The Antiepileptic Valproic Acid Ameliorates Charcot-Marie-Tooth 2W (CMT2W) Disease-Associated HARS1 Mutation-Induced Inhibition of Neuronal Cell Morphological Differentiation Through c-Jun N-terminal Kinase

Neurochemical Research - Hereditary peripheral neuropathies called Charcot-Marie-Tooth (CMT) disease affect the sensory nerves as well as motor neurons. CMT diseases are composed of a heterogeneous...     

Knockdown of p180 Eliminates the Terminal Differentiation of a Secretory Cell Line

We have previously reported that the expression in yeast of an integral membrane protein (p180) of the endoplasmic reticulum (ER), isolated for its ability to mediate ribosome binding, is capable of inducing new membrane biogenesis and an increase in secretory capacity. To demonstrate that p180 is necessary and sufficient for terminal differentiation and acquisition of a secretory phenotype in mammalian cells, we studied the differentiation of a secretory cell line where p180 levels had been significantly reduced using RNAi technology and by transiently expressing p180 in nonsecretory cells. A human monocytic (THP-1) cell line, that can acquire macrophage-like properties, failed to proliferate rough ER when p180 levels were lowered. The Golgi compartment and the secretion of apolipoprotein E (Apo E) were dramatically affected in cells expressing reduced p180 levels. On the other hand, expression of p180 in a human embryonic kidney nonsecretory cell line (HEK293) showed a significant increase in proliferation of rough ER membranes and Golgi complexes. The results obtained from knockdown and overexpression experiments demonstrate that p180 is both necessary and sufficient to induce a secretory phenotype in mammalian cells. These findings support a central role for p180 in the terminal differentiation of secretory cells and tissues.     

Sulfated Glucuronorhamnoxylan from Capsosiphon fulvescens Ameliorates Osteoporotic Bone Resorption via Inhibition of Osteoclastic Cell Differentiation and Function In Vitro and In Vivo

Marine Biotechnology - Excessive osteoclast differentiation and/or bone resorptive function causes a gradual loss of bone, leading to the pathogenesis of bone diseases such as osteoporosis (OP). In...     

Lentiviral Vector-Mediated siRNA Knockdown of c-MYC: Cell Growth Inhibition and Cell Cycle Arrest at G2/M Phase in Jijoye Cells

Inhibition of c-MYC has been considered as a potential therapy for lymphoma treatment. We explored a lentiviral vector-mediated small interfering RNA (siRNA) expression vector to stably reduce c-MYC expression in B cell line Jijoye cells and investigated the effects of c-MYC downregulation on cell growth, cell cycle, and apoptosis in vitro. The expression of c-MYC mRNA and protein levels were inhibited significantly by c-MYC siRNA. The c-MYC downregulation resulted in the inhibition of cell proliferation and cell cycle arrest at G2/M phase, which was associated with decreased expression of cyclin B and cyclin-dependent kinase 1 (CDK1) and increased expression of CDK inhibitor p21 proteins. In addition, downregulation of c-MYC induced cell apoptosis characterized by DNA fragmentation and caspase-3 activation. Taken together, these results suggest that lentiviral vector-mediated siRNA for c-MYC may be a promising approach for targeting c-MYC in the treatment of Burkitt lymphoma.     

StarD7 Knockdown Modulates ABCG2 Expression, Cell Migration, Proliferation, and Differentiation of Human Choriocarcinoma JEG-3 Cells

Background

StAR-related lipid transfer domain containing 7 (StarD7) is a member of the START-domain protein family whose function still remains unclear. Our data from an explorative microarray assay performed with mRNAs from StarD7 siRNA-transfected JEG-3 cells indicated that ABCG2 (ATP-binding cassette sub-family G member 2) was one of the most abundantly downregulated mRNAs.

Methodology/Principal Findings

Here, we have confirmed that knocking down StarD7 mRNA lead to a decrease in the xenobiotic/lipid transporter ABCG2 at both the mRNA and protein levels (−26.4% and −41%, p<0.05, at 48 h of culture, respectively). Also a concomitant reduction in phospholipid synthesis, bromodeoxyuridine (BrdU) uptake and ³H-thymidine incorporation was detected. Wound healing and transwell assays revealed that JEG-3 cell migration was significantly diminished (p<0.05). Conversely, biochemical differentiation markers such as human chorionic gonadotrophin β-subunit (βhCG) protein synthesis and secretion as well as βhCG and syncytin-1 mRNAs were increased approximately 2-fold. In addition, desmoplakin immunostaining suggested that there was a reduction of intercellular desmosomes between adjacent JEG-3 cells after knocking down StarD7.

Conclusions/Significance

Altogether these findings provide evidence for a role of StarD7 in cell physiology indicating that StarD7 modulates ABCG2 multidrug transporter level, cell migration, proliferation, and biochemical and morphological differentiation marker expression in a human trophoblast cell model.     

p100蛋白表达抑制的HepG2肝癌细胞稳定株的建立

目的建立p100表达抑制的HepG2肝癌细胞稳定株,并初步探讨p100在HepG2肝癌细胞中的功能。方法用脂质体将含有真核细胞筛选标记Neo和GFP的p100 shRNA表达质粒转染入HepG2细胞。经G418耐药筛选稳定整合抗药基因的细胞单克隆;荧光镜检GFP阳性细胞单克隆,挑取单克隆;Western检测HepG2细胞稳定株HepG2（p100I）中p100表达的抑制效果;平板细胞克隆形成实验检测细胞克隆形成能力;MTS法检测细胞存活;划痕实验检测细胞迁移能力。结果成功获得了p100表达抑制的HepG2肝癌细胞稳定株HepG2（p100I）,其中p100的表达明显降低。并且实验表明,该p100表达抑制稳定株的克隆形成能力,抵抗化疗药物Cisplatin诱导的细胞死亡的能力和迁移能力明显低于对照组细胞。结论p100表达抑制的HepG2肝癌细胞稳定株的建立为研究p100蛋白在肝癌中的作用提供了体外细胞系模型,基于此稳定株的研究,发现p100能够影响HepG2肝癌细胞的多种细胞功能。     

Inhibition of Endothelial Cell Differentiation on a Glycosylated Reconstituted Basement Membrane Complex

The vascular basement membrane is involved in the regulation of endothelial cell differentiation. The accumulation of advanced glycosylation endproducts (AGEs) has been demonstrated on these basement membranes in patients with diabetes. We examined the effect of AGEs on endothelial cell behavior on reconstituted basement membrane, Matrigel. Human umbilical vein-derived endothelial cells (HUVECs) stopped proliferating and differentiated into capillary-like tube-shaped structures on Matrigel. Laminin antibody partially blocked this process. HUVECs cultured on glycosylated Matrigel, however, proliferated and formed a monolayer without tube formation. The inclusion of aminoguanidine, an inhibitor of AGE formation, during the glycosylation of Matrigel restored HUVEC differentiation. Although the laminin adsorbed onto the plastic culture wells promoted HUVEC attachment and spreading, glycosylated laminin reduced HUVEC attachment by 50% and abolished cellular spreading. These effects were restored by aminoguanidine. HUVEC attachment to glycosylated laminin was further reduced by AGE-modified albumin, poly I, acetylated low-density lipoprotein, or maleylated albumin, ligands for a scavenger receptor. Coating the culture dishes with the laminin peptides RGD, YIGSR, and SIKVAV supported the attachment of HUVECs that was unaffected by glycosylation. Results suggest that AGE accumulation on the basement membranes inhibits endothelial cell differentiation by impairing the normal interactions of endothelial cell receptors with their specific matrix ligands. This process may be involved in diabetic angiopathy.     

THE EFFECTS OF HALOTHANE ON CULTURED MOUSE NEUROBLASTOMA CELLS : I. Inhibition of Morphological Differentiation

Mouse neuroblastoma cells (clone NB2a) were cultured in the presence of 0.3–2.1% halothane in the gas phase for up to 72 h. Halothane inhibited neurite extension dose dependently and virtually abolished microspike formation even at the lowest concentration tested. These effects were completely reversible. Electron microscopy demonstrated that microfilaments measuring 40–80 Å in diameter are the only fibrous organelles visible within microspikes. When the cells were exposed to halothane, no microfilamentous complexes could be identified in any cells and the subcortical regions of neurites often appeared devoid of individual microfilaments. Microtubules were still present in neurites after exposure to halothane concentrations at which microfilaments disappeared. However, at concentrations above 1.0%, microtubules gradually appeared to decrease in number. Short-term experiments showed that existing neurites and microspikes rapidly retracted when suddenly exposed to culture medium equilibrated with 1.0% halothane and quickly reformed when the halothane was removed. The inhibition of neuroblastoma cell differentiation by halothane appears to be mediated by disruption of 40–80 Å diameter microfilaments.     

Activation of GPER Induces Differentiation and Inhibition of Coronary Artery Smooth Muscle Cell Proliferation

Background

Vascular pathology and dysfunction are direct life-threatening outcomes resulting from atherosclerosis or vascular injury, which are primarily attributed to contractile smooth muscle cells (SMCs) dedifferentiation and proliferation by re-entering cell cycle. Increasing evidence suggests potent protective effects of G-protein coupled estrogen receptor 1 (GPER) activation against cardiovascular diseases. However, the mechanism underlying GPER function remains poorly understood, especially if it plays a potential role in modulating coronary artery smooth muscle cells (CASMCs).

Methodology/Principal Findings

The objective of our study was to understand the functional role of GPER in CASMC proliferation and differentiation in coronary arteries using from humans and swine models. We found that the GPER agonist, G-1, inhibited both human and porcine CASMC proliferation in a concentration- (10⁻⁸ to 10⁻⁵ M) and time-dependent manner. Flow cytometry revealed that treatment with G-1 significantly decreased the proportion of S-phase and G2/M cells in the growing cell population, suggesting that G-1 inhibits cell proliferation by slowing progression of the cell cycle. Further, G-1-induced cell cycle retardation was associated with decreased expression of cyclin B, up-regulation of cyclin D1, and concomitant induction of p21, and partially mediated by suppressed ERK1/2 and Akt pathways. In addition, G-1 induces SMC differentiation evidenced by increased α-smooth muscle actin (α-actin) and smooth muscle protein 22α (SM22α) protein expressions and inhibits CASMC migration induced by growth medium.

Conclusion

GPER activation inhibits CASMC proliferation by suppressing cell cycle progression via inhibition of ERK1/2 and Akt phosphorylation. GPER may constitute a novel mechanism to suppress intimal migration and/or synthetic phenotype of VSMC.     

Inhibition of HeLa Cell apoptosis by storage-protein 2

Apoptosis is a barrier to maintaining high viable cell densities in animal cell culture. Silkworm hemolymph and its 30K protein have been reported to exhibit anti-apoptotic activity in various mammalian and insect cell systems. The 30K protein is thermally unstable at temperatures higher than 60 degrees C; however, the silkworm hemolymph heat-treated at 70-80 degrees C still exhibited anti-apoptotic activity. This indicates that silkworm hemolymph contains another anti-apoptotic compound other than 30K protein. In this article, the anti-apoptotic molecule other than 30K protein was found from the silkworm hemolymph and identified. This molecule was storage-protein 2 (SP2), which has no homology with any known anti-apoptotic protein. This molecule was heat-stable up to 80 degrees C, while 30K protein lost its activity at temperatures higher than 60 degrees C. When apoptosis was induced by staurosporine in HeLa cells, SP2 protein suppressed nuclear fragmentation and apoptotic body formation. Moreover, the generation of reactive oxygen species after apoptosis induction was inhibited, which means the inhibition occurred in an early step of the apoptotic process. Inhibition of apoptosis by the SP2 protein would lead to the minimization of cell death during commercial mammalian cell culture.     

Inhibition of Mitochondrial Complex III Blocks Neuronal Differentiation and Maintains Embryonic Stem Cell Pluripotency

The mitochondrion is emerging as a key organelle in stem cell biology, acting as a regulator of stem cell pluripotency and differentiation. In this study we sought to understand the effect of mitochondrial complex III inhibition during neuronal differentiation of mouse embryonic stem cells. When exposed to antimycin A, a specific complex III inhibitor, embryonic stem cells failed to differentiate into dopaminergic neurons, maintaining high Oct4 levels even when subjected to a specific differentiation protocol. Mitochondrial inhibition affected distinct populations of cells present in culture, inducing cell loss in differentiated cells, but not inducing apoptosis in mouse embryonic stem cells. A reduction in overall proliferation rate was observed, corresponding to a slight arrest in S phase. Moreover, antimycin A treatment induced a consistent increase in HIF-1α protein levels. The present work demonstrates that mitochondrial metabolism is critical for neuronal differentiation and emphasizes that modulation of mitochondrial functions through pharmacological approaches can be useful in the context of controlling stem cell maintenance/differentiation.     

Shikonin Suppresses NLRP3 and AIM2 Inflammasomes by Direct Inhibition of Caspase-1

Shikonin is a highly lipophilic naphtoquinone found in the roots of Lithospermum erythrorhizon used for its pleiotropic effects in traditional Chinese medicine. Based on its reported antipyretic and anti-inflammatory properties, we investigated whether shikonin suppresses the activation of NLRP3 inflammasome. Inflammasomes are cytosolic protein complexes that serve as scaffolds for recruitment and activation of caspase-1, which, in turn, results in cleavage and secretion of proinflammatory cytokines IL-1β and IL-18. NLRP3 inflammasome activation involves two steps: priming, i.e. the activation of NF-κB pathway, and inflammasome assembly. While shikonin has previously been reported to suppress the priming step, we demonstrated that shikonin also inhibits the second step of inflammasome activation induced by soluble and particulate NLRP3 instigators in primed immortalized murine bone marrow-derived macrophages. Shikonin decreased NLRP3 inflammasome activation in response to nigericin more potently than acetylshikonin. Our results showed that shikonin also inhibits AIM2 inflammasome activation by double stranded DNA. Shikonin inhibited ASC speck formation and caspase-1 activation in murine macrophages and suppressed the activity of isolated caspase-1, demonstrating that it directly targets caspase-1. Complexing shikonin with β-lactoglobulin reduced its toxicity while preserving the inhibitory effect on NLRP3 inflammasome activation, suggesting that shikonin with improved bioavailability might be interesting for therapeutic applications in inflammasome-mediated conditions.     

Golgi Phosphoprotein 3 Determines Cell Binding Properties under Dynamic Flow by Controlling Golgi Localization of Core 2 N-Acetylglucosaminyltransferase 1

Core 2 N-acetylglucosaminyltransferase 1 (C2GnT1) is a key enzyme participating in the synthesis of core 2-associated sialyl Lewis x (C2-O-sLe^x), a ligand involved in selectin-mediated leukocyte trafficking and cancer metastasis. To accomplish that, C2GnT1 needs to be localized to the Golgi and this step requires interaction of its cytoplasmic tail (CT) with a protein that has not been identified. Employing C2GnT1 CT as the bait to perform a yeast two-hybrid screen, we have identified Golgi phosphoprotein 3 (GOLPH3) as a principal candidate protein that interacts with C2GnT1 and demonstrated that C2GnT1 binds to GOLPH3 via the LLRRR⁹ sequence in the CT. Confocal fluorescence microscopic analysis shows substantial Golgi co-localization of C2GnT1 and GOLPH3. Upon GOLPH3 knockdown, C2GnT1 is found mainly in the endoplasmic reticulum and decorated with complex-type N-glycans, indicating that the enzyme has been transported to the Golgi but is not retained. Also, we have found that a recombinant protein consisting of C2GnT1 CT^1–16-Leu^17–32-Gly^33–42-GFP is localized to the Golgi although the same construct with mutated CT (AAAAA⁹) is not. The data demonstrate that the C2GnT1 CT is necessary and sufficient for Golgi localization of C2GnT1. Furthermore, GOLPH3 knockdown results in reduced synthesis of C2-O-sLe^x associated with P-selectin glycoprotein ligand-1, reduced cell tethering to and rolling on immobilized P- or E-selectin, and compromised E-selectin-induced activation of spleen tyrosine kinase and cell adhesion to intercellular adhesion molecule-1 under dynamic flow. Our results reveal that GOLPH3 can regulate cell-cell interaction by controlling Golgi retention of C2GnT1.     

Simultaneous Inhibition of T Helper 2 and T Regulatory Cell Differentiation by Small Molecules Enhances Bacillus Calmette-Guerin Vaccine Efficacy against Tuberculosis

Tuberculosis affects nine million individuals and kills almost two million people every year. The only vaccine available, Bacillus Calmette-Guerin (BCG), has been used since its inception in 1921. Although BCG induces host-protective T helper 1 (Th1) cell immune responses, which play a central role in host protection, its efficacy is unsatisfactory, suggesting that additional methods to enhance protective immune responses are needed. Recently we have shown that simultaneous inhibition of Th2 cells and Tregs by using the pharmacological inhibitors suplatast tosylate and D4476, respectively, dramatically enhances Mycobacterium tuberculosis clearance and induces superior Th1 responses. Here we show that treatment with these two drugs during BCG vaccination dramatically improves vaccine efficacy. Furthermore, we demonstrate that these drugs induce a shift in the development of T cell memory, favoring central memory T (Tcm) cell responses over effector memory T (Tem) cell responses. Collectively, our findings provide evidence that simultaneous inhibition of Th2 cells and Tregs during BCG vaccination promotes vaccine efficacy.