ALBERT

Description: Aberrant glycosylation by N -acetylgalactosaminyl transferases (GALNTs) is a well-described pathological alteration that is widespread in hereditary diseases, prominently including human cancers, familial tumoral calcinosis and hyperostosis-hyperphosphatemia. In this study, we integrated different computational tools to perform the in silico analysis of clinically significant mutations (nsSNPs/ single amino acid change) at both functional and structural levels, found in human GALNT3, GALNT8, GALNT12 and GALNT13 genes. From function and structure based insights, mutations encoding R162Q, T359K, C574G, G359D, R297W, Y396C & D313N substitutions were concordantly predicted highly deleterious for relevant GALNTs proteins. From intriguing findings, T359K- GALNT3 was simulated with high contribution for disease susceptibility (tumor calcinosis) as compared to its partner variant T272K [Ichikawa et al., 2006]. Similarly, the prediction of high damaging behavior, evolutionary conservation and structural destabilization for C574G were proposed as major contributing factors to regulate metabolic disorder underlying tumor calcinosis and hyperostosis-hyperphosphatemia syndrome. In case of R297W- GALNT12 , prediction of highly deleterious effect and disruption in ionic interactions were anticipated with reduction in enzymatic activity, associated with bilateral breast cancer and primary colorectal cancers. The second GALNT12 mutation (D303N)-known splice variant- was predicted with disease severity as a result of decrease in charge density and buried behavior neighboring the catalytic B domain. In the lack of adequate in silico data about systematic characterization of clinically significant mutations in GALNTs genes, current study can be used as a significant tool to interpret the role of GALNTs reaction chemistry in disease-association risks in body. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Description: Objective To investigate whether crosstalk between RUNX2 and miRNAs is involved in tooth eruption regulated by dental follicle cells(DFCs) and the possible molecular mechanism. Methods Blood samples and embedded dental follicles were collected from patients with cleidocranial dysplasia (CCD), and RUNX2 gene mutations were analyzed, then RUNX2 +/m DFCs were isolated and identified. The characteristics of RUNX2 +/m DFCs were analyzed. The differential expression of miRNAs was detected between the RUNX2 +/m DFCs and RUNX2 +/+ DFCs by microarray, and target genes were predicted by miRGen. miR-146a was chosen for further investigation, and its effects in DFCs were analyzed by transfecting its mimics and inhibitors, and expression of genes involved in tooth eruption were detected. Results A novel insertion mutation (c.309_310insTG) of RUNX2 gene was identified which had an effect on the characteristics of DFCs. Compared with the RUNX2 +/+ DFCs, there were 69 microRNAs more than 2-fold up-regulated and 54 microRNAs more than 2-fold down-regulated in the RUNX2 +/m DFCs. Among these, miR-146a decreased significantly in RUNX 2 +/m DFCs, and expression of RUNX2, CSF-1,EGFR and OPG was significantly altered when miR-146a was over-expressed or inhibited. Conclusion RUNX2 gene mutation contributes to the characteristic change of dental follicle cells, and the crosstalk between RUNX2 gene and miRNAs may be one of the key regulatory mechanisms of differentiation of dental follicle cells. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Description: Cancer stem cells (CSC) have a central role in driving tumor growth. Since metabolism is becoming an important diagnostic and therapeutic target, characterization of CSC line energetic properties is an emerging need. Embryonic and adult stem cells, compared to differentiated cells, exhibit a reduced mitochondrial activity and a stronger dependence on aerobic glycolysis. Here, we aimed to comparatively analyze bioenergetics features of the human osteosarcoma 3AB-OS CSC-like line, and the parental osteosarcoma MG63 cells, from which 3AB-OS cells have been previously selected. Our results suggest that 3AB-OS cells depend on glycolytic metabolism more strongly than MG63 cells. Indeed, growth in glucose shortage or in presence of galactose or pyruvate -mitochondrial specific substrates- leads to a significant reduction of their proliferation compared to MG63 cells. Accordingly, 3AB-OS cells show an increased expression of lactate dehydrogenase A (LDHA) and a larger accumulation of lactate in the culture medium. In line with these findings 3AB-OS cells as compared to MG63 cells present a reduced mitochondrial respiration, a stronger sensitivity to glucose depletion or glycolysis inhibition and a lessened sensitivity to oxidative phosphorylation inhibitors. Additionally, in contrast to MG63 cells, 3AB-OS display fragmented mitochondria, which become networked as they grow in glucose-rich medium, while almost entirely loose these structures growing in low glucose. Overall, our findings suggest that 3AB-OS CSCs energy metabolism is more similar to normal stem cells and to cancer cells characterized by a glycolytic anaerobic metabolism. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Description: Areca chewing is an important environmental risk factor for development of oral premalignant lesions and cancer. Epidemiological evidence indicates that areca chewing is tightly linked to oral carcinogenesis. However, the pathogenetic impacts of areca nut extract (ANE) on normal human oral keratinocytes (HOKs) are unclear and possibly involve oxidative stress via redox imbalance. Sirtuin 3 (SIRT3) is a member of the sirtuin family of proteins that play an important role in regulating cellular reactive oxygen species (ROS) production. Recent studies have confirmed that ANE and other areca ingredients can induce ROS. In this study, we examined the role of SIRT3 in the regulation of ANE-induced ROS in HOK cells. We examined HOK cell viability following treatment with various ANE concentrations. ANE-induced cytotoxicity increased in a dose-dependent manner and was approximately 48% at a concentration of 50 μg/ml after 24 h. SIRT3 expression and enzyme activity were up-regulated in HOK cells by ANE-induced oxidative stress. Additionally, we identified that SIRT3 controls the enzymatic activity of mitochondrial proteins, such as forkhead box O3a (Foxo3a) transcription factor and antioxidant-encoding gene superoxide dismutase 2 (SOD2), by deacetylation in HOK cells. Moreover, SIRT3-mediated deacetylation and activation of Foxo3a promotes nuclear localization in vivo . These findings suggest that SIRT3 is an endogenous negative regulator in response to ANE-induced oxidative stress and demonstrate an essential role for redox balance in HOK cells. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Description: Collagen is the most abundant structural protein in mammals and is expressed in various tissues. In recent years, sphingosine 1-phosphate receptors (S1PRs) have been proven to play an important role in the regulation of collagen expression. Our previous studies reported that S1PRs are involved in TGF-β1-induced collagen expression via up-regulating S1PR1/3 in mouse bone marrow-derived mesenchymal stem cells (BMSCs), and result in experimental mouse liver fibrogenesis. But it remains unclear whether this process happens in human bone marrow-derived mesenchymal stem cells (hMSCs). In this study, we provide evidences that S1PR1/3, but not S1PR2, negatively regulate the expression of collagen in hMSCs using cellular and molecular approaches in vitro . We find that treatment of hMSCs with TGF-β1 up-regulated collagen expression in a dose- and time-dependent manner. Meanwhile, TGF-β1 inhibited the expression of S1PR1/3, but not S1PR2, in hMSCs in a time-dependent manner. Furthermore, either selective knock-down of S1PR1 or silencing S1PR3 induced collagen α1(I) and collagen α1(III) expression in hMSCs. In contrast, inhibition of S1PR2 by siRNA had no effects on the expression of collagen. Altogether, all these findings demonstrated that collagen expression was negatively regulated by S1PR1 and S1PR3 in hMSCs. This study highlights the differences between hMSCs and mouse BMSCs, provides a new regulation mechanism for collagen expression, and points out the risk of utilizing hMSCs in clinical applications. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Description: Lymph nodes are often the first target of metastatic cancer which can then remetastasize to distant organs. The progression of lymph node metastasis is dependent on sufficient blood supply provided by angiogenesis. In the present study, we have developed a color-coded imaging model to visualize angiogenesis of lymph nodes metastasis using green fluorescent protein (GFP) and red fluorescent protein (RFP). Transgenic mice carrying GFP under the control of the nestin second-intron enhancer (ND-GFP mice) were used as hosts. Nascent blood vessels express GFP in these mice. B16F10-RFP melanoma cells were injected into the efferent lymph vessel of the inguinal lymph node of the ND-GFP nude mice, whereby the melanoma cells trafficked to the axillary lymph node. Three days after melanoma implantation, ND-GFP-expressing nascent blood vessels were imaged in the axillary lymph nodes. Seven days after implantation, ND-GFP-expressing nascent blood vessels formed a network in the lymph nodes. ND-GFP-positive blood vessels surrounded the tumor mass by 14 days after implantation. However, by 28 days after implantation, ND-GFP expression was diminished as the blood vessels matured. Treatment with doxorubicin significantly decreased the mean nascent blood vessel length per tumor volume. These results show that the dual-color ND-GFP blood vessels/RFP-tumor model is a powerful tool to visualize and quantitate angiogenesis of metastatic lymph nodes as well as for evaluation of its inhibition. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Description: The cytoplasmic signaling protein tumor necrosis factor (TNF) receptor-associated factor 5 (TRAF5), which was identified as a signal transducer for members of the TNF receptor super-family, has been implicated in several biological functions in T/B lymphocytes and the innate immune response against viral infection. However, the role of TRAF5 in cardiac hypertrophy has not been reported. In the present study, we investigated the effect of TRAF5 on the development of pathological cardiac hypertrophy induced by transthoracic aorta constriction (TAC) and further explored the underlying molecular mechanisms. Cardiac hypertrophy and function were evaluated with echocardiography, hemodynamic measurements, pathological and molecular analyses. For the first time, we found that TRAF5 deficiency substantially aggravated cardiac hypertrophy, cardiac dysfunction and fibrosis in response to pressure overload after 4 weeks of TAC compared to wild-type (WT) mice. Moreover, the mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)-extracellular signal-regulated kinases 1/2 (ERK1/2) signaling pathway was more activated in TRAF5-deficient mice than WT mice. In conclusion, our results suggest that as an intrinsic cardioprotective factor, TRAF5 plays a crucial role in the development of cardiac hypertrophy through the negative regulation of the MEK-ERK1/2 pathway. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Description: The SON protein is a ubiquitously expressed DNA- and RNA-binding protein primarily localized to nuclear speckles. Although several early studies implicated SON in DNA-binding, tumorigenesis and apoptosis, functional significance of this protein had not been recognized until recent studies discovered SON as a novel RNA splicing co-factor. During constitutive RNA splicing, SON ensures efficient intron removal from the transcripts containing suboptimal splice sites. Importantly, SON-mediated splicing is required for proper processing of selective transcripts related to cell cycle, microtubules/centrosomes maintenance, and genome stability. Moreover, SON regulates alternative splicing of RNAs from the genes involved in apoptosis and epigenetic modification. In addition to the role in RNA splicing, SON has an ability to suppress transcriptional activation at certain promoter/enhancer DNA sequences. Considering the multiple SON target genes which are directly involved in cell proliferation, genome stability and chromatin modifications, SON is an emerging player in gene regulation during cancer development and progression. Here, we summarize available information from several early studies on SON, and highlight recent discoveries describing molecular mechanisms of SON-mediated gene regulation. We propose that our future effort on better understanding of diverse SON functions would reveal novel targets for cancer therapy. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Description: Background Podocytes are a terminally differentiated and highly specialized cell type in the glomerulus that forms a crucial component of the glomerular filtration barrier. Recently, Myo1e was identified in the podocytes of glomeruli. Myo1e podocyte-specific knockout mice exhibit proteinuria, podocyte foot process effacement, glomerular basement membrane disorganization, signs of chronic renal injury, and kidney inflammation. Materials and Methods After overexpression of Myo1e in a conditionally immortalized mouse podocyte cell line (MPC5), podocyte migration was evaluated via transwell assay, endocytosis was evaluated using FITC-transferrin, and adhesion was evaluated using a detachment assay after puromycin aminonucleoside treatment. Results Myo1e overexpression significantly increased the adherence of podocytes. ANOVA analysis indicated significant differences for cell adhesion between the overexpression and control groups (overexpression vs. control, t = 11.3199, P = 0.005; overexpression vs. negative control, t = 12.0570, P = 0.0006). Overexpression of Myo1e inhibited puromycin aminonucleoside-induced podocyte detachment, and the number of cells remaining on the bottom of the culture plate increased. Cell migration was enhanced in Myo1e-overexpressing podocytes in the transwell migration assay. Internalization of FITC-transferrin also increased in Myo1e-overexpressing podocytes relative to control cells. Conclusions Overexpression of Myo1e can enhance podocyte migration ability, endocytosis, and attachment to the glomerular basement membrane. Restoration of Myo1e expression in podocytes may therefore strengthen their functional integrity against environmental and mechanical injury. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Description: Indoleamine 2,3-dioxygenase-1 (IDO1) catabolizes the essential amino acid tryptophan, acting as a modifier of inflammation and immune tolerance. Recent work has implicated IDO1 in many human diseases, including in cancer, chronic infection, autoimmune disorders and neurodegenerative disease, stimulating a major surge in preclinical and clinical studies of its pathogenic functions. In the mouse, IDO1 is expressed widely but in situ detection of the enzyme in murine tissues has been unreliable due to the lack of specific antibodies that do not also react with tissues from animals that are genetically deficient in IDO1. Such probes are crucial to establish cellular mechanisms since IDO1 appears to act in different cell types depending on disease context, but reliable probes have been elusive in the field. In this report, we address this issue with the development of IDO1 monoclonal antibody 4B7 which specifically recognizes the murine enzyme in tissue sections, offering a reliable tool for immunohistology in preclinical disease models. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.