Cells In 96-well Plates Research Articles

A protocol to effectively create single cell suspensions of adherent cells for multiparameter high-throughput flow cytometry

High-throughput flow cytometry of adherent cells is difficult because the creation of single cell suspensions can damage cells and yield artificial results. We describe a protocol to increase the single cell suspension yield of adherent human cells without injury. Doxorubicin, a cytotoxic agent, was administered to adherent human pancreatic carcinoma cell lines (Panc-1 and AsPC-1) to produce alterations in the cell cycle and intracellular protein expression. The cells in 96-well plates were disassociated using a collagenase and trypsin mixture. Fluorescence-activated high-throughput flow cytometry evaluated cellular viability as well as surface and intracellular protein expression. Cell cycle analysis was performed using 7-aminoactinomycin D and intracellular protein characterization was performed using a fluorescein-labeled monoclonal antibody against activated caspase-3. The collagenase-trypsin-based protocol increased single cell events from 31.9 +/- 0.5% using trypsin alone (standard) to a range of 62.1% to 85.5% without adversely affecting viability. High-throughput flow cytometry demonstrated that the addition of collagenase to the disassociation solution not only permitted significantly higher rates of single cell creation, but it did not negatively affect the doxorubicin-induced protein expression. This protocol allows for expedient and effective disassociation of adherent human cells in order to investigate alterations in specific cellular enzymes and pathways.

NDRG4 Is Required for Cell Cycle Progression and Survival in Glioblastoma Cells

NDRG4 is a largely unstudied member of the predominantly tumor suppressive N-Myc downstream-regulated gene (NDRG) family. Unlike its family members NDRG1-3, which are ubiquitously expressed, NDRG4 is expressed almost exclusively in the heart and brain. Given this tissue-specific expression pattern and the established tumor suppressive roles of the NDRG family in regulating cellular proliferation, we investigated the cellular and biochemical functions of NDRG4 in the context of astrocytes and glioblastoma multiforme (GBM) cells. We show that, in contrast to NDRG2, NDRG4 expression is elevated in GBM and NDRG4 is required for the viability of primary astrocytes, established GBM cell lines, and both CD133(+) (cancer stem cell (CSC)-enriched) and CD133(-) primary GBM xenograft cells. While NDRG4 overexpression has no effect on cell viability, NDRG4 knockdown causes G(1) cell cycle arrest followed by apoptosis. The initial G(1) arrest is associated with a decrease in cyclin D1 expression and an increase in p27(Kip1) expression, and the subsequent apoptosis is associated with a decrease in the expression of XIAP and survivin. As a result of these effects on cell cycle progression and survival, NDRG4 knockdown decreases the tumorigenic capacity of established GBM cell lines and GBM CSC-enriched cells that have been implanted intracranially into immunocompromised mice. Collectively, these data indicate that NDRG4 is required for cell cycle progression and survival, thereby diverging in function from its tumor suppressive family member NDRG2 in astrocytes and GBM cells.

Comparison of PC12 and cerebellar granule cell cultures for evaluating neurite outgrowth using high content analysis

Development of high-throughput assays for chemical screening and hazard identification is a pressing priority worldwide. One approach uses in vitro, cell-based assays which recapitulate biological events observed in vivo. Neurite outgrowth is one such critical cellular process underlying nervous system development that can be quantified using automated microscopy and image analysis (high content analysis). The present study characterized and compared the PC-12 cell line (NS-1) and primary cultures of cerebellar granular cells (CGC), as models for assessing chemical effects on neurite outgrowth. High content analysis of neurite outgrowth was performed using the Cellomics ArrayScan V Ti automated epifluorescent imaging system to acquire and analyze images of β-tubulin immunostained cells in 96-well plates. Cell viability was assessed using the CellTiter-Glo ® assay. Culture of NS-1 or CGC in nerve growth factor or serum respectively, rapidly induced neurite outgrowth that increased over four days in vitro. Seven compounds previously shown to affect neurite outgrowth in vitro were tested in both models for changes in total neurite length and cell viability. In NS-1 cells, four chemicals (PKC inhibitor Bis-I, MEK inhibitor U0126, trans-Retinoic acid, methylmercury) inhibited neurite outgrowth, while lead, amphetamine and valproic acid had no effect. In CGC, five chemicals inhibited neurite outgrowth (Bis-I, U0126, lead, methylmercury, and amphetamine), while trans-Retinoic acid decreased cell viability but not neurite outgrowth. Valproic acid was without effect. The sensitivity of the two models was chemical specific: NS-1 cells were more sensitive to Bis-I, methylmercury and trans-Retinoic acid, while CGC were more sensitive to U0126, lead, and amphetamine. For every chemical (except trans-Retinoic acid), neurite outgrowth was equal to or more sensitive than cell viability. In comparison, out of seven chemicals without prior evidence for effects on neurite outgrowth, only one decreased neurite outgrowth (diphenhydramine in CGC). These findings demonstrate that the effects of chemicals on neurite outgrowth may be cell type specific.

Rapid Screening of Serum-Free Media for the Growth of Adherent Vero Cells by Using a Small-Scale and Non-invasive Tool

The paper proposes a rapid screening method for a first step improvement of an animal component-free medium dedicated to the growth of the anchorage-dependent Vero cell line. A new, rapid, and non-invasive technique is presented to specifically monitor cultures of adherent cells in 96-well plates. The operating conditions of an image analyzer are adapted to take into account the decrease of cell size when the attached cell density increases. An experimental design is carried out to assess the influence of ten component groups in the original medium. Two groups including protein extracts, growth factor, insulin, glucose, and pyruvate show significant positive effects. The groups with vitamins and molecules related to nitrogenous bases display a less pronounced influence. The mixture of amino acids, B(1) vitamin, magnesium sulfate, and sodium phosphate as well as the couple sodium citrate and ferric chloride lead to a downward trend. The screening results are proved to be scalable in stirred cultures with cells on microcarriers. An improved serum-free medium, with some component groups being removed or added, can be rapidly formulated to reach respectively similar or 1.6 times higher cell density than in the original medium. The results from this global approach could be helpful to further focus experiments on identified medium components.

The RNA-activated Protein Kinase Enhances the Induction of Interferon-β and Apoptosis Mediated by Cytoplasmic RNA Sensors

Detection of foreign RNA by the innate immune system can trigger the induction of type I interferon (IFN) and apoptosis. Important antiviral defense pathways that result in type I IFN production following the recognition of foreign double-stranded RNA (dsRNA) include the RIG-I family helicases and IPS-1 adaptor cytosolic pathway and the Toll-like receptor 3 and TIR domain-containing adaptor-inducing IFN-beta (TRIF) adaptor membrane-associated pathway, both of which activate IFN regulatory factor 3 (IRF3). In addition to triggering an innate immune response, dsRNAs are widely used to mediate gene-selective silencing in mammalian cells by the RNA interference pathway. We investigated the ability of short interfering RNAs, including T7 phage polymerase-synthesized RNA (PRNA), which like some viral RNAs contains a 5'-triphosphate, to selectively silence gene expression and to cause induction of IFN-beta and apoptosis. We found that PRNA-mediated gene silencing and associated nonspecific pro-apoptotic and IFN-inducing effects were dependent on the cell line and RNA length. Double-stranded PRNAs 50 nucleotides long as well as polyinosinic-polycytidylic acid activated the RNA-dependent protein kinase (PKR) and induced significant levels of IFN-beta and apoptosis, whereas shorter PRNAs and chemically synthesized dsRNAs did not. Effector caspase activation and apoptosis following RNA transfection was enhanced by pretreatment with IFN, and removal of the 5'-phosphate from PRNAs decreased induction of both IFN-beta and apoptosis. PKR, in addition to IPS-1 and IRF3 but not TRIF, was required for maximal type I IFN-beta induction and the induction of apoptosis by both transfected PRNAs and polyinosinic-polycytidylic acid.

Role of HSP70 in cellular thermotolerance

Thermal pretreatment has been shown to condition tissue to a more severe secondary heat stress. In this research we examined the particular contribution of heat shock protein 70 (HSP70) in thermal preconditioning. For optimization of preshock exposures, a bioluminescent Hsp70-luciferase reporter system in NIH3T3 cells tracked the activation of the Hsp70 gene. Cells in 96-well plates were pretreated in a 43 degrees C water bath for 30 minutes, followed 4 hours later with a severe heat shock at 45 degrees C for 50 minutes. Bioluminescence was measured at 2, 4, 6, 8, and 10 hours after preshock only (PS) and at 4 hours after preshock with heatshock (PS+HS). Viability was assessed 48 hours later with a fluorescent viability dye. Preshock induced thermotolerance was then evaluated in hsp70-containing Murine Embryo Fibroblast (+/+) cells and Hsp70-deficient MEF cells (-/-) through an Arrhenius damage model across varying temperatures (44.5-46 degrees C). A time gap of 4 hours between preconditioning and the thermal insult was shown to be the most effective for thermotolerance with statistical confidence of P<0.05. The benefit of preshocking was largely abrogated in Hsp70-deficient cells. The Arrhenius data showed that preshocking leads to increases in the activation energies, E(a), and increases in frequency factors, A. The frequency factor increase was significantly greater in Hsp70-deficient cells. The data shows that HSP70 contributes significantly to cellular thermotolerance but there are other pathways that provide residual thermotolerance in cells deficient in Hsp70.

Characterization of Molecular Interactions between Eosinophil Cationic Protein and Heparin

Eosinophil cationic protein (ECP) is currently used as a biomarker for airway inflammation. It is a heparin-binding ribonuclease released by activated eosinophils. Its cytotoxicity toward cancer cell lines is blocked by heparin. The objective of this study was to locate the heparin binding site of ECP by site-directed mutagenesis and construction of a synthetic peptide derived from this region. Synthetic heparin with > or =5 monosaccharide units showed strong inhibition of ECP binding to the cell surface. Analysis of ECP mt1 (R34A/W35A/R36A/K38A) showed that these charged and aromatic residues were involved in ECP binding to heparin and the cell surface. A potential binding motif is located in the loop L3 region between helix alpha2 and strand beta1, outside the RNA binding domain. The synthetic peptide derived from the loop L3 region displayed strong pentasaccharide binding affinity and blocked ECP binding to cells. In addition, ECP mt1 showed reduced cytotoxicity. Thus, the tight interaction between ECP and heparin acts as the primary step for ECP endocytosis. These results provide new insights into the structure and function of ECP for anti-asthma therapy.

Evaluation of in vitro cytotoxicity and paracellular permeability of intact monolayers with mouse embryonic stem cells

Mouse embryonic stem (mES) cells were induced to form intact monolayers in cell culture inserts, using combinations of extracellular matrix (ECM) components and growth factors (GFs). Progressive formation of intact monolayers was monitored using transepithelial electrical resistance (TEER) and passage of paracellular permeability (PP) markers. The mES cells were initially inoculated on inactivated mouse embryonic fibroblasts (MEFs) plus leukemia inhibitory factor (LIF). At 75% confluence, cells were passaged in the absence of MEF and LIF to stimulate formation of rounded multicellular aggregates (MA). After 4 days, cultures containing MA were transferred to culture inserts coated with ECM components only, and grown in the presence of selected individual GFs. An additional 10-14 days revealed confluent monolayers with TEER values of 500-700 ohms cm2 (Omega cm2). Monolayers grown on inserts coated with ECM components, such as fibronectin or collagen-IV, in the presence of epidermal growth factor or keratinocyte growth factor in the medium, yielded the highest TEER measurements when compared to cultures grown without GFs or ECM. Acute cytotoxicity (AC) studies with confluent monolayers of mES cells in 96-well plates indicated that there is a high correlation (R2=0.91) between cell viability and TEER for 24-h exposure time. Also, decrease in TEER is inversely proportional with increase in PP of markers. In comparison to standardized Registry of Cytotoxicity (RC) data and TEER measurements, MTT IC50 values for mES cells are lower. Thus, at equivalent concentrations for the same chemicals, cell viability decreases before the integrity of the monolayer is compromised. This system represents a novel approach for the manipulation of mES cells toward specific intact monolayers, as an in vitro model for biological monolayer formation, and most importantly, for applications to cytotoxicity testing.

Lysophosphatidic acid receptor subtype‐specific regulation of human oral fibroblast healing responses

Background: LPA1–5 receptors (LPARs) bind lysophosphatidic acid (LPA), a G protein‐coupled lipid mediator involved in wound healing. We have published that LPA regulates the growth and migration of human gingival (GF) and periodontal ligament (PDLF) fibroblasts, and that they express LPA1–3; however, which of these subtype(s) mediate stimulation of MAPK, elevation of intracellular calcium, or collagen gel contraction is not known. Methods: Serum‐starved cells in 24‐well plates were stimulated with LPA or subtype‐specific agonists [NAEPA (LPA1); FAP‐12 (LPA2); OMPT (LPA3)] for 5 min, and activation of p44/p42 MAPK was assessed by SDS‐PAGE and WB. Serum‐starved cells in 96‐well plates were loaded with Fluo‐3 and elevation of intracellular calcium after stimulation with LPA or subtype‐specific agonists was measured using the FlexStation II (Molecular Devices). Cells in collagen gels were stimulated with LPA or subtype‐specific agonists, and gel areas were measured over time using Sorcerer (Optomax). Results: For GF and PDLF, LPA1–3 agonists stimulate MAPK, but LPA1 and LPA3 mediate elevation of intracellular calcium and collagen gel contraction. The responses mediated through LPA1 and LPA3 were inhibited by Ki16425, an LPA⅓ antagonist. Conclusion: LPA signals through different LPAR subtypes to control specific wound healing‐related responses of GF and PDLF. Support: NIH/NIDCR 1 R15 DE016855‐02 (D.R.C).

Preparation of Cells in 96-Well Plates for siRNA Transfection and High-Content Analysis

INTRODUCTIONRNA interference using siRNA libraries is a powerful technology for elucidating gene function by downregulating gene expression at the post-transcriptional level. Phenotypic changes associated with siRNA knockdown can be monitored using cell lines expressing fluorescent reporter proteins. Test siRNAs are transiently transfected into the reporter cell line and behavior of the fluorescent reporter probe is monitored using a high-content imaging system. A range of cell features and additional fluorescent probes can be monitored to assess the effects of knockdown. This article details how to perform siRNA transfection in multiwell plates for subsequent high-content analysis.

The Farnesyl-diphosphate/Geranylgeranyl-diphosphate Synthase of Toxoplasma gondii Is a Bifunctional Enzyme and a Molecular Target of Bisphosphonates

Farnesyl-diphosphate synthase (FPPS) catalyzes the synthesis of farnesyl diphosphate, an important precursor of sterols, dolichols, ubiquinones, and prenylated proteins. We report the cloning and characterization of two Toxoplasma gondii farnesyl-diphosphate synthase (TgFPPS) homologs. A single genetic locus produces two transcripts, TgFPPS and TgFPPSi, by alternative splicing. Both isoforms were heterologously expressed in Escherichia coli, but only TgFPPS was active. The protein products predicted from the nucleotide sequences have 646 and 605 amino acids and apparent molecular masses of 69.5 and 64.5 kDa, respectively. Several conserved sequence motifs found in other prenyl-diphosphate synthases are present in both TgFPPSs. TgFPPS was also expressed in the baculovirus system and was biochemically characterized. In contrast to the FPPS of other eukaryotic organisms, TgFPPS is bifunctional, catalyzing the formation of both farnesyl diphosphate and geranylgeranyl diphosphate. TgFPPS localizes to the mitochondria, as determined by the co-localisation of the affinity-purified antibodies against the protein with MitoTracker, and in accord with the presence of an N-terminal mitochondria-targeting signal in the protein. This enzyme is an attractive target for drug development, because the order of inhibition of the enzyme by a number of bisphosphonates is the same as that for inhibition of parasite growth. In summary, we report the first bifunctional farnesyl-diphosphate/geranylgeranyl-diphosphate synthase identified in eukaryotes, which, together with previous results, establishes this enzyme as a valid target for the chemotherapy of toxoplasmosis.

A rapid DNA hybridization assay for the evaluation of antiviral compounds against Epstein-Barr virus

There is a need for additional therapies for Epstein-Barr virus (EBV) infections, but the routine screening of large numbers of potential inhibitors has been difficult due to the laborious nature of traditional assays. A new rapid assay was developed to evaluate compounds for antiviral activity against this virus that is both rapid and robust. Test compounds are added to cultures of Akata cells in 96-well plates that have been induced to undergo a lytic infection. Viral DNA produced during the infection is transferred to a membrane and quantified using a non-radioactive DNA hybridization assay. This assay was validated using a set of compounds with known activity against EBV and results compared favorably to an established real-time PCR assay. Subsequent experience with this assay has confirmed that it offers improved efficiency and robustness compared to other assays used routinely to evaluate candidate compounds for antiviral activity against EBV.

Evaluation of an automated in vitro micronucleus assay in CHO-K1 cells

In this paper, we describe the evaluation of an automated in vitro micronucleus assay using CHO-K1 cells in 96-well plates. CHO-K1 cells were pre-loaded with a cell dye that stains the cytoplasm, after which the cells were treated with the test compounds for either 3 h (for the +S9 condition) or 24 h (for the −S9 condition). A total of 10 concentrations were tested, of which the top five concentrations were scored (limited by either cytotoxicity or solubility). At the end of the incubation period the cells were fixed and their DNA was stained with Hoechst. The visualization and scoring of the cells was done using an automated fluorescent microscope coupled with proprietary automated image analysis software provided by Cellomics (Pittsburg, PA). A total of 46 compounds were used in this evaluation, including 8 aneugens and 25 clastogens with varied mechanisms of action. Thirteen non-genotoxic compounds were also included. The automated scoring had a sensitivity of 88% and a specificity of 100%, with a predictive value positive of 100% and a predictive value negative of 76%, compared to data from the literature that was obtained with manual scoring. We also describe the incorporation of a metabolic activation system using rat liver S9 homogenates, and the use of cell number counts as a cytotoxicity index which is complementary to the CBPI- (cytokinesis-block proliferation index) based index. Finally, we also discuss the potential for artefactual findings due to fluorescent precipitate, which should be carefully monitored to prevent false positive results. In conclusion, the automated in vitro micronucleus scoring is a valid alternative to the manual scoring of slides, and it has the advantage of generating data in a rapid and consistent manner, and with low compound requirements, which makes it well suited as a screening assay in the early stages of compound development.

A Rapid DNA Hybridization Assay for the Evaluation of Antiviral Compounds Against Epstein-Barr Virus

There is a need for additional therapies for Epstein-Barr virus (EBV) infections, but the routine screening of large numbers of potential inhibitors has been difficult due to the laborious nature of traditional assays. A new rapid assay was developed to evaluate compounds for antiviral activity against this virus that is both rapid and robust. Test compounds are added to cultures of Akata cells in 96-well plates that have been induced to undergo a lytic infection. Viral DNA produced during the infection is transferred to a membrane and quantified using a non-radioactive DNA hybridization assay. This assay was validated using a set of compounds with known activity against EBV and results compared favorably to an established real-time PCR assay. Subsequent experience with this assay has confirmed that it offers improved efficiency and robustness compared to other assays used routinely to evaluate candidate compounds for antiviral activity against EBV.

MUTATIONS IN THE VON HIPPEL-LANDAU TUMOR SUPPRESSOR PROTEIN CONFER RESISTANCE TO IONIZING RADIATION.

Purpose Renal cell carcinoma (RCC) has historically been regarded as a radiation-resistant malignancy, and radiotherapy has no current role in the management of primary tumors. The von Hippel-Landau (VHL) tumor suppressor gene has been shown to have a role in UV-radiation protection and is frequently mutated in a high percentage of RCCs. This study investigated the radiation sensitivity of RCC cell lines with point mutations in the VHL gene associated with disease to determine if VHL mutational status is associated with radiation resistance. Methods The renal cell line 786-0 was stably transfected with wild-type VHL, or V84L, Y98H, F119S, L188Q, or L188V VHL mutations. Radiation sensitivity was determined by irradiating cells in 96-well plates at 0, 2, 4, 6, or 8 Gy, followed by a 5-day incubation and assaying viability with thiazolyl blue tetrazolium bromide reduction. VHL protein expression was determined by immunoblotting after radiation treatment. Results Radiation had similar effects on all cell lines regardless of VHL status, except for L188V, which was significantly more radiation resistant than the other cell lines. VHL protein expression was down-regulated in a dose-dependent manner with radiation only in the L188V mutant line. Conclusions Preliminary data suggest that the response of RCC cell lines to radiation may depend on the specific VHL mutation. Studies are currently in progress to determine if radiation induces microRNAs that may be associated with differences in VHL expression.

Streptococcus pneumoniae Sheds Syndecan-1 Ectodomains through ZmpC, a Metalloproteinase Virulence Factor

Several microbial pathogens stimulate the ectodomain shedding of host cell surface proteins to promote their pathogenesis. We reported previously that Pseudomonas aeruginosa and Staphylococcus aureus activate the ectodomain shedding of syndecan-1 and that syndecan-1 shedding promotes P. aeruginosa pathogenesis in mouse models of lung and burned skin infections. However, it remains to be determined whether activation of syndecan-1 shedding is a virulence mechanism broadly used by pathogens. Here we show that Streptococcus pneumoniae stimulates syndecan-1 shedding in cell culture-based assays. S. pneumoniae-induced syndecan-1 shedding was repressed by peptide hydroxamate inhibitors of metalloproteinases but not by inhibitors of intracellular signaling pathways previously found to be essential for syndecan-1 shedding caused by P. aeruginosa, S. aureus, or other shedding agonists. A 170-kDa protein fraction with a peptide hydroxamate-sensitive shedding activity was purified by ammonium sulfate precipitation, DEAE chromatography, and size exclusion chromatography. Mass spectrometry analyses revealed that the 170-kDa fraction is composed of ZmpB and ZmpC, two metalloproteinase virulence factors of S. pneumoniae. Both the purified 170-kDa ZmpB/ZmpC fraction and unfractionated S. pneumoniae culture supernatant generated syndecan-1 ectodomains that are smaller than those released by endogenous shedding. Further, a mutant S. pneumoniae strain deficient in zmpC, but not zmpB, lost its capacity to stimulate syndecan-1 shedding. These data demonstrate that S. pneumoniae directly sheds syndecan-1 ectodomains through the action of ZmpC.

Syndecan-1 Expression in Epithelial Cells Is Induced by Transforming Growth Factor β through a PKA-dependent Pathway

Syndecans comprise a major family of cell surface heparan sulfate proteoglycans (HSPGs). Syndecans bind and modulate a wide variety of biological molecules through their heparan sulfate (HS) moiety. Although all syndecans contain the ligand binding HS chains, they likely perform specific functions in vivo because their temporal and spatial expression patterns are different. However, how syndecan expression is regulated has yet to be clearly defined. In this study, we examined how syndecan-1 expression is regulated in epithelial cells. Our results showed that among several bioactive agents tested, only forskolin and three isoforms of TGFbeta (TGFbeta1-TGFbeta3) significantly induced syndecan-1, but not syndecan-4, expression on various epithelial cells. Steady-state syndecan-1 mRNA was not increased by TGFbeta treatment and cycloheximide did not inhibit syndecan-1 induction by TGFbeta, indicating that TGFbeta induces syndecan-1 in a post-translational manner. However, TGFbeta induction of syndecan-1 was inhibited by transient expression of a dominant-negative construct of protein kinase A (PKA) and by specific inhibitors of PKA. Further (i) syndecan-1 cytoplasmic domains were Ser-phosphorylated when cells were treated with TGFbeta and this was inhibited by a PKA inhibitor, (ii) PKA was co-immunoprecipitated from cell lysates by anti-syndecan-1 antibodies, (iii) PKA phosphorylated recombinant syndecan-1 cytoplasmic domains in vitro, and (iv) expression of a syndecan-1 construct with its invariant Ser(286) replaced with a Gly was not induced by TGFbeta. Together, these findings define a regulatory mechanism where TGFbeta signals through PKA to phosphorylate the syndecan-1 cytoplasmic domain and increases syndecan-1 expression on epithelial cells.

Freezing Embryonic Stem (ES) Cells in 96-Well Plates

Freezing Embryonic Stem (ES) Cells in 96-Well Plates

Intracellular Zinc Release and ERK Phosphorylation Are Required Upstream of 12-Lipoxygenase Activation in Peroxynitrite Toxicity to Mature Rat Oligodendrocytes

Peroxynitrite toxicity has been implicated in the pathogenesis of white matter injury. The mechanisms of peroxynitrite toxicity to oligodendrocytes (OLs), the major cell type of the white matter, are unknown. Using primary cultures of mature OLs that express myelin basic protein, we found that 3-morpholinosydnonimine, a peroxynitrite generator, caused toxicity to OLs. N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine, a zinc chelator, completely blocked peroxynitrite-induced toxicity. Use of FluoZin-3, a specific fluorescence zinc indicator, demonstrated the liberation of zinc from intracellular stores by peroxynitrite. Peroxynitrite caused the sequential activation of extracellular signal-regulated kinase 42/44 (ERK42/44), 12-lipoxygenase, and generation of reactive oxygen species, which were all dependent upon the intracellular release of zinc. The same cell death pathway was also activated when exogenous zinc was used. These results suggest that in addition to preventing the formation of peroxynitrite, useful strategies in preventing disease progression in pathologies in which peroxynitrite toxicity plays a critical role might include maintaining intracellular zinc homeostasis, blocking phosphorylation of ERK42/44, inhibiting activation of 12-lipoxygenase, and eliminating the accumulation of reactive oxygen species.

Tumor Necrosis Factor Promotes Runx2 Degradation through Up-regulation of Smurf1 and Smurf2 in Osteoblasts

Tumor necrosis factor (TNF) plays an important role in the pathogenesis of inflammatory bone loss through stimulation of osteoclastic bone resorption and inhibition of osteoblastic bone formation. Compared with the well established role of TNF in osteoclastogenesis, mechanisms by which TNF inhibits osteoblast function have not been fully determined. Runx2 is an osteoblast-specific transcription factor whose steady-state protein levels are regulated by proteasomal degradation, mediated by the E3 ubiquitin ligases, Smurf1 and Smurf2. We hypothesized that TNF inhibits osteoblast function through Smurf-mediated Runx2 degradation. We treated C2C12 and 2T3 osteoblast precursor cell lines and primary osteoblasts with TNF and found that TNF, but not interleukin-1, significantly increased Smurf1 and Smurf2 expression. TNF increased the degradation of endogenous or transfected Runx2 protein, which was blocked by treating cells with a proteasomal inhibitor or by infecting cells with small interfering (si)RNA against Smurf1 or Smurf2. TNF inhibited the expression of bone morphogenetic protein and transforming growth factor-beta signaling reporter constructs, and the inhibition of each was blocked by Smurf1 siRNA and Smurf2 siRNA, respectively. Overexpression of Smurf1 and/or Smurf2 siRNAs prevented the inhibitory effect of TNF on Runx2 reporter. Consistent with these in vitro findings, bones from TNF transgenic mice or TNF-injected wild type mice had increased Smurf1 and decreased Runx2 protein levels. We propose that one of the mechanisms by which TNF inhibits bone formation in inflammatory bone disorders is by promoting Runx2 proteasomal degradation through up-regulation of Smurf1 and Smurf2 expression.