oxidative stress. Surprisingly, NER capacities were not differentially improved following a typical NER-triggering of ultraviolet C (UVC) stress. Oxidative stress generates a differential and specific DNA damage response in the presence of Aβ. We hypothesized that the release of NER components such as DNA damage binding protein 2 (DDB2) and Xeroderma Pigmentosum complementation group C protein (XPC) following oxidative stress might putatively involve their apoptotic role rather than DNA repair function.     

Structures and Functions of Qβ Replicase: Translation Factors beyond Protein Synthesis

Qβ replicase is a unique RNA polymerase complex, comprising Qβ virus-encoded RNA-dependent RNA polymerase (the catalytic β-subunit) and three host-derived factors: translational elongation factor (EF) -Tu, EF-Ts and ribosomal protein S1. For almost fifty years, since the isolation of Qβ replicase, there have been several unsolved, important questions about the mechanism of RNA polymerization by Qβ replicase. Especially, the detailed functions of the host factors, EF-Tu, EF-Ts, and S1, in Qβ replicase, which are all essential in the Escherichia coli (E. coli) host for protein synthesis, had remained enigmatic, due to the absence of structural information about Qβ replicase. In the last five years, the crystal structures of the core Qβ replicase, consisting of the β-subunit, EF-Tu and Ts, and those of the core Qβ replicase representing RNA polymerization, have been reported. Recently, the structure of Qβ replicase comprising the β-subunit, EF-Tu, EF-Ts and the N-terminal half of S1, which is capable of initiating Qβ RNA replication, has also been reported. In this review, based on the structures of Qβ replicase, we describe our current understanding of the alternative functions of the host translational elongation factors and ribosomal protein S1 in Qβ replicase as replication factors, beyond their established functions in protein synthesis.     

Mechanisms Mediating the Regulation of Peroxisomal Fatty Acid Beta-Oxidation by PPARα

In mammalian cells, two cellular organelles, mitochondria and peroxisomes, share the ability to degrade fatty acid chains. Although each organelle harbors its own fatty acid β-oxidation pathway, a distinct mitochondrial system feeds the oxidative phosphorylation pathway for ATP synthesis. At the same time, the peroxisomal β-oxidation pathway participates in cellular thermogenesis. A scientific milestone in 1965 helped discover the hepatomegaly effect in rat liver by clofibrate, subsequently identified as a peroxisome proliferator in rodents and an activator of the peroxisomal fatty acid β-oxidation pathway. These peroxisome proliferators were later identified as activating ligands of Peroxisome Proliferator-Activated Receptor α (PPARα), cloned in 1990. The ligand-activated heterodimer PPARα/RXRα recognizes a DNA sequence, called PPRE (Peroxisome Proliferator Response Element), corresponding to two half-consensus hexanucleotide motifs, AGGTCA, separated by one nucleotide. Accordingly, the assembled complex containing PPRE/PPARα/RXRα/ligands/Coregulators controls the expression of the genes involved in liver peroxisomal fatty acid β-oxidation. This review mobilizes a considerable number of findings that discuss miscellaneous axes, covering the detailed expression pattern of PPARα in species and tissues, the lessons from several PPARα KO mouse models and the modulation of PPARα function by dietary micronutrients.     

CCN2 Increases TGF-β Receptor Type II Expression in Vascular Smooth Muscle Cells: Essential Role of CCN2 in the TGF-β Pathway Regulation

The cellular communication network factor 2 (CCN2/CTGF) has been traditionally described as a mediator of the fibrotic responses induced by other factors including the transforming growth factor β (TGF-β). However, several studies have defined a direct role of CCN2 acting as a growth factor inducing oxidative and proinflammatory responses. The presence of CCN2 and TGF-β together in the cellular context has been described as a requisite to induce a persistent fibrotic response, but the precise mechanisms implicated in this relation are not described yet. Considering the main role of TGF-β receptors (TβR) in the TGF-β pathway activation, our aim was to investigate the effects of CCN2 in the regulation of TβRI and TβRII levels in vascular smooth muscle cells (VSMCs). While no differences were observed in TβRI levels, an increase in TβRII expression at both gene and protein level were found 48 h after stimulation with the C-terminal fragment of CCN2 (CCN2(IV)). Cell pretreatment with a TβRI inhibitor did not modify TβRII increment induced by CCN2(VI), demonstrating a TGF-β-independent response. Secondly, CCN2(IV) rapidly activated the SMAD pathway in VSMCs, this being crucial in the upregulation of TβRII since the preincubation with an SMAD3 inhibitor prevented it. Similarly, pretreatment with the epidermal growth factor receptor (EGFR) inhibitor erlotinib abolished TβRII upregulation, indicating the participation of this receptor in the observed responses. Our findings suggest a direct role of CCN2 maintaining the TGF-β pathway activation by increasing TβRII expression in an EGFR-SMAD dependent manner activation.     

Association between the Hypomethylation of Osteopontin and Integrin β3 Promoters and Vascular Smooth Muscle Cell Phenotype Switching in Great Saphenous Varicose Veins

Lower extremity varicose veins are a common condition in vascular surgery and proliferation of vascular smooth muscle cells (VSMCs) in the intima is a significant pathological feature of varicosity. However, the pathogenesis of varicose veins is not fully understood. Osteopontin (OPN) could promote the migration and adhesion of VSMCs through the cell surface receptor integrin β3 and the cooperation of OPN and integrin β3 is involved in many vascular diseases. However, the role of OPN and integrin β3 in varicosity remains unclear. In the current study, we found that the methylation levels in the promoter regions of OPN and integrin β3 genes in the VSMCs of varicose veins are reduced and the protein expression of OPN and integrin β3 are increased, compared with normal veins. Furthermore, it was observed that VSMCs in the neointima of varicose veins were transformed into the synthetic phenotype. Collectively, hypomethylation of the promoter regions for OPN and integrin β3 genes may increase the expression of these genes in varicosity, which is closely related to VSMC phenotype switching. Hypomethylation of the promoter regions for OPN and integrin β3 genes may be a key factor in the pathogenesis of varicosity.     

The Effect and Mechanism of Tamoxifen-Induced Hepatocyte Steatosis in Vitro

The aim of this study was to determine the effect and mechanism of tamoxifen (TAM)-induced steatosis in vitro. HepG 2 (Human hepatocellular liver carcinoma cell line) cells were treated with different concentrations of TAM for 72 h. Steatosis of hepatocytes was determined after Oil Red O staining and measurement of triglyceride (TG) concentration. The expressions of genes in the TG homeostasis pathway, including sterol regulatory element-binding protein-1c (SREBP-1c), peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), stearoyl-CoA desaturase (SCD), carnitine palmitoyltransferase 1 (CPT1) and microsomal triglyceride transfer protein (MTP), were examined using quantitative real-time PCR and Western blot analysis. Cell proliferation was examined using the cell counting kit-8 (CCK-8) assay. We found that hepatocytes treated with TAM had: (1) induced hepatocyte steatosis and increased hepatocyte TG; (2) upregulation of SREBP-1c, FAS, ACC, SCD and MTP mRNA expressions (300%, 600%, 70%, 130% and 160%, respectively); (3) corresponding upregulation of protein expression; and (4) no difference in HepG 2 cell proliferation. Our results suggest that TAM can induce hepatocyte steatosis in vitro and that the enhancement of fatty acid synthesis through the upregulations of SREBP-1c and its downstream target genes (FAS, ACC and SCD) may be the key mechanism of TAM-induced hepatocyte steatosis.