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Lysophosphatidic Acid-induced ERK Activation and Chemotaxis in MC3T3-E1 Preosteoblasts are Independent of EGF Receptor Transactivation  

SciTech Connect

Growing evidence indicates that bone-forming osteoblasts and their progenitors are target cells for the lipid growth factor lysophosphatidic acid (LPA) which is produced by degranulating platelets at sites of injury. LPA is a potent inducer of bone cell migration, proliferation and survival in vitro and an attractive candidate to facilitate preosteoblast chemotaxis during skeletal regeneration in vivo, but the intracellular signaling pathways mediating the effects of this lipid on bone cells are not defined. In this study we measured the ability of LPA to stimulate extracellular signal-related kinase (ERK1/2) in MC3T3-E1 preosteoblastic cells and determined the contribution of this pathway to LPA-stimulated chemotaxis. LPA-treated cells exhibited a bimodal activation of ERK1/2 with maximal phosphorylation at 5 and 60 minutes. The kinetics of ERK1/2 phosphorylation were not coupled to Ras activation or LPA-induced elevations in cytosolic Ca2+. While LPA is coupled to the transactivation of the EGF receptor in many cell types, LPA-stimulated ERK1/2 activation in MC3T3-E1 cells was unaffected by inhibition of EGF receptor function. ERK isoforms rapidly accumulated at nuclear sites in LPA-treated cells, a process that was blocked if ERK1/2 phosphorylation was prevented with the MEK1 inhibitor U0126. Blocking ERK1/2 phosphorylation with U0126 also diminished MC3T3-E1 cell migration and altered the normal disassembly of LPA-induced stress fibers, while the inhibition of EGF receptor function had no effect on LPA-coupled preosteoblast motility. Our results identify ERK1/2 activation as a mediatora mediator of LPA-stimulated MC3T3-E1 cell migration that may be relevant to preosteoblast motility during bone repair in vivo.

Karagiosis, Sue A.; Chrisler, William B.; Bollinger, Nikki; Karin, Norman J.



PDGF-induced receptor phosphorylation and phosphoinositide hydrolysis are unaffected by protein kinase C activation in mouse swiss 3T3 and human skin fibroblasts  

SciTech Connect

Short (1-10 min) pretreatment of intact cells with activators of protein kinase C (e.g. phorbol-12 myristate, 13-acetate, PMA) affects the activity of a variety of surface receptors (for growth factors, hormones and neurotransmitters), with inhibition of transmembrane signal generation. In two types of fibroblasts it is demonstrated that the PDGF receptor is unaffected by PMA. Exposure to PMA at concentrations up to 100 nM for 10 min failed to inhibit either one of the agonist-induced, receptor-coupled responses of PDGF: the autophosphorylation of receptor molecules at tyrosine residues, and the hydrolysis of membrane polyphosphoinositides. In contrast, the EGF receptor autophosphorylation (in A 431 cells) and the bombesin-induced phosphoinositide hydrolysis were readily inhibited by PMA.

Sturani, E.; Vicentini, L.M.; Zippel, R.; Toschi, L.; Pandiella-Alonso, A.; Comoglio, P.M.; Meldolesi, J.



Increased tyrosine phosphorylation of the insulin receptor, the insulin receptor substrate-1 and a 73 kDa protein associated with insulin-induced mitogenesis in SV40-transformed 3T3T cells  

Microsoft Academic Search

Insulin selectively induces mitogenesis in quiescent SV40 large T antigen-transformed murine 3T3T (CSV3-1) cells but not in quiescent nontransformed 3T3T cells. This mitogenic effect induced by insulin in CSV3-1 cells requires an induction of AP-1 activity associated with c-Jun and JunB. To further investigate the mechanisms that are involved in insulin-induced mitogenesis in CSV3-1 cells, the current experiments were performed.

Hanlin Wang



Phosphatidylcholine induces apoptosis of 3T3-L1 adipocytes  

PubMed Central

Background Phosphatidylcholine (PPC) formulation is used for lipolytic injection, even though its mechanism of action is not well understood. Methods The viability of 3T3-L1 pre-adipocytes and differentiated 3T3-L1 cells was measured after treatment of PPC alone, its vehicle sodium deoxycholate (SD), and a PPC formulation. Western blot analysis was performed to examine PPC-induced signaling pathways. Results PPC, SD, and PPC formulation significantly decreased 3T3-L1 cell viability in a concentration-dependent manner. PPC alone was not cytotoxic to CCD-25Sk human fibroblasts at concentrations <1 mg/ml, whereas SD and PPC formulation were cytotoxic. Western blot analysis demonstrated that PPC alone led to the phosphorylation of the stress signaling proteins, such as p38 mitogen-activated protein kinase and c-Jun N-terminal kinase, and activated caspase-9, -8, -3 as well as cleavage of poly(ADP-ribose) polymerase. However, SD did not activate the apoptotic pathways. Instead, SD and PPC formulation induced cell membrane lysis, which may lead to necrosis of cells. Conclusions PPC results in apoptosis of 3T3-L1 cells.



Characterization of thyroid hormone (T3) receptors in three osteosarcoma cell lines of distinct osteoblast phenotype: interactions among T3, vitamin D3, and retinoid signaling.  


T3 is required for normal skeletal development, but its cellular targets in bone are unknown. T3 regulates target gene transcription via a specific nuclear receptor (T3R), which can heterodimerize with 9-cis-retinoic acid, 1 alpha, 25-dihydroxyvitamin D3, or retinoic acid receptors to modify T3 responsiveness. Serum-free cultures were developed to investigate hormone interactions in three osteosarcoma cell lines, ROS25/1, UMR106, and ROS17/2.8, that express fibroblast-like, preosteoblast, and mature osteoblast phenotypes. ROS25/1 expressed T3R alpha 1, but only low levels of T3R beta 1, whereas UMR106 and ROS17/2.8 cells expressed both receptor proteins. All cells expressed c-erb-A alpha 2 protein and equal levels of 1 alpha,25-dihydroxyvitamin D3 receptor, 9-cis-retinoic acid receptor, and retinoic acid receptor messenger RNAs. Endogenous T3R activity and the effects of D3 and 9-cis-RA on T3 responsiveness were determined in transfections using reporter genes containing T3 response elements from rat malic enzyme or alpha-myosin heavy chain genes. Cell-specific T3 responses were associated with differing patterns of T3R gene expression and stages of osteoblast phenotype expression. A change in T3R beta 1 gene expression during osteoblast phenotype differentiation may modify T3 action in developing bone. PMID:7988420

Williams, G R; Bland, R; Sheppard, M C



Depletion of the p43 Mitochondrial T3 Receptor Increases Sertoli Cell Proliferation in Mice.  


Among T3 receptors, TR?1 is ubiquitous and its deletion or a specific expression of a dominant-negative TR?1 isoform in Sertoli cell leads to an increase in testis weight and sperm production. The identification of a 43-kDa truncated form of the nuclear receptor TR?1 (p43) in the mitochondrial matrix led us to test the hypothesis that this mitochondrial transcription factor could regulate Sertoli cell proliferation. Here we report that p43 depletion in mice increases testis weight and sperm reserve. In addition, we found that p43 deletion increases Sertoli cell proliferation in postnatal testis at 3 days of development. Electron microscopy studies evidence an alteration of mitochondrial morphology observed specifically in Sertoli cells of p43-/- mice. Moreover, gene expression studies indicate that the lack of p43 in testis induced an alteration of the mitochondrial-nuclear cross-talk. In particular, the up-regulation of Cdk4 and c-myc pathway in p43-/- probably explain the extended proliferation recorded in Sertoli cells of these mice. Our finding suggests that T3 limits post-natal Sertoli cell proliferation mainly through its mitochondrial T3 receptor p43. PMID:24040148

Fumel, Betty; Roy, Stéphanie; Fouchécourt, Sophie; Livera, Gabriel; Parent, Anne-Simone; Casas, François; Guillou, Florian



Identical Gene Regulation Patterns of T3 and Selective Thyroid Hormone Receptor Modulator GC-1  

PubMed Central

Synthetic selective thyroid hormone (TH) receptor (TR) modulators (STRM) exhibit beneficial effects on dyslipidemias in animals and humans and reduce obesity, fatty liver, and insulin resistance in preclinical animal models. STRM differ from native TH in preferential binding to the TR? subtype vs. TR?, increased uptake into liver, and reduced uptake into other tissues. However, selective modulators of other nuclear receptors exhibit important gene-selective actions, which are attributed to differential effects on receptor conformation and dynamics and can have profound influences in animals and humans. Although there are suggestions that STRM may exhibit such gene-specific actions, the extent to which they are actually observed in vivo has not been explored. Here, we show that saturating concentrations of the main active form of TH, T3, and the prototype STRM GC-1 induce identical gene sets in livers of euthyroid and hypothyroid mice and a human cultured hepatoma cell line that only expresses TR?, HepG2. We find one case in which GC-1 exhibits a modest gene-specific reduction in potency vs. T3, at angiopoietin-like factor 4 in HepG2. Investigation of the latter effect confirms that GC-1 acts through TR? to directly induce this gene but this gene-selective activity is not related to unusual T3-response element sequence, unlike previously documented promoter-selective STRM actions. Our data suggest that T3 and GC-1 exhibit almost identical gene regulation properties and that gene-selective actions of GC-1 and similar STRM will be subtle and rare.

Yuan, Chaoshen; Lin, Jean Z.H.; Sieglaff, Douglas H.; Ayers, Steven D.; DeNoto-Reynolds, Frances; Baxter, John D.



Characterization of melanocortin receptor subtype expression in murine adipose tissues and in the 3T3-L1 cell line.  


It has been known for many years that adipocytes express high affinity ACTH and alpha-melanocyte stimulating hormone (MSH) binding sites, and that ACTH, alpha-MSH, and beta-lipotropin are potent lipolytic hormones. We show here that the adipocyte response to the melanocortin peptides results from the expression of both the MC2 (ACTH) receptor as well as the newly discovered MC5 receptor. Using RT-PCR and Northern blot hybridization, high levels of MC2 receptor messenger RNA (mRNA) were found in all adipose tissues examined in the mouse, whereas MC5 receptor mRNA was found in a subset of these. Both receptors mRNAs were also found in the 3T3-L1 cell line but only after the cells had been induced to differentiate into adipocytes. This cell line was then used to characterize the pharmacological properties of the MC2 and MC5 receptor sites in situ. The MC2 receptor exhibits properties similar to the ACTH receptor characterized in adrenocortical cells, coupling to activation of adenylyl cyclase with an EC50 of approximately 1 nM. An MSH binding site characterized in these cells is presumably the MC5 receptor, based on the observation that this is the only other melanocortin receptor mRNA detected in these cells. The MC5 receptor in the 3T3-L1 adipocyte activated adenylyl cyclase in response to alpha-MSH stimulation. Interestingly, Nle4, D-Phe7-alpha-MSH (NDP-MSH), a commonly used synthetic alpha-MSH agonist, was a potent antagonist of the MC5 receptor expressed in the 3T3-L1 cell line. Although the agouti signaling peptide is a potent antagonist of NDP-MSH binding to the MC1 and MC4 melanocortin receptors, agouti was unable to block NDP-MSH binding in the 3T3-L1 adipocyte. PMID:8612546

Boston, B A; Cone, R D



Thyroid hormone receptor binding to DNA and T3-dependent transcriptional activation are inhibited by uremic toxins.  


BACKGROUND: There is a substantial clinical overlap between chronic renal failure (CRF) and hypothyroidism, suggesting the presence of hypothyroidism in uremic patients. Although CRF patients have low T3 and T4 levels with normal thyroid-stimulating hormone (TSH), they show a higher prevalence of goiter and evidence for blunted tissue responsiveness to T3 action. However, there are no studies examining whether thyroid hormone receptors (TRs) play a role in thyroid hormone dysfunction in CRF patients. To evaluate the effects of an uremic environment on TR function, we investigated the effect of uremic plasma on TRbeta1 binding to DNA as heterodimers with the retinoid X receptor alpha (RXRalpha) and on T3-dependent transcriptional activity. RESULTS: We demonstrated that uremic plasma collected prior to hemodialysis (Pre-HD) significantly reduced TRbeta1-RXRalpha binding to DNA. Such inhibition was also observed with a vitamin D receptor (VDR) but not with a peroxisome proliferator-activated receptor gamma (PPARgamma). A cell-based assay confirmed this effect where uremic pre-HD ultrafiltrate inhibited the transcriptional activation induced by T3 in U937 cells. In both cases, the inhibitory effects were reversed when the uremic plasma and the uremic ultrafiltrate were collected and used after hemodialysis (Post-HD). CONCLUSION: These results suggest that dialyzable toxins in uremic plasma selectively block the binding of TRbeta1-RXRalpha to DNA and impair T3 transcriptional activity. These findings may explain some features of hypothyroidism and thyroid hormone resistance observed in CRF patients. PMID:15807894

Santos, Guilherme M; Pantoja, Carlos J; Costa E Silva, Aluízio; Rodrigues, Maria C; Ribeiro, Ralff C; Simeoni, Luiz A; Lomri, Noureddine; Neves, Francisco Ar



Ligand Activation of Overexpressed Epidermal Growth Factor Receptors Transforms NIH 3T3 Mouse Fibroblasts  

NASA Astrophysics Data System (ADS)

The cell surface receptor for the mitogenic peptide epidermal growth factor (EGF) is involved in control of normal cell growth and may play a role in the genesis of human neoplasia such as squamous carcinoma and glioblastoma. Soft-agar growth and focus-formation experiments with NIH 3T3 mouse fibroblasts transfected with an expression plasmid demonstrated the ligand-dependent transforming potential of the human EGF receptor without structural alterations. Activation of overexpressed normal receptor alone appears to be sufficient for transformation of NIH 3T3 cells in vitro.

Riedel, Heimo; Massoglia, Sharon; Schlessinger, Joseph; Ullrich, Axel



Lysophosphatidic acid induces chemotaxis in MC3T3-E1 osteoblastic cells  

SciTech Connect

Lysophosphatidic acid (LPA) is a bioactive lipid that has pleiotropic effects on a variety of cell types and enhances the migration of endothelial and cancer cells, but it is not known if this lipid can alter osteoblast motility. We performed transwell migration assays using MC3T3-E1 osteoblastic cells and found LPA to be a potent chemotactic agent. Quantitative time-lapse video analysis of osteoblast migration after wounds were introduced into cell monolayers indicated that LPA stimulated both migration velocity and the average migration distance per cell. LPA also elicited substantial changes in cell shape and actin cytoskeletal structure; lipid-treated cells contained fewer stress fibers and displayed long membrane processes that were enriched in F-actin. Quantitative RT-PCR analysis showed that MC3T3-E1 cells express all four known LPA-specific G protein-coupled receptors (LPA1-LPA4) with a relative mRNA abundance of LPA1 > LPA4 > LPA2 >> LPA3. LPA-induced changes in osteoblast motility and morphology were antagonized by both pertussis toxin and Ki16425, a subtype-specific blocker of LPA1 and LPA3 receptor function. Cell migration in many cell types is linked to changes in intracellular Ca2+. Ki16425 also inhibited LPA-induced Ca2+ signaling in a dose-dependent manner, suggesting a link between LPA-induced Ca2+ transients and osteoblast chemotaxis. Our data show that LPA stimulates MC3T3-E1 osteoblast motility via a mechanism that is linked primarily to the G protein-coupled receptor LPA1.

Masiello, Lisa M.; Fotos, Joseph S.; Galileo, Deni S.; Karin, Norm J.



Lysophosphatidic acid induces chemotaxis in MC3T3-E1 osteoblastic cells.  


Lysophosphatidic acid (LPA) is a bioactive lipid that has pleiotropic effects on a variety of cell types and enhances the migration of endothelial and cancer cells, but it is not known if this lipid can alter osteoblast motility. We performed transwell migration assays using MC3T3-E1 osteoblastic cells and found LPA to be a potent chemotactic agent. Quantitative time-lapse video analysis of osteoblast migration after wounds were introduced into cell monolayers indicated that LPA stimulated both migration velocity and the average migration distance per cell. LPA also elicited substantial changes in cell shape and actin cytoskeletal structure; lipid-treated cells contained fewer stress fibers and displayed long membrane processes that were enriched in F-actin. Quantitative RT-PCR analysis showed that MC3T3-E1 cells express all four known LPA-specific G-protein-coupled receptors (LPA1-LPA4) with a relative mRNA abundance of LPA1>LPA4>LPA2>LPA3. LPA-induced changes in osteoblast motility and morphology were antagonized by both pertussis toxin and Ki16425, a subtype-specific blocker of LPA1 and LPA3 receptor function. Cell migration in many cell types is linked to changes in intracellular Ca2+. Ki16425 also inhibited LPA-induced Ca2+ signaling in a dose-dependent manner, suggesting a link between LPA-induced Ca2+ transients and osteoblast chemotaxis. Our data show that LPA stimulates MC3T3-E1 osteoblast motility via a mechanism that is linked primarily to the G-protein-coupled receptor LPA1. PMID:16487757

Masiello, Lisa M; Fotos, Joseph S; Galileo, Deni S; Karin, Norman J



Dexamethasone regulates beta adrenergic receptors in 3T3-L1 fibroblasts  

SciTech Connect

3T3-L1 fibroblasts, containing ..beta..-adrenergic receptors (..beta..AR) of predominantly the ..beta../sub 1/AR subtype, express only ..beta../sub 2/AR after 48 hr treatment with 250 nM dexamethasone (dex). A two-fold increase in ..beta..AR number was also observed upon dex treatment. ..beta..AR were assayed in membranes using the radioligand (/sup 125/I)-cyanopindolol and the ..beta../sub 2/AR selective antagonist ICI. The relative potency of agonists in stimulating whole cell adenylate cyclase activity was consistent with the subtypes determined by binding experiments. The ability of compounds to cause the subtype switch correlates with glucocorticoid activity since these compounds were more effective in facilitating the subtype conversion and the increase in ..beta..AR number than mineralocorticoids. Sex steroids and thyroid hormone were ineffective at concentrations up to in causing these changes. RNA and protein synthesis appear to be required for the ..beta..AR subtype switch and increase in ..beta..AR number since these changes were not observed with dex treatment in the presence of actinomycin D or cyclohexamide. This study suggests that glucocorticoids may induce gene activation in 3T3-L1 fibroblasts that results in an increase in ..beta..AR number and a shift in subtype.

Nakada, M.T.; Stadel, J.M.; Poksay, K.S.; Crooke, S.T.



Midkine induces the transformation of NIH3T3 cells.  

PubMed Central

Midkine (MK) is a heparin-binding growth factor and is frequently expressed at high levels in many human carcinomas. To investigate further the roles of MK in the regulation of cell growth, we introduced MK expression in NIH3T3 cells. A mixture of transfectants of an MK expression vector, but not a control vector, formed colonies in soft agar, showed an elevated cell number at confluence, and formed tumours in nude mice. An interesting characteristic of the transformed cells was that they became spontaneously detached from the culture dish substratum. In the transformed cells, MK was not only secreted, but also localized, in the perinuclear region as spots. The present data indicate that MK has the potential to transform NIH3T3 cells and suggest that overexpression of the MK gene may promote unregulated cell growth in vivo. Images Figure 2 Figure 3 Figure 4

Kadomatsu, K.; Hagihara, M.; Akhter, S.; Fan, Q. W.; Muramatsu, H.; Muramatsu, T.



Transformation of NIH3T3 fibroblasts by the c-Kit receptor tyrosine kinase: effect of receptor density and ligand-requirement  

Microsoft Academic Search

Ectopic expression of the normal murine receptor tyrosine kinase, c-Kit, in NIH3T3 cells induced many phenotypic changes characteristic of transformation including anchorage-independent growth, focus formation and tumorigenicity in nude mice. Although transformation was largely dependent on the presence of recombinant murine Steel Factor (SLF), the ligand to the c-Kit receptor, anchorage independent growth did occur at a low frequency in

Georgina Caruana; Antony C Cambareri; Thomas J Gonda; Leonie K Ashman



Mechanisms of Resveratrol-Induced Inhibition of Clonal Expansion and Terminal Adipogenic Differentiation in 3T3-L1 Preadipocytes.  


We show that resveratrol prevents clonal expansion and terminal adipogenesis in 3T3-L1 preadipocytes. An early resveratrol effect was the inhibition of AKT and mitogen-activated protein kinase signaling, accompanied by down regulation of cyclin D1 expression, abrogation of retinoblastoma protein hyperphosphorylation, and subsequent inhibition of cell cycle reentry and clonal expansion, as indicated by cyclin A2 repression. Resveratrol inhibited terminal adipogenesis at the level of peroxisome proliferator-activated receptor-?2 expression and activity. This was independent from the preceding inhibition of clonal expansion. Peroxisome proliferator-activated receptor-?2 overexpression and activation partially restored fatty acid-binding protein 4 induction in resveratrol-treated 3T3-L1. Resveratrol activated AMP-activated protein kinase (AMPK) but did not induce PPAR-? co-activator 1? (PGC1?) and mitochondrial biogenesis in 3T3-L1. Treatment with the Sirt1 inhibitor splitomicin augmented downregulation of peroxisome proliferator-activated receptor-?2 and fatty acid-binding protein 4 expressions in resveratrol-treated 3T3-L1 and did not prevent the inhibition of terminal adipogenesis. Moreover, splitomicin could not obviate resveratrol-induced cyclin D1 repression, retinoblastoma protein hypophosphorylation, and inhibition of clonal expansion. Our data suggest that resveratrol inhibits clonal expansion and terminal adipogenesis in 3T3-L1 by several mechanisms. PMID:23525482

Mitterberger, Maria C; Zwerschke, Werner



Triac regulation of transcription is T(3) receptor isoform- and response element-specific.  


3,5,3'-triiodothyroacetic acid (Triac) is a naturally occurring triiodothyronine (T(3)) analog, which has been used on an empirical basis to treat the syndrome of resistance to thyroid hormone (RTH). The aim of our studies was to compare the effects of Triac and T(3) on negative and positive thyroid hormone response elements (TREs). We used transient transfections with luciferase reporter genes to show that on palindromic, inverted palindrome and human TRH reporters, Triac is more potent than T(3) for transcriptional regulation by TRbeta1 and TRbeta2 isoforms, while regulation by TRalpha1 is equivalent for both ligands. Other TREs (direct repeat, hTSHalpha and hTSHbeta) are not regulated differently by Triac and T(3). Dose-response curves show that the difference between Triac and T(3) is maximal in the 1-10 nM range. Receptor-binding studies reveal a greater affinity of Triac than T(3) for TRbeta1 and TRbeta2 isoforms, which could explain its isoform-specific effects. These data suggest that the TRE- and TR isoform-specific effects of Triac favor its use in RTH. PMID:10940484

Messier, N; Langlois, M F



Interaction of human beta 1 thyroid hormone receptor and its mutants with DNA and retinoid X receptor beta. T3 response element-dependent dominant negative potency.  

PubMed Central

Mutations in the human beta thyroid hormone receptor (h-TR beta) gene are associated with the syndrome of generalized resistance to thyroid hormone. We investigated the interaction of three h-TR beta 1 mutants representing different types of functional impairment (kindreds ED, OK, and PV) with different response elements for 3,3',5-triiodothyronine (T3) and with retinoid X receptor beta (RXR beta). The mutant receptors showed an increased tendency to form homodimers on a palindromic T3-response element (TREpal), a direct repeat (DR + 4), and an inverted palindrome (TRElap). On TRElap, wild type TR binding was decreased by T3, while the mutant receptors showed a variably decreased degree of dissociation from TRElap in response to T3. The extent of dissociation was proportional to their T3 binding affinities. RXR beta induced the formation of h-TR beta 1:RXR beta heterodimers equally well for mutants and the wild type h-TR beta 1 on these T3 response elements. However, the T3-dependent increase in heterodimerization with RXR beta was absent or reduced for the mutant TRs. Transient transfection studies indicated that the dominant negative potency was several-fold more pronounced on the TRElap as compared to TREpal or DR + 4. In CV-1 and HeLa cells, transfection of RXR beta could not reverse the dominant negative action. These results demonstrate that the binding of mutant h-TRs to DNA, as well as their dominant negative potency, are TRE dependent. In addition, competition for DNA binding, rather than for limiting amounts of RXR beta, is likely to mediate the dominant negative action. Images

Meier, C A; Parkison, C; Chen, A; Ashizawa, K; Meier-Heusler, S C; Muchmore, P; Cheng, S Y; Weintraub, B D



Fluoroaluminate Induces Pertussis Toxin-Sensitive Protein Phosphorylation: Differences in MC3T3-E1 Osteoblastic and NIH3T3 Fibroblastic Cells  

Microsoft Academic Search

Fluoride is an acknowledged bone-forming agent that may act through stimulation of osteoblast proliferation. Fluoride's action on osteoblasts and bone is potentiated by aluminum, which can form a complex with fluoride (fluoroaluminate) and activate heterotrimeric G proteins. Here we examined signaling pathways activated by fluoroaluminate in MC3T3-E1 osteoblastic and in NIH3T3 fibroblastic cells. In MC3T3-E1 cells, fluoroaluminate induced a decrease

Mira Šuša; Gesche J. R. Standke; Margit Jeschke; Daisy Rohner



Ligand Activation of Overexpressed Epidermal Growth Factor Receptors Transforms NIH 3T3 Mouse Fibroblasts  

Microsoft Academic Search

The cell surface receptor for the mitogenic peptide epidermal growth factor (EGF) is involved in control of normal cell growth and may play a role in the genesis of human neoplasia such as squamous carcinoma and glioblastoma. Soft-agar growth and focus-formation experiments with NIH 3T3 mouse fibroblasts transfected with an expression plasmid demonstrated the ligand-dependent transforming potential of the human

Heimo Riedel; Sharon Massoglia; Joseph Schlessinger; Axel Ullrich



Chemotaxis of 3T3 and SV3T3 cells to fibronectin is mediated through the cell-attachment site in fibronectin and a fibronectin cell surface receptor  

PubMed Central

Fibronectin (FN) is a multidomain extracellular matrix protein that induces attachment and chemotactic migration of fibroblastic cells. In this study we analyzed the molecular determinants involved in the FN- induced chemotactic migration of normal and SV40-transformed 3T3 cells. Two different monoclonal antibodies to the cell-binding site of FN blocked chemotaxis to a 140-kD FN fragment (Ca 140) containing the cell- binding domain. A monoclonal antibody to a determinant distant from the cell-binding site did not affect chemotaxis. A synthetic tetrapeptide, RGDS, which represents the major cell-attachment sequence, was able to compete with FN and the Ca 140 fragment in chemotaxis assays, but this peptide itself had no significant chemotactic activity. A larger peptide encompassing this sequence, GRGDSP, was chemotactic, while the peptide GRGESP, where a glutamic acid residue was substituted for aspartic acid, was inactive. Chemotactic migration could be prevented in a dose-dependent manner by a rabbit polyclonal antiserum to a 140-kD cell surface FN receptor. This antibody was more effective on normal than on transformed 3T3 cells. Neither the anti-FN receptor antiserum nor a monoclonal antibody to the cell-binding site of FN blocked migration induced by another potent chemoattractant, platelet-derived growth factor. These data indicate that FN-induced chemotaxis of 3T3 and SV3T3 cells is mediated via the RGDS cell-attachment site of FN and the 140-kD cell surface FN receptor. The interaction is specific and can be altered by transformation.



Curcumin induces apoptosis in immortalized NIH 3T3 and malignant cancer cell lines  

Microsoft Academic Search

Curcumin, which is a widely used dietary pigment and spice, has been demonstrated to be an effective inhibitor of tumor promotion in mouse skin carcinogenesis. We report that curcumin induces cell shrinkage, chromatin condensation, and DNA fragmentation, characteristics of apoptosis, in immortalized mouse embryo fibroblast NIH 3T3 erb B2 oncogene?transformed NIH 3T3, mouse sarcoma S180, human colon cancer cell HT?29,

Ming?Chung Jiang



The T3-induced gene KLF9 regulates oligodendrocyte differentiation and myelin regeneration.  


Hypothyroidism is a well-described cause of hypomyelination. In addition, thyroid hormone (T3) has recently been shown to enhance remyelination in various animal models of CNS demyelination. What are the ways in which T3 promotes the development and regeneration of healthy myelin? To begin to understand the mechanisms by which T3 drives myelination, we have identified genes regulated specifically by T3 in purified oligodendrocyte precursor cells (OPCs). Among the genes identified by genomic expression analyses were four transcription factors, Kruppel-like factor 9 (KLF9), basic helix-loop-helix family member e22 (BHLHe22), Hairless (Hr), and Albumin D box-binding protein (DBP), all of which were induced in OPCs by both brief and long term exposure to T3. To begin to investigate the role of these genes in myelination, we focused on the most rapidly and robustly induced of these, KLF9, and found it is both necessary and sufficient to promote oligodendrocyte differentiation in vitro. Surprisingly, we found that loss of KLF9 in vivo negligibly affects the formation of CNS myelin during development, but does significantly delay remyelination in cuprizone-induced demyelinated lesions. These experiments indicate that KLF9 is likely a novel integral component of the T3-driven signaling cascade that promotes the regeneration of lost myelin. Future analyses of the roles of KLF9 and other identified T3-induced genes in myelination may lead to novel insights into how to enhance the regeneration of myelin in demyelinating diseases such as multiple sclerosis. PMID:22472204

Dugas, Jason C; Ibrahim, Adiljan; Barres, Ben A



Intracellular calcium mobilization induces period genes via MAP kinase pathways in NIH3T3 cells  

Microsoft Academic Search

Mammalian period genes have a pivotal role in generating circadian rhythms and are rapidly induced by several stimuli in mammalian cells. In the present study, we revealed that treatment with thapsigargin significantly induced transcripts of mouse period 1 and 2 (mPer1 and mPer2) but not mPer3 among circadian related genes in NIH3T3 cells. Thapsigargin-induced mPer1 and mPer2 mRNA expressions took

Kentaro Oh-hashi; Yoshihisa Naruse; Masaki Tanaka



Heat Shock Induces the Release of Fibroblast Growth Factor 1 from NIH 3T3 Cells  

Microsoft Academic Search

Fibroblast growth factor 1 (FGF-1) is a potent angiogenic and neurotrophic factor whose structure lacks a classical signal sequence for secretion. Although the initiation of these biological activities involves the interaction between FGF-1 and cell surface receptors, the mechanism responsible for the regulation of FGF-1 secretion is unknown. We report that murine NIH 3T3 cells transfected with a synthetic gene

Anthony Jackson; Stanley Friedman; Xi Zhan; Kurt A. Engleka; Reza Forough; Thomas Maciag



Mice Lacking the p43 Mitochondrial T3 Receptor Become Glucose Intolerant and Insulin Resistant during Aging  

PubMed Central

Thyroid hormones (TH) play an important regulatory role in energy expenditure regulation and are key regulators of mitochondrial activity. We have previously identified a mitochondrial triiodothyronine (T3) receptor (p43) which acts as a mitochondrial transcription factor of the organelle genome, which leads in vitro and in vivo, to a stimulation of mitochondrial biogenesis. Recently, we generated mice carrying a specific p43 invalidation. At 2 months of age, we reported that p43 depletion in mice induced a major defect in insulin secretion both in vivo and in isolated pancreatic islets, and a loss of glucose-stimulated insulin secretion. The present study was designed to determine whether p43 invalidation influences life expectancy and modulates blood glucose and insulin levels as well as glucose tolerance or insulin sensitivity during aging. We report that from 4 months old onwards, mice lacking p43 are leaner than wild-type mice. p43?/? mice also have a moderate reduction of life expectancy compared to wild type. We found no difference in blood glucose levels, excepted at 24 months old where p43?/? mice showed a strong hyperglycemia in fasting conditions compared to controls animals. However, the loss of glucose-stimulated insulin secretion was maintained whatever the age of mice lacking p43. If up to 12 months old, glucose tolerance remained unchanged, beyond this age p43?/? mice became increasingly glucose intolerant. In addition, if up to 12 months old p43 deficient animals were more sensitive to insulin, after this age we observed a loss of this capacity, culminating in 24 months old mice with a decreased sensitivity to the hormone. In conclusion, we demonstrated that during aging the depletion of the mitochondrial T3 receptor p43 in mice progressively induced an increased glycemia in the fasted state, glucose intolerance and an insulin-resistance several features of type-2 diabetes.

Bertrand, Christelle; Blanchet, Emilie; Pessemesse, Laurence; Annicotte, Jean Sebastien; Feillet-Coudray, Christine; Chabi, Beatrice; Levin, Jonathan; Fajas, Lluis; Cabello, Gerard; Wrutniak-Cabello, Chantal; Casas, Francois



Effect of different thyroid state on ornithine decarboxylase activity and receptors of T3 in rat liver.  


The effects of thyreoidectomy, of the administration of 3,5,3'-triiodothyronine (T3), of thyroxine (T4) together with the inhibitor of iodothyronine 5'-deiodinase activity 6-propyl-2-thiouracil (PTU) and of PTU alone on rat liver ornithine decarboxylase (ODC) activity was studied. 28 days after thyroidectomy there was a decrease in ODC activity, whereas T3 treatment of normal rats increased enzyme activity. PTU decreased ODC activity in intact rats and inhibited ODC activity in animals treated with T4. Neither thyroidectomy T3, T4 given in vivo nor the polyamines spermine and spermidine added in vitro influenced the binding of 125I-T3 to its receptor in liver nuclei. Only addition of PTU increased Ka significantly (P less than 0.01). It is concluded that ODC activity in the liver is directly correlated with the thyroid state but not to parameters of thyroid hormone receptor in liver nuclei. PMID:2630312

Knopp, J; Brtko, J



Identification of a T3Associated gamma delta T Cell Receptor on Thy-I+ Dendritic Epidermal Cell Lines  

Microsoft Academic Search

The murine epidermis contains a subpopulation of bone marrow-derived lymphocytes that have a dendritic morphology and that express Thy-1 and T3 cell-surface antigens but not other markers (L3T4 or Lyt-2) characteristic of mature peripheral T lymphocytes. An alternative type of T cell receptor was earlier identified on a subpopulation of murine thymocytes with a similar phenotype (T3+, L3T4-, Lyt-2-), but

Frits Koning; Georg Stingl; Wayne M. Yokoyama; Hidekazu Yamada; W. Lee Maloy; Erwin Tschachler; Ethan M. Shevach; John E. Coligan



Morphological transformation induced by glass fibers in BALB/c-3T3 cells.  


Studies were conducted to determine whether 1) glass fibers can induce morphological transformation in BALB/c-3T3 cells, 2) the transforming activity of glass fibers is related to fiber size, and 3) transformed cells induced by glass fibers possess neoplastic properties. In the transformation assay, BALB/c-3T3 cells were treated with three different types of glass fibers: Manville code 100 (JM-100, Manville Corp., Denver, CO), Owens-Corning AAA-10 (AAA-10, Owens-Corning Corp., Toledo, OH), and Owens-Corning general building insulation (ISL, Owens-Corning Corp.) fibers. The neoplastic properties were investigated using the soft agar cloning and gene transfection methods. All three different glass fibers were cytotoxic at high concentrations and induced dose-related increases in morphological transformation. The transforming activity was inversely related to fiber size, with AAA-10 showing higher activity than JM-100 and JM-100 showing higher activity than ISL fiber. Transformed cells induced by glass fibers exerted anchorage-independent growth (90%) and DNA transfection-mediated transformation (100%). These results indicate that glass fibers are capable of transforming mammalian (BALB/c-3T3) cells in vitro as a function of their physical properties and that glass fiber-induced transformed cells possess preneoplastic characteristics. PMID:8525469

Gao, H G; Whong, W Z; Jones, W G; Wallace, W E; Ong, T



Human c-fgr induces a monocyte-specific enzyme in NIH 3T3 cells  

SciTech Connect

The mutant c-fgr protein (p58{sup c-fgr/F523}) containing Phe-523 instead of Tyr-523 exhibited transforming activity in NIH 3T3 cells like other protein-tyrosine kinases of the src family, but normal p58{sup c-fgr} (p58{sup c-fgr/wt}) did not. The mutant protein showed tyrosine kinase activity threefold higher than that of the normal protein in vitro. Surprisingly, transfection of the normal c-fgr gene into NIH 3T3 cells resulted in induction of sodium fluoride (NaF)-sensitive {alpha}-naphthyl butyrate esterase ({alpha}-NBE), marker enzyme of cells of monocytic origin, which was not induced in v-src-, v-fgr-, or lyn-transfected NIH 3T3 cells. The NaF-sensitive {alpha}-NBE induced in c-fgr transfectants was shown by isoelectric focusing to have a pI of 5.2 to 5.4, a range which was the same as those for thioglycolate-induced murine peritoneal macrophages and 1{alpha}, 25-dihydroxyvitamin D{sub 3}-treated WEHI-3B cells. Immunoblotting studies with antophosphotyrosine antibodies revealed that 58-, 62-, 75-, 120-, 200-, and 230-kDa proteins were commonly phosphorylated at tyrosine residues in NIH 3T3 cells transfected with normal and mutated c-fgr, while 95-kDa protein was significantly phosphorylated at tyrosine residues in NIH 3T3 cells transfected with normal and mutated c-fgr, while 95-kDa protein was significantly phosphorylated at tyrosine residues in cells transfected with the mutated c-fgr. These findings suggest that tyrosine phosphorylation of specific cellular substrate proteins is important in induction of NaF-sensitive {alpha}-NBE and cell transformation by p58{sup c-fgr}.

Inoue, Kazushi; Akiyama, Tetsu; Toyoshima, Kumao (Osaka Univ. (Japan)); Wongsasant, Budsaba (Univ. of Tokyo (Japan))



Regulation of gonadotropin alpha subunit gene expression by dopamine D(2) receptor agonist in clonal mouse gonadotroph alphaT3-1 cells.  


Pituitary prolactin biosynthesis is negatively regulated by hypothalamic dopamine through D(2) receptors in pituitary lactotrophs, but little is known about the direct effect of dopamine on gonadotrophs. In this study, the clonal gonadotroph-derived cell line, alphaT3-1, was used to examine whether gene expression of the pituitary gonadotropin alpha subunit, stimulated with GnRH or pituitary adenylate cyclase-activating polypeptide (PACAP), was controlled by dopamine D(2) receptor. Western blotting and reverse transcription-polymerase chain reaction analysis demonstrated the presence of dopamine D(2) receptors in alphaT3-1 cells. Both GnRH and PACAP increased alpha subunit gene expression. GnRH-induced alpha subunit gene expression was not affected by quinpirol, a specific dopamine D(2) receptor agonist. In contrast, PACAP-induced gene expression was significantly lower in the presence of quinpirol. The roles of extracellular signal-regulated kinase (ERK) and cAMP in the expression of the alpha subunit gene were examined. GnRH activated ERK, but PACAP did not, and the activation was not inhibited by quinpirol. GnRH-induced alpha subunit gene expression was completely inhibited by an ERK inhibitor, PD098059. Cyclic AMP accumulation in alphaT3-1 cells was increased by treatment with PACAP, and quinpirol inhibited this effect. GnRH did not affect cAMP production in these cells. These results suggest that in alphaT3-1 cells, dopamine D(2) receptors negatively regulate pituitary alpha subunit gene expression in association with the cAMP-dependent pathway, but not with the ERK pathway. PMID:12297539

Kanasaki, Haruhiko; Yonehara, Toshie; Yamada, Yoko; Takahashi, Kentaro; Hata, Kohkichi; Fujiwaki, Rituto; Yamamoto, Hideyuki; Takeuchi, Yusuke; Fukunaga, Kohji; Miyamoto, Eishichi; Miyazaki, Kohji



Reactive Oxygen Species Regulate Swelling-induced Taurine Efflux in NIH3T3 Mouse Fibroblasts  

Microsoft Academic Search

NIH3T3 mouse fibroblasts generate reactive oxygen species (ROS) and release taurine following exposure to hypotonic medium and to isotonic medium containing the lipase activator melittin. The swelling-induced taurine release is potentiated by H2O2, the calmodulin antagonist W7, and ATP, but inhibited by the antioxidant butulated hydroxytoluene (BHT), the NAD(P)H oxidase inhibitor diphenylene iodonium (DI), and the iPLA2 inhibitor bromoenol lactone

I. H. Lambert



Butyrate modulates the expression of. beta. -adrenergic receptor subtype in 3T3-L1 cells  

SciTech Connect

In mouse 3T3-L1 fibroblasts, the glucocorticoid dexamethasone (dex) affects a switch in ..beta..-adrenergic receptor (..beta..AR) subtype expression from ..beta../sub 1/AR to ..beta../sub 2/AR and increases total ..beta..AR number. They now demonstrate a similar effect by sodium butyrate (B) and find that the combined effect of these two gene-activating agents is greater than additive suggesting different mechanisms of action on the ..beta..AR. ..beta..AR are assayed in membranes prepared from 3T3-L1 cells using the radiolabeled ..beta..AR-specific antagonist (/sup 125/I)-cyanopindolol. ..beta..AR subtype is determined by competition binding of the ..beta../sub 2/AR-selective antagonist ICI 118.551 for the radioligand. B (2-10mM) causes a dose-dependent increase in total ..beta..AR number (up to 2-fold over control) and the proportion of ..beta../sub 2/AR. B (5mM) causes a time-dependent increase in total ..beta..AR number (2-fold) and the proportion of ..beta../sub 2/AR up to 24 hr. Dex maximally increases total ..beta..AR number (2-fold) when treated for 48 hr at concentrations greater than or equal to 100nM. B (2 or 5mM) together with dex (250nM) have a greater than additive effect on total ..beta..AR number at 24 hr (1.7-fold) and at 48 hr (1.4-2.4-fold, using 5 or 10mM B and dex greater than or equal to 10nM). The proportion of ..beta../sub 2/AR is also greater when both compounds are added together. In comparison with proprionate and valerate, B increases total ..beta..AR number and the proportion of ..beta../sub 2/AR to a greater extent and at lower concentrations. To determine a functional correlate to these findings, cells were pre-treated for 48 hr with B and/or dex, intracellular ATP labeled with /sup 3/H-adenine, followed by treatment with forskolin ( and ..beta..AR agonists. B caused a dramatic increase in /sup 3/H-cAMP produced compared to control and dex treatments and a greater than additive effect was again achieved when B and dex were added together.

Poksay, K.S.; Nakada, M.T.; Crooke, S.T.; Stadel, J.M.



Human papillomavirus type 16 DNA-induced malignant transformation of NIH 3T3 cells  

SciTech Connect

A biological function for human papillomavirus 16 (HPV 16) DNA was demonstrated by transformation of NIH 3T3 cells. HPV 16 DNA has been found frequently in genital cancer and has been classified as a papillomavirus on the basis of DNA homology. A recombinant HPV 16 DNA (pSHPV16d), which contains a head-to-tail dimer of the full-length HPV 16 genome, induced morphologic transformation; the transformed cells were tumorigenic in nude mice. Expression of transforming activity was unique because of the long latency period (more than 4 weeks) required for induction of morphologic transformation and because the transfected DNA existed primarily in a multimeric form with some rearrangement. Furthermore, virus-specific RNAs were expressed in the transformants. The transformation of NIH 3T3 cells provides a model for analyzing the functions of HPV 16, which is associated with cervical carcinomas.

Yasumoto, S.; Burkhardt, A.L.; Doniger, J.; DiPaolo, J.A.



Drinking water disinfection byproduct iodoacetic acid induces tumorigenic transformation of NIH3T3 cells.  


Iodoacetic acid (IAA) and iodoform (IF) are unregulated iodinated disinfection byproducts (DBPs) found in drinking water. Their presence in the drinking water of China has not been documented. Recently, the carcinogenic potential of IAA and IF has been a concern because of their mutagenicity in bacteria and genotoxicity in mammalian cells. Therefore, we measured their concentrations in Shanghai drinking water and assessed their cytotoxicity, genotoxicity, and ability to transform NIH3T3 cells to tumorigenic lines. The concentrations of IAA and IF in Shanghai drinking water varied between summer and winter with maximum winter levels of 2.18 ?g/L IAA and 0.86 ?g/L IF. IAA with a lethal concentration 50 (LC50) of 2.77 ?M exhibited more potent cytotoxicity in NIH3T3 cells than IF (LC50 = 83.37 ?M). IAA, but not IF, induced a concentration-dependent DNA damage measured by ?-H2AX staining and increased tail moment in single-cell gel electrophoresis. Neither IAA nor IF increased micronucleus frequency. Prolonged exposure of NIH3T3 cells to IAA increased the frequencies of transformed cells with anchorage-independent growth and agglutination with concanavalin A. IAA-transformed cells formed aggressive fibrosarcomas after inoculation into Balb/c nude mice. This study demonstrated that IAA has a biological activity that is consistent with a carcinogen and human exposure should be of concern. PMID:23641915

Wei, Xiao; Wang, Shu; Zheng, Weiwei; Wang, Xia; Liu, Xiaolin; Jiang, Songhui; Pi, Jingbo; Zheng, Yuxin; He, Gengsheng; Qu, Weidong



Triac regulation of transcription is T 3 receptor isoform- and response element-specific  

Microsoft Academic Search

3,5,3?-triiodothyroacetic acid (Triac) is a naturally occurring triiodothyronine (T3) analog, which has been used on an empirical basis to treat the syndrome of resistance to thyroid hormone (RTH). The aim of our studies was to compare the effects of Triac and T3 on negative and positive thyroid hormone response elements (TREs). We used transient transfections with luciferase reporter genes to

N Messier; M. F Langlois



Prednisolone induces the Wnt signalling pathway in 3T3-L1 adipocytes  

PubMed Central

Synthetic glucocorticoids are potent anti-inflammatory drugs but show dose-dependent metabolic side effects such as the development of insulin resistance and obesity. The precise mechanisms involved in these glucocorticoid-induced side effects, and especially the participation of adipose tissue in this are not completely understood. We used a combination of transcriptomics, antibody arrays and bioinformatics approaches to characterize prednisolone-induced alterations in gene expression and adipokine secretion, which could underlie metabolic dysfunction in 3T3-L1 adipocytes. Several pathways, including cytokine signalling, Akt signalling, and Wnt signalling were found to be regulated at multiple levels, showing that these processes are targeted by prednisolone. These results suggest that mechanisms by which prednisolone induce insulin resistance include dysregulation of wnt signalling and immune response processes. These pathways may provide interesting targets for the development of improved glucocorticoids.

Fleuren, Wilco W. M.; Linssen, Margot M. L.; Toonen, Erik J. M.; van der Zon, Gerard C. M.; Guigas, Bruno; de Vlieg, Jacob; Dokter, Wim H. A.; Ouwens, D. Margriet



Isolation of cDNA clones encoding the 20K non-glycosylated polypeptide chain of the human T-cell receptor\\/T3 complex  

Microsoft Academic Search

The antigen receptor on human T lymphocytes consists of two variable immunoglobulin-like glycoproteins, alpha and beta, which occur in association with three invariable T3 membrane proteins1-3. In humans two of these proteins, T3-gamma and T3-delta, are glycoproteins of relative molecular mass (Mr) 25,000 (25K) and 20,000 (20K), respectively, while the third, T3-?, is a 20K non-glycosylated protein4-6. On the surface

Daniel P. Gold; Jennifer M. Puck; Carolyn L. Pettey; Mildred Cho; John Coligan; James N. Woody; Cox Terhorst



Role of Epidermal Growth Factor and ErbB2 Receptors in 3T3-L1 Adipogenesis  

Microsoft Academic Search

Objective: Epidermal growth factor (EGF) stimulates proliferation in 3T3-L1 preadipocytes, but EGF action in differentiation is less clear. EGF promotes differentiation at concentrations <1 nM but inhibits differentiation at higher concentrations, suggesting a dual role in adipogenesis. We hypothesized that differences in EGF receptor activation and downstream signaling mediate distinct biological effects of EGF at low vs. high abundance.Research Methods

Molly Harrington; Sunthorn Pond-Tor; Charlotte M. Boney



Human Ornithine Decarboxylase-overproducing NIH3T3 Cells Induce Rapidly Growing, Highly Vascularized Tumors in Nude Mice1  

Microsoft Academic Search

Overexpressionof human ornithine decarboxylase(ODC) under the control of strong promoters induces morphological transformation of immortalized NIH3T3 and Rat.! fibroblasts (M. Auvinen et aL, Nature (Lond.), 360: 355â€\\

Merja Auvinen; Aire Lame; Aino Paasinen-Sohns; Anneli Kangas; Lauri Kangas; Leif C. Andersson


Anandamide induced PPAR? transcriptional activation and 3T3-L1 preadipocyte differentiation  

Microsoft Academic Search

We investigated the effects of anandamide on peroxisome proliferator-activated receptor ? (PPAR?) activity. In two different transactivation systems using either full-length or only the ligand binding domain of PPAR?, we showed that anandamide, but not palmitoylethanolamide induced transcriptional activation of PPAR? in a dose dependent manner with an EC50 of 8 ?M. In addition, competition binding experiments showed that anandamide

Monsif Bouaboula; Sandrine Hilairet; Jean Marchand; Lluis Fajas; Gerard Le Fur; Pierre Casellas



Induction of Peroxisome Proliferator-Activated Receptor g during the Conversion of 3T3 Fibroblasts into Adipocytes Is Mediated by C\\/EBPb, C\\/EBPd, and Glucocorticoids  

Microsoft Academic Search

The differentiation of 3T3 preadipocytes into adipocytes is accompanied by a transient induction of C\\/EBPb and C\\/EBPd expression in response to treatment of the cells with methylisobutylxanthine (MIX) and dexa- methasone (DEX), respectively. In this report, we demonstrate that peroxisome proliferator-activated receptor g (PPARg) expression in 3T3-L1 preadipocytes is induced by MIX and DEX, suggesting that C\\/EBPb and C\\/EBPdmay be




Erythropoietin improves insulin resistance via the regulation of its receptor-mediated signaling pathways in 3T3L1 adipocytes.  


Recombinant human erythropoietin (rHuEPO) reduces serum insulin levels, increases insulin sensitivity, and reduces insulin resistance (IR). However, the mechanisms behind these effects are unclear. This study aimed to investigate the mechanism by which rHuEPO effects IR in 3T3L1 adipocytes. After treatment with different concentrations of rHuEPO, glucose consumption, and tumor necrosis factor (TNF-?), adiponectin, and leptin levels were assayed with a commercial enzyme-linked immunosorbent assays. Endogenous erythropoietin receptor (EPOR) expression was inhibited using small interfering RNA (siRNA). EPOR protein and mRNA expression was detected via immunofluorescence and real-time PCR analyses, respectively. The expression of pAKT/AKT and p-STAT5/STAT5 was determined via Western blot analysis. rHuEPO treatment improved glucose uptake, increased adiponectin levels, and reduced TNF-? and leptin levels in 3T3L1 adipocytes with dexamethasone-induced IR. Whereas EPOR protein and gene expression was absent in preadipocytes, it was observed in mature 3T3L1 adipocytes. However, the expression of EPOR in insulin resistant 3T3L1 adipocytes was significantly decreased (p<0.05). rHuEPO increased the expression of EPOR, and upregulated the expression of pAKT/AKT and pSTAT5/STAT5 in 3T3L1 adipocytes (p<0.05), which was blocked by siEPOR, the phosphatidylinositol-3-kinase (PI3K) inhibitor, LY294002, and a STAT5 inhibitor, respectively. In summary, rHuEPO reduced IR in adipocytes by increasing glucose uptake and improving the adipokine profile. rHuEPO-induced EPOR protein expression and subsequent induction of pAKT and pSTAT5 suggest that the EPO-EPOR system may play a role in glucose metabolism within adipocytes. PMID:23313788

Pan, Yu; Shu, Jin Lian; Gu, Hui Fang; Zhou, Dong Chi; Liu, Xiao Li; Qiao, Qing Yan; Fu, Shun Kun; Gao, Feng Hou; Jin, Hui Min



Bromocriptine inhibits adipogenesis and lipogenesis by agonistic action on ?2-adrenergic receptor in 3T3-L1 adipocyte cells.  


The primary goals of the present study were to investigate the inhibitory effects of bromocriptine (BC) on adipogenesis and lipogenesis in 3T3-L1 adipocyte cells as well as to elucidate its molecular mechanism of action. Adipogenic and lipogenic capacity of BC-treated cells was evaluated by oil red-O staining, triglyceride content assay, real-time RT-PCR and immunoblotting. To determine the mechanism responsible for the anti-obesity effect of BC, we applied two methods. Firstly, we knocked down dopamine D2 receptor (D2R) up to 50% using siRNA. Secondly, we blocked the activity of ?2-adrenergic receptor (?2-AR) by yohimbine treatment and monitored its effects on adipogenic and lipogenic events in 3T3-L1 cells. BC decreased the expression levels of adipogenic activators, including Ppar?, Ppar?, and Cebp?, as well as major lipogenic target genes, including Me1, Acc1, 6Pgd, Fasn, and Prkaa1. Moreover, BC markedly reduced intracellular nitric oxide formation in a dose-dependent manner and expression of pro-inflammatory genes, Tnf? and Il6, which reflects attenuated pro-inflammatory responses. Further, upon treatment with BC, D2R-deficient cells displayed a significant decrease in lipogenic activity compared to control cells, whereas yohimbine-treated cells exhibited no reduction in lipogenic activity. BC can effectively attenuate adipogenesis and lipogenesis in 3T3-L1 cells by downregulating the expression of lipogenic genes and proteins. Our current experimental data collectively establish that the anti-obesity effects of BC are not D2R-dependent but result from the action of ?2-AR in 3T3-L1 adipocytes. PMID:23271132

Mukherjee, Rajib; Yun, Jong Won



Expression of progesterone receptor B is associated with G0/G1 arrest of the cell cycle and growth inhibition in NIH3T3 cells  

SciTech Connect

Previously, we found a significant reduction of progesterone receptor B (PR-B) expression levels in the Ras-mediated NIH3T3 cell transformation, and re-expression of exogenous PR-B eliminated the tumorigenic potential. We hypothesized that this reduction is of biological significance in cell transformation. In the present study, we determined the correlation between PR-B expression and cell cycle progression. In synchronized NIH3T3 cells, we found an increase in PR-B protein and p27 CDK inhibitor levels in the G0/G1 phase and a reduction due to redistribution in the S and G2/M phases. The MEK inhibitor or cAMP stimulation arrested NIH3T3 cells in the G0/G1 phase of the cell cycle. The expression of PR-B and p27 CDK inhibitors was up-regulated by treatment with both the MEK inhibitor and cAMP. Treatment of synchronized cells with a PKA inhibitor in the presence of 1% calf serum resulted in a significant reduction in both PR-B and p27 levels. The decrease in the PR-B levels caused by anti-sense oligomers or siRNA corresponded to the reduction in p27 levels. PR-B overexpression by adenovirus infection induced p27 and suppressed cell growth. Finally, we showed that PR-B modulation involved in the regulation of NIH3T3 cell proliferation was independent of nuclear estrogen receptor (ER) activity but dependent on non-genomic ER activity.

Horiuchi, Shinji [Department of Molecular Genetics, Division of Molecular and Cell Therapeutics, Medical Institute of Bioregulation, Kyushu University, Tsurumihara 4546, Beppu, Oita, 874-0838 (Japan) and Department of Obstetrics and Gynecology, School of Medicine, Fukuoka University, Jyounanku Fukuoka 814-0180 (Japan); Kato, Kiyoko [Department of Molecular Genetics, Division of Molecular and Cell Therapeutics, Medical Institute of Bioregulation, Kyushu University, Tsurumihara 4546, Beppu, Oita, 874-0838 (Japan)]. E-mail:; Suga, Shin [Department of Molecular Genetics, Division of Molecular and Cell Therapeutics, Medical Institute of Bioregulation, Kyushu University, Tsurumihara 4546, Beppu, Oita, 874-0838 (Japan); Takahashi, Akira [Department of Molecular Genetics, Division of Molecular and Cell Therapeutics, Medical Institute of Bioregulation, Kyushu University, Tsurumihara 4546, Beppu, Oita, 874-0838 (Japan); Ueoka, Yousuke [Department of Molecular Genetics, Division of Molecular and Cell Therapeutics, Medical Institute of Bioregulation, Kyushu University, Tsurumihara 4546, Beppu, Oita, 874-0838 (Japan); Arima, Takahiro [Department of Molecular Genetics, Division of Molecular and Cell Therapeutics, Medical Institute of Bioregulation, Kyushu University, Tsurumihara 4546, Beppu, Oita, 874-0838 (Japan); Nishida, Jun-ichi [Department of Molecular Genetics, Division of Molecular and Cell Therapeutics, Medical Institute of Bioregulation, Kyushu University, Tsurumihara 4546, Beppu, Oita, 874-0838 (Japan); Hachisuga, Toru; Kawarabayashi, Tatsuhiko [Department of Obstetrics and Gynecology, School of Medicine, Fukuoka University, Jyounanku Fukuoka 814-0180 (Japan); Wake, Norio [Department of Molecular Genetics, Division of Molecular and Cell Therapeutics, Medical Institute of Bioregulation, Kyushu University, Tsurumihara 4546, Beppu, Oita, 874-0838 (Japan)



Primary structure of the T3 gamma subunit of the T3/T cell antigen receptor complex deduced from cDNA sequences: evolution of the T3 gamma and delta subunits.  

PubMed Central

cDNA clones, whose fusion proteins were recognised by an anti-(T3 gamma chain) serum, were isolated from a lambda gt11 expression library prepared from the human T leukaemia cell line J6. The clones encoded a unique sequence related to that of the T3 delta chain, and hybridised to two mRNA transcripts of 0.8 and 3.5 kb in size, whose expression was restricted to T lymphocytes. The 182 amino acid sequence deduced from the cDNA revealed a typical signal peptide, a predominantly hydrophilic 89 amino residue domain with two N-glycosylation sites, a hydrophobic domain with a centrally located glutamic acid residue and a 44-residue domain with at least one potential serine phosphorylation site for protein kinase C. Given this arrangement the T3 gamma polypeptide most probably has a transmembrane orientation with the N-terminal domain exposed on the cell surface. The amino acid and nucleotide sequences showed marked homology with those of the T3 delta chain, suggesting that the respective genes arose by duplication about 200 million years ago. The intracellular and membrane-proximal half of the extracellular domains were especially well conserved. Images Fig. 1. Fig. 5.

Krissansen, G W; Owen, M J; Verbi, W; Crumpton, M J



Critical role of leukotriene B4 receptor signaling in mouse 3T3-L1 preadipocyte differentiation  

PubMed Central

Background Various inflammatory mediators related to obesity might be closely related to insulin resistance. Leukotrienes (LTs) are involved in inflammatory reactions. However, there are few reports regarding the role of LTs in adipocyte differentiation. Therefore, we investigated the role of leukotriene B4 (LTB4)-leukotriene receptor (BLT) signaling in mouse 3T3-L1 fibroblastic preadipocyte differentiation to mature adipocytes. Methods Mouse 3T3-L1 preadipocytes were treated with lipoxygenase (LOX) inhibitors, BLT antagonist, and small interfering RNA (siRNA) for BLT1 and BLT2 to block the LTB4-BLT signaling pathway, then the adipocyte differentiation such as lipid accumulation and the increase in triglyceride was evaluated. Results Blockade of BLT signaling by treatment with a LOX inhibitor or a BLT antagonist suppressed preadipocyte differentiation into mature adipocytes. In addition, knockdown of BLT1 and BLT2 by siRNAs dramatically inhibited differentiation. These results indicate the LTB4-BLT signaling pathway may positively regulate preadipocyte differentiation and be a rate-limiting system to control adipocyte differentiation. Conclusions The LTB4-BLT signaling pathway provides a potent regulatory signal that accelerates the differentiation of mouse 3T3-L1 preadipocytes. Further investigations are necessary to confirm the exact role of LTB4 and BLTs signaling pathways in preadipocyte differentiation.



Environmental endocrine disruptors promote adipogenesis in the 3T3-L1 cell line through glucocorticoid receptor activation.  


The burgeoning obesity and diabetes epidemics threaten health worldwide, yet the molecular mechanisms underlying these phenomena are incompletely understood. Recently, attention has focused on the potential contributions of environmental pollutants that act as endocrine disrupting chemicals (EDCs) in the pathogenesis of metabolic diseases. Because glucocorticoid signaling is central to adipocyte differentiation, the ability of EDCs to stimulate the glucocorticoid receptor (GR) and drive adipogenesis was assessed in the 3T3-L1 cell line. Various EDCs were screened for glucocorticoid-like activity using a luciferase reporter construct, and four (bisphenol A (BPA), dicyclohexyl phthalate (DCHP), endrin, and tolylfluanid (TF)) were shown to significantly stimulate GR without significant activation of the peroxisome proliferator-activated receptor-gamma. 3T3-L1 preadipocytes were then treated with EDCs and a weak differentiation cocktail containing dehydrocorticosterone (DHC) in place of the synthetic dexamethasone. The capacity of these compounds to promote adipogenesis was assessed by quantitative oil red O staining and immunoblotting for adipocyte-specific proteins. The four EDCs increased lipid accumulation in the differentiating adipocytes and also upregulated the expression of adipocytic proteins. Interestingly, proadipogenic effects were observed at picomolar concentrations for several of the EDCs. Because there was no detectable adipogenesis when the preadipocytes were treated with compounds alone, the EDCs are likely promoting adipocyte differentiation by synergizing with agents present in the differentiation cocktail. Thus, EDCs are able to promote adipogenesis through the activation of the GR, further implicating these compounds in the rising rates of obesity and diabetes. PMID:19927138

Sargis, Robert M; Johnson, Daniel N; Choudhury, Rashikh A; Brady, Matthew J



Mouse osteoblastic cell line (MC3T3-E1) expresses extracellular calcium (Ca2+o)-sensing receptor and its agonists stimulate chemotaxis and proliferation of MC3T3-E1 cells.  


The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Osteoblasts appear at sites of osteoclastic bone resorption during bone remodeling in the "reversal" phase following osteoclastic resorption and preceding bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for osteoblasts in the vicinity, leading us to determine whether such osteoblasts express the CaR. In this study, we used the mouse osteoblastic, clonal cell line MC3T3-E1. Both immunocytochemistry and Western blot analysis, using an antiserum specific for the CaR, detected CaR protein in MC3T3-E1 cells. We also identified CaR transcripts in MC3T3-E1 cells by Northern analysis using a CaR-specific riboprobe and by reverse transcription-polymerase chain reaction with CaR-specific primers, followed by nucleotide sequencing of the amplified products. Exposure of MC3T3-E1 cells to high Ca2+o (up to 4.8 mM) or the polycationic CaR agonists, neomycin and gadolinium (Gd3+), stimulated both chemotaxis and DNA synthesis in MC3T3-E1 cells. Therefore, taken together, our data strongly suggest that the osteoblastic cell line MC3T3-E1 possesses both CaR protein and mRNA very similar, if not identical, to those in parathyroid and kidney. Furthermore, the CaR in these osteoblasts could play a key role in regulating bone turnover by stimulating the proliferation and migration of such cells to sites of bone resorption as a result of local release of Ca2+o. PMID:9783541

Yamaguchi, T; Chattopadhyay, N; Kifor, O; Butters, R R; Sugimoto, T; Brown, E M



SirT3 suppresses hypoxia inducible factor 1? and tumor growth by inhibiting mitochondrial ROS production  

PubMed Central

It has become increasing clear that alterations in cellular metabolism have a key role in the generation and maintenance of cancer. Some of the metabolic changes can be attributed to the activation of oncogenes or loss of tumor suppressors. Here, we show that the mitochondrial sirtuin, SirT3, acts as a tumor suppressor via its ability to suppress reactive oxygen species (ROS) and regulate hypoxia inducible factor 1? (HIF-1?). Primary mouse embryo fibroblasts (MEFs) or tumor cell lines expressing SirT3 short-hairpin RNA exhibit a greater potential to proliferate, and augmented HIF-1? protein stabilization and transcriptional activity in hypoxic conditions. SirT3 knockdown increases tumorigenesis in xenograft models, and this is abolished by giving mice the anti-oxidant N-acetyl cysteine. Moreover, overexpression of SirT3 inhibits stabilization of HIF-1? protein in hypoxia and attenuates increases in HIF-1? transcriptional activity. Critically, overexpression of SirT3 decreases tumorigenesis in xenografts, even when induction of the sirtuin occurs after tumor initiation. These data suggest that SirT3 acts to suppress the growth of tumors, at least in part through its ability to suppress ROS and HIF-1?.

Bell, E L; Emerling, B M; Ricoult, S J H; Guarente, L



High glucose and glucosamine induce insulin resistance via different mechanisms in 3T3-L1 adipocytes.  


Sustained hyperglycemia induces insulin resistance, but the mechanism is still incompletely understood. Glucosamine (GlcN) has been extensively used to model the role of the hexosamine synthesis pathway (HSP) in glucose-induced insulin resistance. 3T3-L1 adipocytes were preincubated for 18 h in media +/- 0.6 nmol/l insulin containing either low glucose (5 mmol/l), low glucose plus GlcN (0.1-2.5 mmol/l), or high glucose (25 mmol/l). Basal and acute insulin-stimulated (100 nmol/l) glucose transport was measured after re-equilibration in serum and insulin-free media. Preincubation with high glucose or GlcN (1-2.5 mmol/l) inhibited basal and acute insulin-stimulated glucose transport only if insulin was present during preincubation. However, only preincubation with GlcN plus insulin inhibited insulin-stimulated GLUT4 translocation. GLUT4 and GLUT1 protein expression were not affected. GlcN (2.5 mmol/l) increased cellular UDP-N-acetylhexosamines (UDP-HexNAc) by 400 and 900% without or with insulin, respectively. High glucose plus insulin increased UDP-HexNAc by 30%. GlcN depleted UDP-hexoses, whereas high glucose plus insulin increased them. Preincubation with 0.5 mmol/l GlcN plus insulin maximally increased UDP-HexNAc without affecting insulin-stimulated or basal glucose transport. GlcN plus insulin (but not high glucose plus insulin) caused marked GlcN dose-dependent accumulation of GlcN-6-phosphate, which correlated with insulin resistance of glucose transport (r = 0.935). GlcN plus insulin (but not high glucose plus insulin) decreased ATP (10-30%) and UTP (>50%). GTP was not measured, but GDP increased. Neither high glucose plus insulin nor GlcN plus insulin prevented acute insulin stimulation (approximately 20-fold) of insulin receptor substrate 1-associated phosphatidylinositol (PI)-3 kinase. We have come to the following conclusions. 1) Chronic exposure to high glucose or GlcN in the presence of low insulin caused insulin resistance of glucose transport by different mechanisms. 2) GlcN inhibited GLUT4 translocation, whereas high glucose impaired GLUT4 "intrinsic activity" or membrane intercalation. 3) Both agents may act distally to PI-3 kinase. 4) GlcN has metabolic effects not shared by high glucose. GlcN may not model HSP appropriately, at least in 3T3-L1 adipocytes. PMID:10866051

Nelson, B A; Robinson, K A; Buse, M G



The requirements for viral entry differ from those for virally induced syncytium formation in NIH 3T3/DTras cells exposed to Moloney murine leukemia virus.  


Moloney murine leukemia virus (Mo-MuLV) has the unique ability to infect different cells via either a low-pH-dependent or a pH-independent entry pathway. Only the pH-independent mechanism of Mo-MuLV entry has been associated with Mo-MuLV-induced syncytium formation. We have now identified a transformed cell line (NIH 3T3/DTras) which efficiently forms syncytia when exposed to Mo-MuLV, yet is low pH dependent for Mo-MuLV entry. Treatment of NIH 3T3/DTras cells with chloroquine, an agent which raises endosomal pH, blocks Mo-MuLV entry, but not Mo-MuLV-induced syncytium formation. This demonstrates that fusion which accompanies viral entry and fusion which is responsible for syncytium formation occur as independent processes in these cells. In addition, we determined that neither inherent differences in the Mo-MuLV receptor nor reduced affinity for Mo-MuLV gp70 can account for resistance of NIH 3T3 cells to Mo-MuLV-induced syncytium formation. PMID:1433518

Wilson, C A; Marsh, J W; Eiden, M V



Characterization of Wnt1 and Wnt2 induced growth alterations and signaling pathways in NIH3T3 fibroblasts  

Microsoft Academic Search

Members of the Wnt family induce mouse mammary tumors and partially transform mammary epithelial cells in culture. However, their mechanism of transformation remains to be elucidated. In NIH3T3 mouse embryo fibroblasts, a standard transformation model, Wnt-1 and Wnt-2 were shown to induce altered properties including increased saturation density and growth in soft agar. Such cells also exhibited increased cell-cell adhesiveness.

Anna Bafico; Arnona Gazit; Sidney S Wu-Morgan; Abraham Yaniv; Stuart A Aaronson



Expression of an Exogenous Eukaryotic DNA Methyltransferase Gene Induces Transformation of NIH 3T3 Cells  

Microsoft Academic Search

Abnormal regional increases in DNA methylation, which have potential for causing gene inactivation and chromosomal instability, are consistently found in immortalized and tumorigenic cells. Increased DNA methyltransferase activity, which is also a characteristic of such cells, is a candidate to mediate these abnormal DNA methylation patterns. We now show that, in NIH 3T3 mouse fibroblasts, constitutive overexpression of an exogenous

Jianjun Wu; Jean-Pierre Issa; James Herman; Douglas E. Bassett Jr.; Barry D. Nelkin; Stephen B. Baylin



Bis(alpha-furancarboxylato)oxovanadium(IV) prevents and improves dexamethasone-induced insulin resistance in 3T3-L1 adipocytes.  


Previous studies showed that bis(alpha-furancarboxylato)oxovanadium(IV) (BFOV), an orally active anti-diabetic organic vanadium complex, could improve insulin resistance in animals with type 2 diabetes. The present study has been carried out to evaluate the effects of BFOV on insulin-resistant glucose metabolism using dexamethasone-treated 3T3-L1 adipocytes as an in-vitro model of insulin resistance. The results showed that BFOV, similar to vanadyl sulfate and rosiglitazone, caused a concentration-dependent increase in glucose consumption by insulin-resistant adipocytes. Moreover, BFOV enhanced the action of insulin and completely prevented the development of insulin resistance induced by dexamethasone, leading to glucose consumption equal to that by normal cells. In addition, dexamethasone reduced the mRNA expression of insulin receptor substrate 1 (IRS-1) and glucose transporter 4 (GLUT4) in 3T3-L1 adipocytes, while BFOV normalized the expression of IRS-1 and GLUT4. These findings suggest that BFOV prevents and improves dexamethasone-induced insulin resistance in 3T3-L1 adipocytes by enhancing expression of IRS-1 and GLUT4 mRNA. PMID:18812026

Zuo, Yi-Qing; Liu, Wei-Ping; Niu, Yan-Fen; Tian, Chang-Fu; Xie, Ming-Jin; Chen, Xi-Zhu; Li, Ling



Lysophosphatidic acid induces chemotaxis in MC3T3-E1 osteoblastic cells  

Microsoft Academic Search

Lysophosphatidic acid (LPA) is a bioactive lipid that has pleiotropic effects on a variety of cell types and enhances the migration of endothelial and cancer cells, but it is not known if this lipid can alter osteoblast motility. We performed transwell migration assays using MC3T3-E1 osteoblastic cells and found LPA to be a potent chemotactic agent. Quantitative time-lapse video analysis

Lisa M. Masiello; Joseph S. Fotos; Deni S. Galileo; Norman J. Karin



Conjugated linoleic acid suppresses triglyceride accumulation and induces apoptosis in 3T3-L1 preadipocytes  

Microsoft Academic Search

Four sets of experiments were conducted to examine the influence of conjugated linoleic acid (CLA) isomers during proliferation\\u000a and differentiation of cultures of 3T3-L1 preadipocytes using physiological culturing conditions. Cultures treated with either\\u000a albumin [bovine serum albumin (BSA) vehicle] or linoleic acid (LA) served as controls. For the proliferation study (Expt.\\u000a 1), cells were cultured in media containing a crude

M. Evans; C. Geigerman; J. Cook; L. Curtis; B. Kuebler; M. McIntosh



Microconstituent-Induced Pitting Corrosion in Aluminum Alloy 2024-T3  

Microsoft Academic Search

Free corrosion immersion experiments were conducted on a commercial airframe material, Al 2024-T3 (UNS A92024), in 0.5 M sodium chloride (NaCl) solution to investigate the role of microconstituents in pitting corrosion. The alloy was found to contain numerous constituent particles (> 300,000 per cm [> 2 million per in.]), and pitting corrosion essentially was attributable to these particles. Two types

G. S. Chen; M. S. Gao; R. P. Wei



Grape skin extract reduces adipogenesis- and lipogenesis-related gene expression in 3T3-L1 adipocytes through the peroxisome proliferator-activated receptor-? signaling pathway.  


We previously reported that grape skin ethanol extract (GSE) decreases adipogenic transcription factor gene expression, inhibiting triglyceride accumulation in 3T3-L1 adipocytes. In this study, we hypothesized that GSE may induce differential expression profiles in adipocytes, thus providing protection against obesity. Thirty-five genes involved in the peroxisome proliferator-activated receptor-? (PPAR?) signaling pathway, lipid metabolism, or adipogenesis were identified through microarray analysis of adipocytes treated with GSE. Expression of the genes involved in PPAR? signaling, Adipoq, Scd1, Nr1h3, Fabp5, Scd2, and Pparg decreased with GSE treatment, whereas expression of Ppargc1a increased. Lipid metabolism-associated genes Mlxp1, Stat5a, Hsl, Plin1, and Vdr were down-regulated. Interestingly, GSE also affected expression of genes related to the mitogen-activated protein kinases pathway. GSE extract treatment decreased expression of aP2, Fas, and Tnfa, known markers of adipogenesis, as measured by real-time polymerase reaction. These findings demonstrate the antiadipogenic effects of GSE on 3T3-L1 adipocytes at the genetic level, primarily on the PPAR? signaling pathway. PMID:22901559

Jeong, Yoo Seok; Hong, Joo Heon; Cho, Kyung Hyun; Jung, Hee Kyoung



Glucocorticoid Receptor and Sequential P53 Activation by Dexamethasone Mediates Apoptosis and Cell Cycle Arrest of Osteoblastic MC3T3-E1 Cells  

PubMed Central

Glucocorticoids play a pivotal role in the proliferation of osteoblasts, but the underlying mechanism has not been successfully elucidated. In this report, we have investigated the molecular mechanism which elucidates the inhibitory effects of dexamethasone on murine osteoblastic MC3T3-E1 cells. It was found that the inhibitory effects were largely attributed to apoptosis and G1 phase arrest. Both the cell cycle arrest and apoptosis were dependent on glucocorticoid receptor (GR), as they were abolished by GR blocker RU486 pre-treatment and GR interference. G1 phase arrest and apoptosis were accompanied with a p53-dependent up-regulation of p21 and pro-apoptotic genes NOXA and PUMA. We also proved that dexamethasone can’t induce apoptosis and cell cycle arrest when p53 was inhibited by p53 RNA interference. These data demonstrate that proliferation of MC3T3-E1 cell was significantly and directly inhibited by dexamethasone treatment via aberrant GR activation and subsequently P53 activation.

Li, Hui; Qian, Wenwei; Weng, Xisheng; Wu, Zhihong; Li, Huihua; Zhuang, Qianyu; Feng, Bin; Bian, Yanyan



Recombinant tissue metalloproteinase inhibitor-3 protein induces apoptosis of murine osteoblast MC3T3-E1.  


Tissue inhibitor of metalloproteinases (TIMPs) plays an essential role in the regulation of bone metabolism. Here we report that recombinant tissue metalloproteinase inhibitor-3 (TIMP-3) protein induces the apoptosis of MC3T3-E1 osteoblasts. Cell apoptosis was detected by sandwich-enzyme-immunoassay. Fas and Fasl protein levels were determined by Western blot analysis. The enzyme substrate was used to assess the activation of caspase-3 and caspase-8. The phosphorylation of JNK, p38 and ERK1/2 was examined by Western blot analysis. The ELISA suggested that TIMP-3 promoted MC3T3-E1 cell apoptosis. TIMP-3 treatment induced the expression of Fas and Fasl proteins, and the activation of caspase-8 and caspase-3. TIMP-3 treatment induced p38 and ERK phosphorylation. SB203580 and PD98059, the inhibitor of p38 and ERK, respectively, abolished the TIMP-3 effect on osteoblast apoptosis. In conclusion, the signal pathway through which TIMP-3 induces MC3T3-E1 cell apoptosis, mediated by Fas and involves the p38 and ERK signal transduction pathways. PMID:18157585

Yuan, L-Q; Liu, Y-S; Luo, X-H; Guo, L-J; Xie, H; Lu, Y; Wu, X-P; Liao, E-Y



Calcium-sensing receptor-mediated activation of phospholipase C-?1 is downstream of phospholipase C-? and protein kinase C in MC3T3-E1 osteoblasts  

Microsoft Academic Search

Elevated extracellular calcium (Cae) stimulates both chemotaxis and mitogenesis of MC3T3-E1 osteoblasts via a calcium-sensing receptor (CasR). Cae-mediated chemotaxis of these bone-forming cells is dependent on phospholipase C (PLC) and blocked by the Gi-protein inhibitor pertussis toxin. In this study, we examine the signaling mechanisms by which the CasR stimulates PLC activity in MC3T3-E1 osteoblasts. We found that elevated Cae

S. L Godwin; S. P Soltoff



Deoxyactein Isolated from Cimicifuga racemosa protects osteoblastic MC3T3-E1 cells against antimycin A-induced cytotoxicity.  


Deoxyactein is one of the major constituents isolated from Cimicifuga racemosa. In the present study, we investigated the protective effects of deoxyactein on antimycin A (mitochondrial electron transport inhibitor)-induced toxicity in osteoblastic MC3T3-E1 cells. Exposure of MC3T3-E1 cells to antimycin A caused significant cell viability loss, as well as mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, intracellular calcium ([Ca(2+) ]i ) elevation and oxidative stress. Pretreatment with deoxyactein prior to antimycin A exposure significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, [Ca(2+) ]i elevation and oxidative stress. Moreover, deoxyactein increased the activation of PI3K (phosphoinositide 3-kinase), Akt (protein kinase B) and CREB (cAMP-response element-binding protein) inhibited by antimycin A. All these data indicate that deoxyactein may reduce or prevent osteoblasts degeneration in osteoporosis or other degenerative disorders. PMID:22180388

Choi, Eun Mi



Adhesion to fibronectin's EDbdomain induces tyrosine phosphorylation of focal adhesion proteins in Balb\\/c 3T3 cells  

Microsoft Academic Search

Balb\\/c 3T3 cell adhesion on substrata coated with fibronectin's (FN) alternatively-spliced EDb, implicated in some tumor cell systems, and its neighboring type III repeats (III7 and III8) induced intracellular signaling coincident with morphological responses. These events were analysed using minigene-expressed proteins containing various permutations of type III repeats of FN. Cells adherent to the tri-repeat protein 7-EDb-8 were compared to

Wensheng Chen; Lloyd A. Culp



RA induces the neural-like cells generated from epigenetic modified NIH/3T3 cells.  


Recently, differentiated somatic cells had been reprogrammed to pluripotential state in vitro, and various tissue cells had been elicited from those cells. Epigenetic modifications allow differentiated cells to perpetuate the molecular memory needed for the cells to retain their identity. DNA methylation and histone deacetylation are important patterns involved in epigenetic modification, which take critical roles in regulating DNA expression. In this study, we dedifferentiated NIH/3T3 fibroblasts by 5-aza-2-deoxycytidine (5-aza-dC) and Trichstatin A (TSA) combination, and detected gene expression pattern, DNA methylation level, and differentiation potential of reprogrammed cells. As the results, embryonic marker Sox2, klf4, c-Myc and Oct4 were expressed in reprogrammed NIH/3T3 fibroblasts. Total DNA methylation level was significant decreased after the treatment. Moreover, exposure of the reprogrammed cells to all trans-retinoic acid (RA) medium elicited the generation of neuronal class IIIbeta-tubulin-positive, neuron-specific enolase (NSE)-positive, nestin-positive, and neurofilament light chain (NF-L)-positive neural-like cells. PMID:19263240

Zhang, Xi-Mei; Li, Qiu-Ming; Su, Dong-Ju; Wang, Ning; Shan, Zhi-Yan; Jin, Lian-Hong; Lei, Lei



Dexamethasone-induced insulin resistance in 3T3-L1 adipocytes is due to inhibition of glucose transport rather than insulin signal transduction.  


Glucocorticoids reportedly induce insulin resistance. In this study, we investigated the mechanism of glucocorticoid-induced insulin resistance using 3T3-L1 adipocytes in which treatment with dexamethasone has been shown to impair the insulin-induced increase in glucose uptake. In 3T3-L1 adipocytes treated with dexamethasone, the GLUT1 protein expression level was decreased by 30%, which possibly caused decreased basal glucose uptake. On the other hand, dexamethasone treatment did not alter the amount of GLUT4 protein in total cell lysates but decreased the insulin-stimulated GLUT4 translocation to the plasma membrane, which possibly caused decreased insulin-stimulated glucose uptake. Dexamethasone did not alter tyrosine phosphorylation of insulin receptors, and it significantly decreased protein expression and tyrosine phosphorylation of insulin receptor substrate (IRS)-1. Interestingly, however, protein expression and tyrosine phosphorylation of IRS-2 were increased. To investigate whether the reduced IRS-1 content is involved in insulin resistance, IRS-1 was overexpressed in dexamethasone-treated 3T3-L1 adipocytes using an adenovirus transfection system. Despite protein expression and phosphorylation levels of IRS-1 being normalized, insulin-induced 2-deoxy-D-[3H]glucose uptake impaired by dexamethasone showed no significant improvement. Subsequently, we examined the effect of dexamethasone on the glucose uptake increase induced by overexpression of GLUT2-tagged p110alpha, constitutively active Akt (myristoylated Akt), oxidative stress (30 mU glucose oxidase for 2 h), 2 mmol/l 5-aminoimidazole-4-carboxamide ribonucleoside for 30 min, and osmotic shock (600 mmol/l sorbitol for 30 min). Dexamethasone treatment clearly inhibited the increases in glucose uptake produced by these agents. Thus, in conclusion, the GLUT1 decrease may be involved in the dexamethasone-induced decrease in basal glucose transport activity, and the mechanism of dexamethasone-induced insulin resistance in glucose transport activity (rather than the inhibition of phosphatidylinositol 3-kinase activation resulting from a decreased IRS-1 content) is likely to underlie impaired glucose transporter regulation. PMID:11016454

Sakoda, H; Ogihara, T; Anai, M; Funaki, M; Inukai, K; Katagiri, H; Fukushima, Y; Onishi, Y; Ono, H; Fujishiro, M; Kikuchi, M; Oka, Y; Asano, T



A shift in the ligand responsiveness of thyroid hormone receptor alpha induced by heterodimerization with retinoid X receptor alpha.  

PubMed Central

Thyroid hormone (T3) receptors (T3Rs) are ligand-modulated transcription factors that bind to thyroid hormone response elements (T3REs) and mediate either positive or negative transcriptional regulation of target genes. In addition, in response to ligand binding, T3Rs can interfere with AP-1 activity and thereby inhibit transcription of AP-1-responsive genes. T3Rs were recently shown to form heterodimers with retinoid X receptors (RXRs), leading to increased binding to T3REs in vitro and potentiation of transcriptional responses in vivo. Here we demonstrate that T3R alpha forms stable heterodimers with RXR alpha in living cells. Most important, we describe a new role for RXR alpha in modulating ligand-dependent T3R alpha activity: heterodimerization with RXR alpha greatly increases transcriptional interference with AP-1 activity, augments T3-dependent transcriptional activation, and potentiates the reversal of ligand-independent activation by T3R alpha. In all three cases, the responses occur at substantially lower T3 concentrations when elicited by T3R alpha plus RXR alpha than by T3R alpha alone. In vitro, the binding of T3 decreases the DNA-binding activity of T3R alpha homodimers but does not affect DNA binding by T3R alpha:RXR alpha heterodimers. We provide evidence that increased activities of T3R alpha at lower T3 concentrations are not due to changes in its T3 binding properties. Instead, the altered response could be mediated by either RXR alpha-induced conformational changes, increased stability of heterodimers over homodimers, especially following T3 binding, or both.

Claret, F X; Antakly, T; Karin, M; Saatcioglu, F



?-Mangostin Induces Apoptosis and Suppresses Differentiation of 3T3-L1 Cells via Inhibiting Fatty Acid Synthase  

PubMed Central

?-Mangostin, isolated from the hulls of Garcinia mangostana L., was found to have in vitro cytotoxicity against 3T3-L1 cells as well as inhibiting fatty acid synthase (FAS, EC Our studies showed that the cytotoxicity of ?-mangostin with IC50 value of 20 µM was incomplicated in apoptotic events including increase of cell membrane permeability, nuclear chromatin condensation and mitochondrial membrane potential (??m) loss. This cytotoxicity was accompanied by the reduction of FAS activity in cells and could be rescued by 50 µM or 100 µM exogenous palmitic acids, which suggested that the apoptosis of 3T3-L1 preadipocytes induced by ?-mangostin was via inhibition of FAS. Futhermore, ?-mangostin could suppress intracellular lipid accumulation in the differentiating adipocytes and stimulated lipolysis in mature adipocytes, which was also related to its inhibition of FAS. In addition, 3T3-L1 preadipocytes were more susceptible to the cytotoxic effect of ?-mangostin than mature adipocytes. Further studies showed that ?-mangostin inhibited FAS probably by stronger action on the ketoacyl synthase domain and weaker action on the acetyl/malonyl transferase domain. These findings suggested that ?-mangostin might be useful for preventing or treating obesity.

Quan, Xiaofang; Wang, Yi; Ma, Xiaofeng; Liang, Yan; Tian, Weixi; Ma, Qingyun; Jiang, Hezhong; Zhao, Youxing



Contrasting Signaling Pathways of  1A- and  1B-Adrenergic Receptor Subtype Activation of Phosphatidylinositol 3Kinase and Ras in Transfected NIH3T3 Cells  

Microsoft Academic Search

Activation of protein kinases is an important inter- mediate step in signaling pathways of many G pro- tein-coupled receptors including a1-adrenergic re- ceptors. The present study was designed to investigate the capacity of the three cloned sub- types of human a1-receptors, namely, a1A, a1B and a1D, to activate phosphatidylinositol 3-kinase (PI 3-kinase) and p21ras in transfected NIH3T3 cells. Norepinephrine activated

Zhuo-Wei Hu; Xiao-You Shi; Richard Z. Lin; Brian B. Hoffman



Epidermal Growth Factor Receptor (EGFR) Test Utilization in the United States: A Case Study of T3 Translational Research  

PubMed Central

Purpose We examined hospital use of the epidermal growth factor receptor (EGFR) assay for lung cancer patients. Our goal was to inform the development of a model to predict T3 translation of guideline-directed, molecular diagnostic tests. Methods This was a retrospective observational study. Using logistic regression, we analyzed the association between likelihood to order the EGFR assay and hospital’s institutional and regional characteristics. Results Significant institutional predictors included: Affiliation with an academic medical center (odds ratio [OR], 1.48; 95% CI, 1.20–1.83), Participation in an NCI clinical research cooperative group (OR, 2.06, 1.66–2.55), PET scan (OR, 1.44, 1.07–1.94) and cardio thoracic surgery (OR, 1.90, 1.52–2.37) services. Significant regional predictors included: Metropolitan county (OR, 2.08, 1.48 to 2.91), Above average education (OR, 1.46, 1.09 to 1.96), Above average income (OR, 1.46, 1.04–2.05). Distance from an NCI cancer center was a negative predictor (OR, 0.996, 0.995–0.998), a 34% decrease in likelihood for every 100 miles. Conclusion In 2010, 12% of US acute care hospitals ordered the EGFR assay, suggesting most lung cancer patients did not have access to this test. This case study illustrated the need for: 1) Increased dissemination and implementation research. 2) Interventions to improve adoption of guideline-directed, molecular diagnostic tests by community hospitals.

Lynch, Julie A.; Khoury, Muin J.; Borzecki, Ann; Cromwell, Jerry; Hayman, Laura L.; Ponte, Pat Reid; Miller, Glenn A.; Lathan, Christopher S.



A Nanodot Array Modulates Cell Adhesion and Induces an Apoptosis-Like Abnormality in NIH-3T3 Cells  

PubMed Central

Micro-structures that mimic the extracellular substratum promote cell growth and differentiation, while the cellular reaction to a nanostructure is poorly defined. To evaluate the cellular response to a nanoscaled surface, NIH 3T3 cells were grown on nanodot arrays with dot diameters ranging from 10 to 200 nm. The nanodot arrays were fabricated by AAO processing on TaN-coated wafers. A thin layer of platinum, 5 nm in thickness, was sputtered onto the structure to improve biocompatibility. The cells grew normally on the 10-nm array and on flat surfaces. However, 50-nm, 100-nm, and 200-nm nanodot arrays induced apoptosis-like events. Abnormality was triggered after as few as 24 h of incubation on a 200-nm dot array. For cells grown on the 50-nm array, the abnormality started after 72 h of incubation. The number of filopodia extended from the cell bodies was lower for the abnormal cells. Immunostaining using antibodies against vinculin and actin filament was performed. Both the number of focal adhesions and the amount of cytoskeleton were decreased in cells grown on the 100-nm and 200-nm arrays. Pre-coatings of fibronectin (FN) or type I collagen promoted cellular anchorage and prevented the nanotopography-induced programed cell death. In summary, nanotopography, in the form of nanodot arrays, induced an apoptosis-like abnormality for cultured NIH 3T3 cells. The occurrence of the abnormality was mediated by the formation of focal adhesions.



GH induced lipolysis stimulation in 3T3-L1 adipocytes stably expressing hGHR: analysis on signaling pathway and activity of 20K hGH  

Microsoft Academic Search

We have constructed a cell line of 3T3-L1 which can efficiently express human GHR (3T3-L1-hGHR) after differentiation to adipocytes. The expressed hGHR was detected as two bands with approximate molecular sizes of 120K by Western analysis using hGHR specific monoclonal antibody. Maximum lipolytic activity induced by hGH in the 3T3-L1-hGHR was enhanced 10-fold as compared to that in 3T3-L1, suggesting

N Asada; Y Takahashi; M Wada; N Naito; H Uchida; M Ikeda; M Honjo



CREB activation induced by mitochondrial dysfunction triggers triglyceride accumulation in 3T3-L1 preadipocytes  

Microsoft Academic Search

Several mitochondrial pathologies are characterized by lipid redistribution and microvesicular cell phenotypes resulting from triglyceride accumulation in lipid- metabolizing tissues. However, the molecular mechanisms underlying abnormal fat distribution induced by mitochondrial dysfunction remain poorly understood. In this study, we show that inhibition of respiratory complex III by antimycin A as well as inhibition of mitochondrial protein synthesis trigger the accumulation

Sébastien Vankoningsloo; Aurélia De Pauw; Andrée Houbion; Silvia Tejerina; Catherine Demazy; Françoise de Longueville; Vincent Bertholet; Patricia Renard; José Remacle; Paul Holvoet; Martine Raes; Thierry Arnould



?-Tocotrienol induced cell cycle arrest and apoptosis via activating the Bax-mediated mitochondrial and AMPK signaling pathways in 3T3-L1 adipocytes.  


This study aimed to examine the anti-proliferative effects of ?-, ?- and ?-tocotrienols (?T3, ?T3 and ?T3), and ?-tocopherol on 3T3-L1 adipocytes. Results showed that compared with other vitamin E analogues, ?T3 demonstrated the most potent anti-proliferative effect on 3T3-L1 cells. It significantly caused a reduction in mitochondrial membrane potential (??m) and an increase in ROS formation, as well as inducing cell apoptosis and cell cycle arrest at S phase. Further studies showed that it down-regulated Bcl-2 and PPAR-? expression, suppressed Akt and ERK activation and phosphorylation, and caused cytochrome c release from mitochondria to cytosol, whereas it up-regulated CD95 (APO-1/CD95) and Bax expression, and caused caspase-3 and JNK activation, PARP cleavage and AMPK phosphorylation. Pretreatments with caspase-3 (z-DEVD-fmk) and AMPK (CC) inhibitors significantly suppressed the ?T3-induced ROS production and cell death. Caspase-3 inhibitor also efficiently blocked CD95 (APO-1/CD95) and Bax expression, caspase-3 activation and PARP cleavage, whereas antioxidant N-acetyl-l-cysteine, AMPK inhibitor and AMPK siRNA effectively blocked the AMPK phosphorylation. Taken together, these results conclude that the potent anti-proliferative and anti-adipogenic effects of ?T3 on 3T3-L1 adipocytes could be through the Bax-mediated mitochondrial and AMPK signaling pathways. PMID:23816832

Wu, Shu-Jing; Huang, Guang-Yu; Ng, Lean-Teik



The gene expression profile induced by Wnt 3a in NIH 3T3 fibroblasts  

PubMed Central

Wnt proteins play important roles in regulating cell differentiation, proliferation and polarity. Wnts have been proposed to play roles in tissue repair and fibrosis, yet the gene expression profile of fibroblasts exposed to Wnts has not been examined. We use Affymetrix genome-wide expression profiling to show that a 6-h treatment of fibroblasts of Wnt3a results in the induction of mRNAs encoding known Wnt targets such as the fibrogenic pro-adhesive molecule connective tissue growth factor (CTGF, CCN2). Wnt3a also induces mRNAs encoding potent pro-fibrotic proteins such as TGF? and endothelin-1 (ET-1). Moreover, Wnt3a promotes genes associated with cell adhesion and migration, vasculature development, cell proliferation and Wnt signaling. Conversely, Wnt3a suppresses gene associated with skeletal development, matrix degradation and cell death. Results were confirmed using real-time polymerase chain reaction of cells exposed to Wnt3a and Wnt10b. These results suggest that Wnts induce genes promoting fibroblast differentiation towards angiogenesis and matrix remodeling, at the expense of skeletal development.

Chen, Shaoqiong; McLean, Sarah; Carter, David E.



Macrophage-conditioned medium inhibits differentiation-induced Rb phosphorylation in 3T3-L1 preadipocytes  

SciTech Connect

This study examines the mechanisms underlying the anti-adipogenic effect of macrophage-secreted products. 3T3-L1 preadipocytes were induced to differentiate over 8 days in medium conditioned by murine J774 macrophages (MacCM). The inhibitory effect on lipid accumulation and expression of adipogenic markers was diminished when addition of MacCM was delayed to day 2 of differentiation. Clonal expansion, an early event required for 3T3-L1 adipogenesis, was reduced in the presence of MacCM (89%; n = 3; p < 0.001), and BrdU incorporation was impaired by 55% (n = 3; p < 0.01). Activation of ERK1/2 was not affected by MacCM, and neither was the expression of p27{sup kip1}, a cyclin-dependent kinase inhibitor. However, phosphorylation of the retinoblastoma protein (Rb), required for cell cycle progression, was impaired by MacCM (94% inhibition; n = 3; p < 0.01). Differentiation-dependent expression, nuclear localization, and DNA binding ability of C/EBP{beta} were not inhibited by MacCM. Alterations in cell cycle-associated proteins may be important with respect to the anti-adipogenic action of MacCM.

Yarmo, Michelle N.; Landry, Anne; Molgat, Andre S.D.; Gagnon, AnneMarie [Department of Medicine, University of Ottawa, Chronic Disease Program, Ottawa Health Research Institute, Ottawa, Ontario (Canada); Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Chronic Disease Program, Ottawa Health Research Institute, Ottawa, Ontario (Canada); Sorisky, Alexander [Department of Medicine, University of Ottawa, Chronic Disease Program, Ottawa Health Research Institute, Ottawa, Ontario (Canada); Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Chronic Disease Program, Ottawa Health Research Institute, Ottawa, Ontario (Canada)], E-mail:



JNK and tumor necrosis factor-alpha mediate free fatty acid-induced insulin resistance in 3T3-L1 adipocytes.  


Lipid infusion and high fat feeding are established causes of systemic and adipose tissue insulin resistance. In this study, we treated 3T3-L1 adipocytes with a mixture of free fatty acids (FFAs) to investigate the molecular mechanisms underlying fat-induced insulin resistance. FFA treatment impaired insulin receptor-mediated signal transduction and decreased insulin-stimulated GLUT4 translocation and glucose transport. FFAs activated the stress/inflammatory kinases c-Jun N-terminal kinase (JNK) and IKKbeta, and the suppressor of cytokine signaling protein 3, increased secretion of the inflammatory cytokine tumor necrosis factor (TNF)-alpha, and decreased secretion of adiponectin into the medium. RNA interference-mediated down-regulation of JNK blocked JNK activation and prevented most of the FFA-induced defects in insulin action. Blockade of TNF-alpha signaling with neutralizing antibodies to TNF-alpha or its receptors or with a dominant negative TNF-alpha peptide had a partial effect to inhibit FFA-induced cellular insulin resistance. We found that JNK activation by FFAs was not inhibited by blocking TNF-alpha signaling, whereas the FFA-induced increase in TNF-alpha secretion was inhibited by RNA interference-mediated JNK knockdown. Together, these results indicate that 1) JNK can be activated by FFAs through TNF-alpha-independent mechanisms, 2) activated JNK is a major contributor to FFA-induced cellular insulin resistance, and 3) TNF-alpha is an autocrine/paracrine downstream effector of activated JNK that can also mediate insulin resistance. PMID:16085647

Nguyen, M T Audrey; Satoh, Hiroaki; Favelyukis, Svetlana; Babendure, Jennie L; Imamura, Takeshi; Sbodio, Juan I; Zalevsky, Jonathan; Dahiyat, Bassil I; Chi, Nai-Wen; Olefsky, Jerrold M



DMSO is a strong inducer of DNA hydroxymethylation in pre-osteoblastic MC3T3-E1 cells.  


Artificial induction of active DNA demethylation appears to be a possible and useful strategy in molecular biology research and therapy development. Dimethyl sulfoxide (DMSO) was shown to cause phenotypic changes in embryonic stem cells altering the genome-wide DNA methylation profiles. Here we report that DMSO increases global and gene-specific DNA hydroxymethylation levels in pre-osteoblastic MC3T3-E1 cells. After 1 day, DMSO increased the expression of genes involved in DNA hydroxymethylation (TET) and nucleotide excision repair (GADD45) and decreased the expression of genes related to DNA methylation (Dnmt1, Dnmt3b, Hells). Already 12 hours after seeding, before first replication, DMSO increased the expression of the pro-apoptotic gene Fas and of the early osteoblastic factor Dlx5, which proved to be Tet1 dependent. At this time an increase of 5-methyl-cytosine hydroxylation (5-hmC) with a concomitant loss of methyl-cytosines on Fas and Dlx5 promoters as well as an increase in global 5-hmC and loss in global DNA methylation was observed. Time course-staining of nuclei suggested euchromatic localization of DMSO induced 5-hmC. As consequence of induced Fas expression, caspase 3/7 and 8 activities were increased indicating apoptosis. After 5 days, the effect of DMSO on promoter- and global methylation as well as on gene expression of Fas and Dlx5 and on caspases activities was reduced or reversed indicating down-regulation of apoptosis. At this time, up regulation of genes important for matrix synthesis suggests that DMSO via hydroxymethylation of the Fas promoter initially stimulates apoptosis in a subpopulation of the heterogeneous MC3T3-E1 cell line, leaving a cell population of extra-cellular matrix producing osteoblasts.  PMID:22507896

Thaler, Roman; Spitzer, Silvia; Karlic, Heidrun; Klaushofer, Klaus; Varga, Franz



PPAR? agonist fenofibrate attenuates TNF-?-induced CD40 expression in 3T3-L1 adipocytes via the SIRT1-dependent signaling pathway.  


The ligand-activated transcription factor peroxisome proliferator-activated receptor-? (PPAR?) participates in the regulation of cellular inflammation. More recent studies indicated that sirtuin1 (SIRT1), a NAD(+)-dependent deacetylase, regulates the inflammatory response in adipocytes. However, whether the role of PPAR? in inflammation is mediated by SIRT1 remains unclear. In this study, we aimed to determine the effect of PPAR? agonist fenofibrate on the expressions of SIRT1 and pro-inflammatory cytokine CD40 and underlying mechanisms in 3T3-L1 adipocytes. We found that fenofibrate inhibited CD40 expression and up-regulated SIRT1 expression in tumor necrosis factor-? (TNF-?)-stimulated adipocytes, and these effects of fenofibrate were reversed by PPAR? antagonist GW6471. Moreover, SIRT1 inhibitors sirtinol/nicotinamide (NAM) or knockdown of SIRT1 could attenuate the effect of fenofibrate on TNF-?-induced CD40 expression in adipocytes. Importantly, NF-?B inhibitor pyrrolidine dithiocarbamate (PDTC) augmented the effect of fenofibrate on CD40 expression in adipocytes. Further study found that fenofibrate decreased the expression of acetylated-NF-?B p65 (Ac-NF-?B p65) in TNF-?-stimulated adipocytes, and the effect of fenofibrate was abolished by SIRT1 inhibition. In addition, fenofibrate up-regulated SIRT1 expression through AMPK in TNF-?-stimulated adipocytes. Taken together, these findings indicate that PPAR? agonist fenofibrate inhibits TNF-?-induced CD40 expression in 3T3-L1 adipocytes via the SIRT1-dependent signaling pathway. PMID:23603572

Wang, Weirong; Lin, Qinqin; Lin, Rong; Zhang, Jiye; Ren, Feng; Zhang, Jianfeng; Ji, Meixi; Li, Yanxiang



Gene expression changes in BALB/3T3 transformants induced by poly(L-lactic acid) or polyurethane films.  


We performed DNA microarray analysis on two BALB/3T3 transformants (A5 and A6) induced by polyurethane (PU) film, two (L11 and L21) induced by biodegradable poly(L-lactic acid) (PLLA) film, and the parental cells. The transforming ability of the cells was in the order A5 < A6 < L21 < L11. In all, 1176 cancer-related genes were up- or down-regulated in at least one transformant. Those that were markedly up-regulated were c-fos protooncogene, FBJ osteosarcoma oncogene B, and Jun oncogene; those markedly down-regulated were pleiotrophin, histidine triad nucleotide-binding protein, protein kinase C iota, and large multifunctional protease 7. A common function of proteins encoded by genes that underwent marked expression changes was bone formation. The genes were c-fos, FBJ osteosarcoma, Jun, pleiotrophin, a disintegrin-like and metalloprotease with TS-1 motif protein 1. This finding was consistent with the tumor formation in the 2-year PLLA or PU subcutaneous implantation into rats. The number of genes that underwent marked expression change in each transformant was consistent with its malignancy. PLLA induced more malignant transformants than PU, especially in relation to osteosarcoma-like gene expression. PMID:14704980

Matsuoka, Atsuko; Tsuchiya, Toshie



Angiotensin II Requires Zinc and Downregulation of the Zinc Transporters ZnT3 and ZnT10 to Induce Senescence of Vascular Smooth Muscle Cells  

PubMed Central

Senescence, a hallmark of mammalian aging, is associated with the onset and progression of cardiovascular disease. Angiotensin II (Ang II) signaling and zinc homeostasis dysfunction are increased with age and are linked to cardiovascular disease, but the relationship among these processes has not been investigated. We used a model of cellular senescence induced by Ang II in vascular smooth muscle cells (VSMCs) to explore the role of zinc in vascular dysfunction. We found that Ang II-induced senescence is a zinc-dependent pathway mediated by the downregulation of the zinc transporters ZnT3 and ZnT10, which work to reduce cytosolic zinc. Zinc mimics Ang II by increasing reactive oxygen species (ROS), activating NADPH oxidase activity and Akt, and by downregulating ZnT3 and ZnT10 and inducing senescence. Zinc increases Ang II-induced senescence, while the zinc chelator TPEN, as well as overexpression of ZnT3 or ZnT10, decreases ROS and prevents senescence. Using HEK293 cells, we found that ZnT10 localizes in recycling endosomes and transports zinc into vesicles to prevent zinc toxicity. Zinc and ZnT3/ZnT10 downregulation induces senescence by decreasing the expression of catalase. Consistently, ZnT3 and ZnT10 downregulation by siRNA increases ROS while downregulation of catalase by siRNA induces senescence. Zinc, siZnT3 and siZnT10 downregulate catalase by a post-transcriptional mechanism mediated by decreased phosphorylation of ERK1/2. These data demonstrate that zinc homeostasis dysfunction by decreased expression of ZnT3 or ZnT10 promotes senescence and that Ang II-induced senescence is a zinc and ROS-dependent process. Our studies suggest that zinc might also affect other ROS-dependent processes induced by Ang II, such as hypertrophy and migration of smooth muscle cells.

Patrushev, Nikolay; Seidel-Rogol, Bonnie; Salazar, Gloria



Structure of the human receptor tyrosine phosphatase gamma gene (PTPRG) and relation to the familial RCC t(3;8) chromosome translocation  

SciTech Connect

The receptor protein tyrosine phosphatase {gamma} gene, PTP{gamma} (locus name PTPRG), was previously mapped to chromosome region 3p14.2, within a 2- to 4-Mb region centromeric to the 3p14.2 breakpoint of the t(3;8) familial renal cell carcinoma (RCC)-associated constitutional chromosome translocation. Because of its chromosomal position, its enzymatic properties as a receptor phosphatase, which might oppose a growth activating kinase activity, its homozygous deletion in murine L cells, and its transcriptional activity in numerous normal tissues, including kidney, the PTP{gamma} gene was an attractive tumor suppressor gene candidate for renal cell carcinoma. To determine whether the PTP{gamma} gene was a target of loss of heterozygosity or mutation in RCCs and to determine its map position relative to the t(3;8) break at 3p14.2, we have isolated YAC and {lambda} genomic clones for the PTP{gamma} gene and other 3p14.2 markers and determined the relative positions of the t(3;8) break, a 3p14.2 de novo break possibly in a fragile site, and the 5{prime} end of the PTP{gamma} gene. Additionally, the genomic structure, position of the proximal promotor, and intron-exon border sequences of the 30-exon {approximately} 780-kb PTP{gamma} gene have been determined, which will facilitate analysis of the PTP{gamma} gene in tumors. 49 refs., 3 figs., 3 tabs.

Kastury, K.; Ohta, M.; Druck, T.; Huebner, K. [Jefferson Medical College, Philadelphia, PA (United States)] [and others



Beta-Mecaptoethanol Suppresses Inflammation and Induces Adipogenic Differentiation in 3T3-F442A Murine Preadipocytes  

PubMed Central

Preadipocytes are present in adipose tissues throughout adult life that can proliferate and differentiate into mature adipocytes in response to environmental cues. Abnormal increase in adipocyte number or size leads to fat tissue expansion. However, it is now recognized that adipocyte hypertrophy is a greater risk factor for metabolic syndrome whereas fat tissue that continues to produce newer and smaller fat cells through preadipocyte differentiation is “metabolically healthy”. Because adipocyte hypertrophy is often associated with increased oxidant stress and low grade inflammation, both are linked to disturbed cellular redox, we tested how preadipocyte differentiation may be regulated by beta-mercaptoethanol (BME), a pharmacological redox regulator and radical scavenger, using murine 3T3-F442A preadipocytes as the cell model. Effects of BME on adipogenesis were measured by microphotography, real-time PCR, and Western analysis. Our data demonstrated that preadipocyte differentiation could be regulated by extracellular BME. At an optimal concentration, BME enhanced expression of adipogenic gene markers and lipid accumulation. This effect was associated with BME-mediated down-regulation of inflammatory cytokine expression during early differentiation. BME also attenuated TNFalpha-induced activation of NFkappaB in differentiating preadipocytes and partially restored TNFalpha-mediated suppression on adipogenesis. Using a non-adipogenic HEK293 cell line transfected with luciferase reporter genes, we demonstrated that BME reduced basal and TNFalpha-induced NFkappaB activity and increased basal and ciglitazone-induced PPARgamma activity; both may contribute to the pro-adipogenic effect of BME in differentiating F442A preadipocytes.

Guo, Wen; Li, Yahui; Liang, Wentao; Wong, Siu; Apovian, Caroline; Kirkland, James L.; Corkey, Barbara E.



Glycine suppresses TNF-?-induced activation of NF-?B in differentiated 3T3-L1 adipocytes.  


Glycine strongly reduces the serum levels of pro-inflammatory cytokines and increases the levels of anti-inflammatory cytokines. Recently, glycine has been shown to decrease the expression and secretion of pro-inflammatory adipokines in monosodium glutamate-induced obese (MSG/Ob) mice. It has been postulated that these effects may be explained by a reduction in nuclear factor kappa B (NF-?B) activation. NF-?B is a transcription factor, which is crucial to the inflammatory response. Hasegawa et al. (2011 and 2012) recently reported a glycine-dependent reduction in NF-?B levels. Here, we have investigated the role of glycine in the regulation of NF-?B in differentiated 3T3-L1 adipocytes. The results revealed that pretreatment with glycine interfered with the activation of NF-?B, which has been shown to be stimulated by tumor necrosis factor-alpha (TNF-?). Glycine alone stimulated NF-?B activation in an unusual way such that the inhibitor ?B-? (I?B-?) degradation was more significant than that of the inhibitor ?B-? (I?B-?) and led to NF-?B complexes comprised of p50 and p65 subunits; I?B-? degradation did not affect by glycine. These findings suggest that glycine could be used as an alternative treatment for chronic inflammation, which is a hallmark of obesity and other comorbidities, and is characterized by an elevated production of pro-inflammatory cytokines. PMID:22732655

Blancas-Flores, Gerardo; Alarcón-Aguilar, Francisco J; García-Macedo, Rebeca; Almanza-Pérez, Julio C; Flores-Sáenz, José L; Román-Ramos, Rubén; Ventura-Gallegos, José L; Kumate, Jesús; Zentella-Dehesa, Alejandro; Cruz, Miguel



Low molecular weight molecules of oyster nacre induce mineralization of the MC3T3-E1 cells.  


The nacre layer from the pearl oyster shell is considered as a promising osteoinductive biomaterial. Nacre contains one or more signal molecules capable of stimulating bone formation. The identity and the mode of action of these molecules on the osteoblast differentiation were analyzed. Water-soluble molecules from nacre were fractionated according to dialysis, solvent extraction, and reversed-phase HPLC. The activity of a fraction composed of low molecular weight molecules in the mineralization of the MC3T3-E1 extracellular matrix was investigated. Mineralization of the preosteoblast cells was monitored according to alizarin red staining, Raman spectroscopy, scanning electron microscopy, and quantitative RT-PCR. Molecules isolated from nacre, ranging from 50 to 235 Da, induced a red alizarin staining of the preosteoblasts extracellular matrix after 16 days of culture. Raman spectroscopy demonstrated the presence of hydroxyapatite (HA) in samples treated with these molecules. Scanning electron microscopy pictures showed at the surface of the treated cells the occurrence of clusters of spherical particles resembling to HA. The treatment of cells with nacre molecules accelerated expression of collagen I and increased the mRNA expression of Runx2 and osteopontin. This study indicated that the nacre molecules efficient in bone cell differentiation are certainly different from proteins, and could be useful for in vivo bone repair. PMID:17729263

Rousseau, Marthe; Boulzaguet, Hélčne; Biagianti, Julie; Duplat, Denis; Milet, Christian; Lopez, Evelyne; Bédouet, Laurent



Phenylenediamine derivatives induce GDF-15/MIC-1 and inhibit adipocyte differentiation of mouse 3T3-L1 cells.  


Phenylenediamine derivatives can function as a hydrogen donor and reportedly exert various biological actions including cytoprotective effects against oxidative stress, possibly by acting as an antioxidant. Previous studies showed that feeding of such compounds to mice reduced their body weight, but the precise mechanism remains unknown at present. Here, we found that these compounds inhibited the in vitro differentiation of mouse preadipocytes, 3T3-L1 cells, into adipocytes, suggesting that, at least in part, reduced generation of adipocytes might contribute to the observed weight loss in mice. Next, we performed array analysis and found that the expression of GDF-15/MIC-1, which is a TGF? superfamily cytokine, and Trib 3, an intracellular downstream effector of the cytokines, was up-regulated by these derivatives. Thus, we identified the compounds as inducers of GDF-15/MIC-1 and suggest that such induction may have led to inhibition of adipocyte differentiation, which could account for the weight-loss effect of these compounds. PMID:22155240

Yanagitai, Mika; Kitagawa, Tomomi; Okawa, Kyouji; Koyama, Hiroki; Satoh, Takumi



Environmental Endocrine Disruptors Promote Adipogenesis in the 3T3-L1 Cell Line through Glucocorticoid Receptor Activation  

Microsoft Academic Search

The burgeoning obesity and diabetes epidemics threaten health worldwide, yet the molecular mechanisms underlying these phenomena are incompletely understood. Recently, attention has focused on the potential contributions of environmental pollutants that act as endocrine disrupting chemicals (EDCs) in the pathogenesis of metabolic diseases. Because glucocorticoid signaling is central to adipocyte differentiation, the ability of EDCs to stimulate the glucocorticoid receptor

Robert M. Sargis; Daniel N. Johnson; Rashikh A. Choudhury; Matthew J. Brady



Muscarinic receptors transform NIH 3T3 cells through a Ras-dependent signalling pathway inhibited by the Ras-GTPase-activating protein SH3 domain.  

PubMed Central

Expression of certain subtypes of human muscarinic receptors in NIH 3T3 cells provides an agonist-dependent model of cellular transformation by formation of foci in response to carbachol. Although focus formation correlates with the ability of the muscarinic receptors to activate phospholipase C, the actual mitogenic signal transduction pathway is unknown. Through cotransfection experiments and measurement of the activation state of native and epitope-tagged Ras proteins, the contributions of Ras and Ras GTPase-activating protein (Ras-GAP) to muscarinic receptor-dependent transformation were defined. Transforming muscarinic receptors were able to activate Ras, and such activation was required for transformation because focus formation was inhibited by coexpression of either Ras with a dominant-negative mutation or constructs of Ras-GAP that include the catalytic domain. Coexpression of the N-terminal region of GAP or of its isolated SH3 (Src homology 3) domain, but not its SH2 domain, was also sufficient to suppress muscarinic receptor-dependent focus formation. Point mutations at conserved residues in the Ras-GAP SH3 domain reversed its action, leading to an increase in carbachol-dependent transformation. The inhibitory effect of expression of the Ras-GAP SH3 domain occurs proximal to Ras activation and is selective for the mitogenic pathway activated by carbachol, as cellular transformation by either v-Ras or trkA/nerve growth factor is unaffected. Images

Mattingly, R R; Sorisky, A; Brann, M R; Macara, I G



Characterization of the binding of ( sup 125 I) acidic fibroblast growth factor (aFGF) to cloned aFGF receptors in NIH 3T3 cell membranes  

SciTech Connect

In the present studies, the binding of ({sup 125}I)aFGF was characterized in a membrane preparation from NIH 3T3 cells transfected with the human flg gene. The inclusion of heparin was necessary to reduce nonspecific binding which accounted for approximately 20-30% of total binding. However, heparin in concentrations ranging from 0.1-100 U/ml was not found to significantly alter specific binding. Specific binding was maximal in the presence of 200 mM NaCl. Under these conditions, saturation studies indicated that ({sup 125}I) aFGF bound with high affinity to a single class of recognition sites. aFGF potently inhibited the binding of 0.1 nM ({sup 125}I)aFGF. Several glycine-substituted point mutations of aFGF were also found to inhibit binding with the following order of activity: 137G > 134G = 143G. The present results indicate that the specific binding of ({sup 125}I)aFGF to the aFGF receptor in NIH 3T3 cell membranes provides a reliable assay for the further analysis of the contributions of the aFGF receptor to mammalian physiology.

Jarvis, M.F.; Gessner, G.; Dionne, C.; Jaye, M.; Martin, G. (Rhone-Poulenc Rorer Central Research, King of Prussia, PA (United States))



Repression of platelet-derived growth factor beta-receptor expression by mitogenic growth factors and transforming oncogenes in murine 3T3 fibroblasts.  

PubMed Central

Platelet-derived growth factor BB (PDGF-BB) is an important extracellular factor for regulating the G0-S phase transition of murine BALB/c-3T3 fibroblasts. We have investigated the expression of the PDGF beta receptor (PDGF beta R) in these cells. We show that the state of growth arrest in G0, resulting from serum deprivation, is associated with increased expression of the PDGF beta R. When the growth-arrested fibroblasts are stimulated to reenter the cell cycle by the mitogenic action of serum or certain specific combinations of growth factors, PDGF beta R mRNA levels and cell surface PDGF-BB-binding sites are markedly downregualted. Oncogene-transformed 3T3 cell lines, which fail to undergo growth arrest following prolonged serum deprivation, express constitutively low levels of the PDGF beta R mRNA and possess greatly reduced numbers of cell surface PDGF receptors, as determined by PDGF-BB binding and Western blotting (immunoblotting). Nuclear runoff assays indicate the mechanism of repression of PDGF beta R expression to be, at least in large part, transcriptional. These data indicate that expression of the PDGF beta R is regulated in a growth state-dependent manner in fibroblasts and suggest that this may provide a means by which cells can modulate their responsiveness to the actions of PDGF.

Vaziri, C; Faller, D V



The Orphan Receptor TAK1 Acts as a Repressor of RAR-, RXR- and T3R-Mediated Signaling Pathways  

Microsoft Academic Search

Recently, we reported the cloning and characterization of the novel orphan receptor TAK1. In this study, we analyze the interaction of TAK1 with a variety of response elements (RE?s) and demonstrate that TAK1 binds effectively to RE?s composed of the core motif PuGGTCA configured in direct repeats spaced by one or more nucleotides. TAK1 bound poorly to palindromic or inverted

T. Hirose; R. Apfel; M. Pfahl; A. M. Jetten



Invasiveness and Metastasis of NIH 3T3 Cells Induced by Met-Hepatocyte Growth Factor\\/Scatter Factor Autocrine Stimulation  

Microsoft Academic Search

The met protooncogene product, Met, is the tyrosine kinase growth factor receptor for hepatocyte growth factor\\/scatter factor (HGF\\/SF). NIH 3T3 cells express HGF\\/SF endogenously and become tumorigenic in nude mice via an autocrine mechanism when murine Met is expressed ectopically (Metmu cells) or when human Met and human HGF\\/SF are coexpressed (HMH cells). Here, we show that Metmu and HMH

Sing Rong; Shraga Segal; Miriam Anver; James H. Resau; George F. Vande Woude



Eucommia ulmoides Oliv. antagonizes H 2 O 2 -induced rat osteoblastic MC3T3-E1 apoptosis by inhibiting expressions of caspases 3, 6, 7, and 9  

Microsoft Academic Search

Eucommia ulmoides Oliv. (EuO), also known as Duzhong, native to China, has been reported to have antioxidative function, but its cellular mechanism\\u000a is not fully examined yet. We investigated inhibitory effects of EuO leaf ethanol extracts on H2O2-induced apoptosis in rat osteoblastic MC3T3-E1 cells and underlying mechanisms. Locally-grown Duzhong leaves were extracted\\u000a with ethanol. MC3T3-E1 cells were treated with EuO

Jun Lin; Yi-jing Fan; Christian Mehl; Jia-jun Zhu; Hong Chen; Ling-yan Jin; Jing-hong Xu; Hui-ming Wang



Dominant negative ?-subunit of farnesyl- and geranylgeranyl-transferase I inhibits insulin-induced differentiation of 3T3-L1 pre-adipocytes  

Microsoft Academic Search

OBJECTIVE: To investigate whether the expression of a dominant negative (DN) farnesyl- and geranygeranyl-transferase I (FTase\\/GGTase I) ?-subunit in 3T3-L1 pre-adipocytes can inhibit insulin's ability to induce differentiation.DESIGN: 3T3-L1 pre-adipocytes were stably transfected with vector alone or vector expressing a mutated DN FTase\\/GGTase I ?-subunit (S60A)(S62A) and incubated in serum-free medium in the absence and presence of insulin.MEASUREMENTS: Various assays

C S Solomon; J W Leitner; M L Goalstone



Antisense RNA--Induced Reduction in Murine TIMP Levels Confers Oncogenicity on Swiss 3T3 Cells  

Microsoft Academic Search

Mouse 3T3 cell lines capable of constitutively synthesizing an RNA complementary to the messenger RNA encoding TIMP, tissue inhibitor of metalloproteinases, were constructed by transfection with appropriate plasmid constructs. Many of the lines were down-modulated for TIMP messenger RNA levels and secreted less TIMP into the culture medium. In comparison to noninvasive, nontumorigenic controls, these cells not only were invasive

Rama Khokha; Paul Waterhouse; Simcha Yagel; Peeyush K. Lala; Christopher M. Overall; Gill Norton; David T. Denhardt



Associations between Weight Loss-Induced Changes in Plasma Organochlorine Concentrations, Serum T3 Concentration, and Resting Metabolic Rate  

Microsoft Academic Search

Organochlorine compounds are released from body fat into the bloodstream during weight loss. Because these compounds may impair thyroid status, which is implicated in the control of resting metabolic rate (RMR), the aim of this study was to determine if the augmentation in plasma organochlorine concentrations might be associated with the decrease in serum T3 concentration and RMR observed in

Catherine Pelletier; Eric Doucet; Pascal Imbeault; Angelo Tremblay



T3 (Triiodothyronine) Test  


... more information, see the Thyroid Foundation of America's web page Thyroid Problems During and After Pregnancy - Are You At Risk? ^ Back to top 2. What is the T3 uptake test? T3 uptake is also known as T3 Resin ...


The neck of caveolae is a distinct plasma membrane subdomain that concentrates insulin receptors in 3T3-L1 adipocytes.  


Insulin receptors (IRs) segregate on plasma membrane microvilli, but in cells devoid of microvilli, such as adipocytes, the localization of IRs is a matter of controversy. In the present study, we examined the distribution of IRs in the plasma membrane of 3T3-L1 adipocytes. Quantitative electron microscopy indicates that IRs are predominantly associated with the neck, but not the bulb, of caveolae. Caveola necks represent distinct microdomains of the plasma membrane. Indeed, as shown by freeze-fracture analysis, intramembrane particles are concentrated as necklaces around the craters of caveolae. In addition, subcellular fractionation suggests that the neck and the bulb of caveolae present a different resistance to detergent solubility. Finally, cytoskeletal components, including actin, are highly enriched in the membrane area underlying the neck part of caveolae. IRs coimmunoprecipitate with cytoskeletal components, and disruption of the actin cytoskeleton alters IRs expression, localization, and signaling, thus supporting the notion that caveola necks are involved in intracellular signaling by IRs. Together, these results suggest that cytoskeletal proteins anchor IRs to microdomains in the caveola necks of 3T3-L1 adipocytes. By homology with IR localization in other cell types, we suggest that the necks of caveolae may represent the counterpart of microvillar domains in cells poor in microvilli such as adipocytes and that they play an important role as signaling platforms. PMID:17227843

Foti, Michelangelo; Porcheron, Genevičve; Fournier, Margot; Maeder, Christine; Carpentier, Jean-Louis



Simvastatin suppresses leptin expression in 3T3-L1 adipocytes via activation of the cyclic AMP-PKA pathway induced by inhibition of protein prenylation.  


Simvastatin inhibits 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which catalyses conversion of HMG-CoA to mevalonate, a rate-limiting step in cholesterol synthesis. We demonstrated that simvastatin at 1 microM markedly inhibited adipocyte differentiation measured by Oil Red O staining in preadipocyte cells (3T3-L1), while expression of leptin, a marker of adipocyte differentiation, was suppressed by 1 muM simvastatin for up to 12 days of culture. Next, to elucidate mechanisms underlying the reduction of leptin expression induced by simvastatin, differentiated 3T3-L1 adipocytes were treated with various inhibitors with mevalonate or its metabolite in the presence or absence of simvastatin. Simvastatin time- and dose-dependently suppressed leptin mRNA expression. Heterogeneous nuclear RNA related to leptin mRNA was inhibited by 10 muM simvastatin, while stability of the mRNA was not changed by treatment with simvastatin in transcription-arrested 3T3-L1 cells. Simvastatin inhibition of leptin gene transcription was not abrogated by pre-treatment with cycloheximide, an inhibitor of protein synthesis. Addition of mevalonate or geranylgeranyl pyrophosphate (GGPP), a mevalonate metabolite, abolished simvastatin-induced inhibition of leptin expression in 3T3-L1 cells. Suppression of expression was observed upon addition of GGTI-298, a geranylgeranyl transferase I inhibitor, but not FTI-277, a farnesyl transferase inhibitor. Expression was suppressed by treatment with hydroxyfasudil, a protein prenylation inhibitor. Treatment with phosphatidylinositol 3-kinase (PI3K) inhibitors, LY294002 and wortmannin, reduced leptin expression in 3T3-L1 cells. Simvastatin dose-dependently increased intra-cellular cyclic AMP (cAMP) concentrations in 3T3-L1 cells, with maximal stimulation obtained at 10 muM. Addition of GGPP abolished simvastatin-induced stimulation of cAMP accumulation and protein kinase A (PKA) activity. H89, an inhibitor of PKA, completely abolished simvastatin-induced suppression of leptin expression. These results suggested that simvastatin reduced geranylgeranylprotein prenylation followed by deactivation of PI3K, leading to cAMP accumulation and subsequent activation of PKA in differentiated 3T3-L1 adipocytes. Finally, PKA inhibited leptin gene transcription without new protein synthesis. PMID:19254925

Maeda, Toyonobu; Horiuchi, Noboru



Overexpression of phospholipase C?-1 protects NIH3T3 cells from oxidative stress-induced cell death  

Microsoft Academic Search

Oxidative stress has been implicated in a wide range of cellular damage which includes DNA oxidation, membrane lipid peroxidation, and apoptosis. In our study, we found that overexpression of PLC-?1 in NIH3T3 fibroblasts protected them from cell death occuring in response to oxidative stress. Cell death caused by treatment with prooxidant tert-butylhydroperoxide (TBH), H2O2, or CdCl2 was considerably suppressed in

Young Han Lee; Seong-Yong Kim; Jae-Ryong Kim; Kyu-Tong Yoh; Suk-Hwan Baek; Myong Jong Kim; Sung Ho Ryu; Pann-Ghill Suh; Jung-Hye Kim



FATP1 mediates fatty acid-induced activation of AMPK in 3T3-L1 adipocytes  

Microsoft Academic Search

Fatty acid transport proteins are integral membrane acyl-CoA synthetases implicated in adipocyte fatty acid influx and esterification. FATP-dependent production of AMP was evaluated using FATP4 proteoliposomes, and fatty acid-dependent activation of AMP-activated protein kinase (AMPK) was assessed in 3T3-L1 adipocytes. Insulin-stimulated fatty acid influx (palmitate or arachidonate) into cultured adipocytes resulted in an increase in the phosphorylation of AMPK and

Brian M. Wiczer; Sandra Lobo; G. Luke Machen; Lee M. Graves; David A. Bernlohr



Green tea (-)-epigallocatechin gallate inhibits insulin stimulation of 3T3-L1 preadipocyte mitogenesis via the 67-kDa laminin receptor pathway.  


Insulin and (-)-epigallocatechin gallate (EGCG) have been reported to regulate fat cell mitogenesis and adipogenesis, respectively. This study investigated the pathways involved in EGCG modulation of insulin-stimulated mitogenesis in 3T3-L1 preadipocytes. EGCG inhibited insulin stimulation of preadipocyte proliferation in a dose- and time-dependent manner. EGCG also suppressed insulin-stimulated phosphorylation of the insulin receptor-beta, insulin receptor (IR) substrates 1 and 2 (IRS1 and IRS2), and mitogen-activated protein kinase pathway proteins, RAF1, MEK1/2, and ERK1/2, but not JNK. Furthermore, EGCG inhibited the association of IR with the IRS1 and IRS2 proteins, but not with the IRS4 protein. These data suggest that EGCG selectively affects particular types of IRS and MAPK family members. Generally, EGCG was more effective than epicatechin, epicatechin gallate, and epigallocatechin in modulating insulin-stimulated mitogenic signaling. We identified the EGCG receptor [also known as the 67-kDa laminin receptor (67LR)] in fat cells and found that its expression was sensitive to growth phase, tissue type, and differentiation state. Pretreatment of preadipocytes with 67LR antiserum prevented the effects of EGCG on insulin-stimulated phosphorylation of IRS2, RAF1, and ERK1/2 and insulin-stimulated preadipocyte proliferation (cell number and bromodeoxyuridine incorporation). Moreover, EGCG tended to increase insulin-stimulated associations between the 67LR and IR, IRS1, IRS2, and IRS4 proteins. These data suggest that EGCG mediates anti-insulin signaling in preadipocyte mitogenesis via the 67LR pathway. PMID:19176763

Ku, Hui-Chen; Chang, Hsin-Huei; Liu, Hsien-Chun; Hsiao, Chiao-Hsin; Lee, Meng-Jung; Hu, Yu-Jung; Hung, Pei-Fang; Liu, Chi-Wei; Kao, Yung-Hsi



Elucidation of a Signaling Pathway Induced by FGF2 Leading to uPA Gene Expression in NIH 3T3 Fibroblasts  

Microsoft Academic Search

Fibroblast growth factors (FGFs) play a role in biological processes such as cell growth and development, angiogenesis, and wound healing. Several genes have been shown to be induced by FGFs, but the underlying mechanisms have not been elucidated. We investigated the effect of FGF-2 (basic FGF) on the urokinase-type plasminogen activator (uPA) gene in NIH 3T3 fibroblasts. We found that

Daniel Besser; Marco Presta; Yoshikuni Nagamine



Ameliorating Effects of Fermented Rice Bran Extract on Oxidative Stress Induced by High Glucose and Hydrogen Peroxide in 3T3-L1 Adipocytes  

Microsoft Academic Search

In this study, we investigated whether fermented rice bran (FRB) can ameliorate the oxidative stress induced by high glucose\\u000a and hydrogen peroxide (H2O2) in 3T3-L1 adipocytes by analyzing reactive oxygen species (ROS), oil red O staining, as well as the expression of mRNAs\\u000a related to glucose homeostasis and adipogenesis. It was first confirmed that rice bran fermented by Issatchenkia orientalis

Dongyeop Kim; Gi Dong Han


SiRNA against Fabp5 induces 3T3-L1 cells apoptosis during adipocytic induction  

Microsoft Academic Search

Fatty acid-binding protein 5 (Fabp5), exhibits an important role in binding free fatty acids, as well as regulating lipid\\u000a metabolism and transport. The purpose of this study was to evaluate the role of Fabp5 during adipogenesis. 3T3-L1 preadipocytes\\u000a were selected as cell differentiation model and short interfering RNAs (siRNA) against Fabp5 (siFabp5) were prepared. Our\\u000a results showed that two potent

Xi MaXia; Xia Ren; Pengfei Han; Shengdi Hu; Junjun Wang; Jingdong Yin



Reversible bronchial hyperresponsiveness induced by OK-T3/IL-2 administration in a patient with multiple myeloma.  


Severe bronchial hyperresponsiveness to methacholine developed after intravenous therapy with OK-T3 and IL-2 in a patient with multiple myeloma, in whom no factors known to be associated with bronchial hyperresponsiveness were present. A substantial increase and activation of peripheral T-lymphocytes was observed after immunotherapy. Bronchial responsiveness and lymphocyte subsets both returned to normal baseline values 2 months after the patient was shifted to subcutaneous low dose administration of IL-2. The strict association between peripheral T-lymphocytes activation and the development of bronchial hyperresponsiveness suggests a causal relationship. PMID:8578020

Rolla, G; Bucca, C; Brussino, L; Massaia, M; Bergandi, D



Cloning of a G-protein-coupled receptor that shows an activity to transform NIH3T3 cells and is expressed in gastric cancer cells.  


The present study was directed towards the identification of novel factors involved in the transformation process leading to the formation of gastric cancer. A cDNA library from human gastric cancer cells was constructed using a retroviral vector. Functional cloning was performed by screening for transformation activity in transduced NIH3T3 cells. Six cDNA clones were isolated, including one encoding the elongation factor 1alpha subunit, which was already known to play a role in tumorigenesis. One cDNA (clone 56.2), which was repeatedly isolated during the course of screening, encoded a protein identical to a G-protein-coupled receptor protein, GPR35. In addition, another cDNA clone (72.3) was found to be an alternatively spliced product of the GPR35 gene, whereby 31 amino acids were added to the N-terminus of GPR35. Hence, the proteins encoded by clones 56.2 and 72.3 were designated GPR35a and GPR35b, respectively. RT-PCR experiments revealed that GPR35 gene expression is low or absent in surrounding non-cancerous regions, while both mRNAs were present in all of the gastric cancers examined. The level of 72.3-encoded mRNA was consistently significantly higher than that of 56.2 encoded mRNA. An expression pattern similar to that observed in gastric cancers was detected in normal intestinal mucosa. Based on the apparent transformation activities of the two GPR35 clones in NIH3T3 cells, and the marked up-regulation of their expression levels in cancer tissues, it is speculated that these two novel isoforms of GPR35 are involved in the course of gastric cancer formation. PMID:14965362

Okumura, Shun-ichiro; Baba, Hiroko; Kumada, Tatsuro; Nanmoku, Koji; Nakajima, Hirofumi; Nakane, Yasushi; Hioki, Koshiro; Ikenaka, Kazuhiro



Ethnic differences in the lymphocyte proliferative response induced by a murine IgG1 antibody, Leu-4, to the T3 molecule  

PubMed Central

The mitogenic effects of isotypically diverse antibodies to the T3 molecule were examined in genetically diverse population groups. Whereas the OKT3 antibody (IgG2a) was mitogenic for blood mononuclear cells from all individuals tested, the 38.1 antibody (IgM) was consistently nonmitogenic. In contrast, studies of the mitogenic effects of the Leu-4 antibody (IgG1) revealed striking ethnic differences. More than 80% of Caucasians and Negroes were good Leu-4 responders, whereas most individuals of Asian origin, including Indian, Japanese, and Chinese, were either Leu-4 nonresponders or Leu-4 low responders. However, the majority of American Indians, as well as a significant minority of Chinese, were good responders. Cell separation studies confirmed that monocytes govern the different mitogenic effects of the anti-T3 antibodies. The results reveal interesting ethnic differences in monocyte accessory function probably mediated via the Fc- gamma receptor, in the stimulation of T lymphocytes by an IgG1 antibody against the T3 molecule.



Blueberry peel extracts inhibit adipogenesis in 3T3-L1 cells and reduce high-fat diet-induced obesity.  


This study examined the anti-obesity effect and mechanism of action of blueberry peel extracts (BPE) in 3T3-L1 cells and high-fat diet (HFD)-induced obese rats. The levels of lipid accumulation were measured, along with the changes in the expression of genes and proteins associated with adipocyte differentiation in 3T3-L1 cells. Evidenced by Oil-red O staining and triglyceride assay, BPE dose-dependently inhibited lipid accumulation at concentrations of 0, 50, and 200 µg/ml. BPE decreased the expression of the key adipocyte differentiation regulator C/EBP?, as well as the C/EBP? and PPAR? genes, during the differentiation of preadipocytes into adipocytes. Moreover, BPE down-regulated adipocyte-specific genes such as aP2 and FAS compared with control adipocytes. The specific mechanism mediating the effects of BP revealed that insulin-stimulated phosphorylation of Akt was strongly decreased, and its downstream substrate, phospho-GSK3?, was downregulated by BPE treatment in 3T3-L1 cells. Together, these data indicated that BP exerted anti-adipogenic activity by inhibiting the expression of PPAR? and C/EBP? and the Akt signaling pathway in 3T3-L1 adipocytes. Next, we investigated whether BP extracts attenuated HFD-induced obesity in rats. Oral administration of BPE reduced HFD-induced body weight gain significantly without affecting food intake. The epididymal or perirenal adipose tissue weights were lower in rats on an HFD plus BPE compared with the tissue weights of HFD-induced obese rats. Total cholesterol and triglyceride levels in the rats fed BPE were modestly reduced, and the HDL-cholesterol level was significantly increased in HFD plus BP-fed rats compared with those of HFD-fed rats. Taken together, these results demonstrated an inhibitory effect of BP on adipogenesis through the down-regulation of C/EBP?, C/EBP?, and PPAR? and the reduction of the phospho-Akt adipogenic factor in 3T3-L1 cells. Moreover, BPE reduced body weight gain and inhibited fat accumulation in an HFD-induced animal model of obesity. PMID:23936120

Song, Yuno; Park, Hyoung Joon; Kang, Suk Nam; Jang, Sun-Hee; Lee, Soo-Jung; Ko, Yeoung-Gyu; Kim, Gon-Sup; Cho, Jae-Hyeon



Mammalian target of rapamycin complex 1 (mTORC1) plays a role in Pasteurella multocida toxin (PMT)-induced protein synthesis and proliferation in Swiss 3T3 cells.  


Pasteurella multocida toxin (PMT) is a potent mitogen known to activate several signaling pathways via deamidation of a conserved glutamine residue in the ? subunit of heterotrimeric G-proteins. However, the detailed mechanism behind mitogenic properties of PMT is unknown. Herein, we show that PMT induces protein synthesis, cell migration, and proliferation in serum-starved Swiss 3T3 cells. Concomitantly PMT induces phosphorylation of ribosomal S6 kinase (S6K1) and its substrate, ribosomal S6 protein (rpS6), in quiescent 3T3 cells. The extent of the phosphorylation is time and PMT concentration dependent, and is inhibited by rapamycin and Torin1, the two specific inhibitors of the mammalian target of rapamycin complex 1 (mTORC1). Interestingly, PMT-mediated mTOR signaling activation was observed in MEF WT but not in G?(q/11) knock-out cells. These observations are consistent with the data indicating that PMT-induced mTORC1 activation proceeds via the deamidation of G?(q/11), which leads to the activation of PLC? to generate diacylglycerol and inositol trisphosphate, two known activators of the PKC pathway. Exogenously added diacylglycerol or phorbol 12-myristate 13-acetate, known activators of PKC, leads to rpS6 phosphorylation in a rapamycin-dependent manner. Furthermore, PMT-induced rpS6 phosphorylation is inhibited by PKC inhibitor, Gö6976. Although PMT induces epidermal growth factor receptor activation, it exerts no effect on PMT-induced rpS6 phosphorylation. Together, our findings reveal for the first time that PMT activates mTORC1 through the G?(q/11)/PLC?/PKC pathway. The fact that PMT-induced protein synthesis and cell migration is partially inhibited by rapamycin indicates that these processes are in part mediated by the mTORC1 pathway. PMID:23223576

Oubrahim, Hammou; Wong, Allison; Wilson, Brenda A; Chock, P Boon



Analysis of the Rana catesbeiana tadpole tail fin proteome and phosphoproteome during T3-induced apoptosis: identification of a novel type I keratin  

PubMed Central

Background Thyroid hormones (THs) are vital in the maintenance of homeostasis and in the control of development. One postembryonic developmental process that is principally regulated by THs is amphibian metamorphosis. This process has been intensively studied at the genomic level yet very little information at the proteomic level exists. In addition, there is increasing evidence that changes in the phosphoproteome influence TH action. Results Here we identify components of the proteome and phosphoproteome in the tail fin that changed within 48 h of exposure of premetamorphic Rana catesbeiana tadpoles to 10 nM 3,5,3'-triiodothyronine (T3). To this end, we developed a cell and protein fractionation method combined with two-dimensional gel electrophoresis and phosphoprotein-specific staining. Altered proteins were identified using mass spectrometry (MS). We identified and cloned a novel Rana larval type I keratin, RLK I, which may be a target for caspase-mediated proteolysis upon exposure to T3. In addition, the RLK I transcript is reduced during T3-induced and natural metamorphosis which is consistent with a larval keratin. Furthermore, GILT, a protein involved in the immune system, is changed in phosphorylation state which is linked to its activation. Using a complementary MS technique for the analysis of differentially-expressed proteins, isobaric tags for relative and absolute quantitation (iTRAQ) revealed 15 additional proteins whose levels were altered upon T3 treatment. The success of identifying proteins whose levels changed upon T3 treatment with iTRAQ was enhanced through de novo sequencing of MS data and homology database searching. These proteins are involved in apoptosis, extracellular matrix structure, immune system, metabolism, mechanical function, and oxygen transport. Conclusion We have demonstrated the ability to derive proteomics-based information from a model species for postembryonic development for which no genome information is currently available. The present study identifies proteins whose levels and/or phosphorylation states are altered within 48 h of the induction of tadpole tail regression prior to overt remodeling of the tail. In particular, we have identified a novel keratin that is a target for T3-mediated changes in the tail that can serve as an indicator of early response to this hormone.

Domanski, Dominik; Helbing, Caren C



Mechanisms of divergent effects of activated peroxisome proliferator-activated receptor-? on mitochondrial citrate carrier expression in 3T3-L1 fibroblasts and mature adipocytes.  


The citrate carrier (CIC), a nuclear-encoded protein located in the mitochondrial inner membrane, plays an important metabolic role in the transport of acetyl-CoA from the mitochondrion to the cytosol in the form of citrate for fatty acid and cholesterol synthesis. Citrate has been reported to be essential for fibroblast differentiation into fat cells. Because peroxisome proliferator-activated receptor-gamma (PPAR?) is known to be one of the master regulators of adipogenesis, we aimed to study the regulation of CIC by the PPAR? ligand rosiglitazone (BRL) in 3T3-L1 fibroblasts and in adipocytes. We demonstrated that BRL up-regulated CIC mRNA and protein levels in fibroblasts, while it did not elicit any effects in mature adipocytes. The enhancement of CIC levels upon BRL treatment was reversed using the PPAR? antagonist GW9662, addressing how this effect was mediated by PPAR?. Functional experiments using a reporter gene containing rat CIC promoter showed that BRL enhanced CIC promoter activity. Mutagenesis studies, electrophoretic-mobility-shift assay and chromatin-immunoprecipitation analysis revealed that upon BRL treatment, PPAR? and Sp1 are recruited on the Sp1-containing region within the CIC promoter, leading to an increase in CIC expression. In addition, mithramycin, a specific inhibitor for Sp1-DNA binding activity, abolished the PPAR?-mediated up-regulation of CIC in fibroblasts. The stimulatory effects of BRL disappeared in mature adipocytes in which PPAR?/Sp1 complex recruited SMRT corepressor to the Sp1 site of the CIC promoter. Taken together, our results contribute to clarify the molecular mechanisms by which PPAR? regulates CIC expression during the differentiation stages of fibroblasts into mature adipocytes. PMID:23370576

Bonofiglio, Daniela; Santoro, Antonella; Martello, Emanuela; Vizza, Donatella; Rovito, Daniela; Cappello, Anna Rita; Barone, Ines; Giordano, Cinzia; Panza, Salvatore; Catalano, Stefania; Iacobazzi, Vito; Dolce, Vincenza; Andň, Sebastiano



Effect of fluoride on insulin level of rats and insulin receptor expression in the MC3T3-E1 cells.  


Studies on the role of insulin and insulin receptor (InsR) in the process of skeletal fluorosis, especially in osteogenic function, are rare. We evaluated the effect of increasing F? doses on the marker of bone formation, serum insulin level and pancreatic secretion changes in vivo and mRNA expression of InsR and osteocalcin (OCN) in vitro. Wistar rats (n?=?50) were divided into two groups, i.e. a control group and fluoride group. The fluoride groups were treated with fluoride by drinking tap water containing 100 mg F?/L. The fluoride ion-selective electrode measured the fluoride concentrations of femurs. The alkaline phosphatase (ALP), OCN, insulin and glucagon of serum were tested to observe the effect of fluoride action on them. Meantime, the pancreas pathological morphometry analysis via ? cells stained by aldehyde fuchsin showed the action of fluoride on pancreas secretion. MC3T3-E1 cells (derived from newborn mouse calvaria) were exposed to varying concentrations and periods of fluoride. The mRNA expression of InsR and OCN was quantified with real-time PCR. Results showed that 1-year fluoride treatment obviously stimulated ALP activity and OCN level along with increase of bone fluoride concentration of rats, which indicated that fluoride obviously stimulated osteogenic action of rats. In vitro study, the dual effect of fluoride on osteoblast function is shown. On the other hand, there was a significant increase of serum insulin level and a general decrease of glucagon level, and the histomorphometry analysis indicated an elevated insulin-positive area and increase in islet size in rats treated with fluoride for 1 year. In addition, fluoride obviously facilitated the mRNA expression of InsR in vitro. To sum up, there existed a close relationship between insulin secretion and fluoride treatment. The insulin signal pathway might be involved in the underlying occurrence or development of skeletal fluorosis. PMID:22872571

Hu, Chun-Yan; Ren, Li-Qun; Li, Xi-Ning; Wu, Nan; Li, Guang-Sheng; Liu, Qin-Yi; Xu, Hui



Eucommia ulmoides Oliv. antagonizes H2O2-induced rat osteoblastic MC3T3-E1 apoptosis by inhibiting expressions of caspases 3, 6, 7, and 9*  

PubMed Central

Eucommia ulmoides Oliv. (EuO), also known as Duzhong, native to China, has been reported to have antioxidative function, but its cellular mechanism is not fully examined yet. We investigated inhibitory effects of EuO leaf ethanol extracts on H2O2-induced apoptosis in rat osteoblastic MC3T3-E1 cells and underlying mechanisms. Locally-grown Duzhong leaves were extracted with ethanol. MC3T3-E1 cells were treated with EuO (6.25, 12.5, 25, 50, and 100 µg/ml) for 24 h, and then H2O2 (800 µmol/L) for an additional 24 h. Cell survival rate, percentage of apoptosis, and expressions of caspases 3, 6, 7, and 9 were examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, microscopic analysis, Western blotting, and reverse transcription polymerase chain reaction (RT-PCR). The final EuO leaf ethanol extract powder was detected to contain caffeotannic acid at 58 mg/g and geniposide at 3.45 mg/g by high performance liquid chromatography (HPLC). EuO remarkably restrained cell oxidative damage and increased cell survival rate in a dose-dependent manner: 0 µg/ml, 0.21; 6.25 µg/ml, 0. 28; 12.5 µg/ml, 0.31; 25 µg/ml, 0.48; 50 µg/ml, 0.54; and 100 µg/ml, 0.66 (P<0.05), with the half-effective concentration being around 25 µg/ml. MTT results were confirmed by microscopic analysis. Western blotting and RT-PCR analyses showed that the expressions of caspases 3, 6, 7, and 9 were significantly decreased in the EuO-treated cells compared with the control (EuO- and H2O2-free) (P<0.05), with the half-effective concentration of EuO ranging from 12.5 to 25 µg/ml. We conclude that the ethanol-extracted EuO leaf extracts promoted the growth of MC3T3-E1 cells, and suppressed the H2O2-induced apoptosis in a rat MC3T3-E1 osteogenic cell model, likely due to the inhibition of caspases’ activities. The results indicate that EuO is a potent antioxidant, which may contribute to its many cellular protective functions, including the promotion of bone growth.

Lin, Jun; Fan, Yi-jing; Mehl, Christian; Zhu, Jia-jun; Chen, Hong; Jin, Ling-yan; Xu, Jing-hong; Wang, Hui-ming



Relationship of cytosolic ion fluxes and protein kinase C activation to platelet-derived growth factor induced competence and growth in BALB/c-3T3 cells.  


Platelet-derived growth factor (PDGF) and other agents that activate protein kinase C (PKC) rapidly alter cytosolic pH (pHi) and intracellular free calcium ([Ca++]i) in BALB/c-3T3 fibroblasts. To define whether changes in pHi or [Ca++]i are linked to PDGF-stimulated mitogenesis, these parameters were assessed in control and PKC depleted fibroblasts. PDGF addition to BALB/c-3T3 fibroblasts resulted in transient acidification of the cytoplasm followed by prolonged cytosolic alkalinization. Exposure of cells to 12-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester that activates PKC, resulted in cytosolic alkalinization without prior acidification. Overnight incubation with 600 nM TPA decreased the total cell PKC histone phosphorylating activity in BALB/c-3T3 fibroblasts by greater than 90%. In PKC-deficient fibroblasts, TPA, and PDGF-induced alkalinization was abolished. In addition, the transient drop in pHi seen initially in control cells treated with PDGF is sustained to the point where pHi is fully 0.6-0.7 pH units below control cell values for up to 30 minutes. PDGF increased [Ca++]i threefold; this transient rise in [Ca++]i was only minimally affected (less than 15%) by lowering of the extracellular calcium level with ethylene glycol bis(b-aminoethyl ether)0 N,N,N' tetraacetic acid (EGTA) or blocking calcium influx with CoCl2. In contrast, 8-(diethylamine)-octyl-3,4,5-trimethoxybenzoate (TMB-8), an agent thought to inhibit calcium release from intracellular stores, substantially inhibited the rise in [Ca++]i caused by PDGF. TPA and 1-oleoyl-2-acetylglycerol (OAG) increased [Ca++]i but in contrast to PDGF this effect was blocked by pretreatment of cells with EGTA or CoCl2. In PKC-deficient fibroblasts, PDGF still increased [Ca++]i and stimulated DNA synthesis as effectively as in controls. TPA and OAG however, no longer increased [Ca++]i. The continued ability of PDGF to stimulate DNA synthesis in the face of sustained acidification and the absence of PKC activity suggests that cytosolic alkalinization and PKC activation are not essential for PDGF-induced competence in BALB/c-3T3 fibroblasts. PMID:2708452

Zagari, M; Stephens, M; Earp, H S; Herman, B



Eucommia ulmoides Oliv. antagonizes H2O2-induced rat osteoblastic MC3T3-E1 apoptosis by inhibiting expressions of caspases 3, 6, 7, and 9.  


Eucommia ulmoides Oliv. (EuO), also known as Duzhong, native to China, has been reported to have antioxidative function, but its cellular mechanism is not fully examined yet. We investigated inhibitory effects of EuO leaf ethanol extracts on H(2)O(2)-induced apoptosis in rat osteoblastic MC3T3-E1 cells and underlying mechanisms. Locally-grown Duzhong leaves were extracted with ethanol. MC3T3-E1 cells were treated with EuO (6.25, 12.5, 25, 50, and 100 µg/ml) for 24 h, and then H(2)O(2) (800 µmol/L) for an additional 24 h. Cell survival rate, percentage of apoptosis, and expressions of caspases 3, 6, 7, and 9 were examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, microscopic analysis, Western blotting, and reverse transcription polymerase chain reaction (RT-PCR). The final EuO leaf ethanol extract powder was detected to contain caffeotannic acid at 58 mg/g and geniposide at 3.45 mg/g by high performance liquid chromatography (HPLC). EuO remarkably restrained cell oxidative damage and increased cell survival rate in a dose-dependent manner: 0 µg/ml, 0.21; 6.25 µg/ml, 0. 28; 12.5 µg/ml, 0.31; 25 µg/ml, 0.48; 50 µg/ml, 0.54; and 100 µg/ml, 0.66 (P<0.05), with the half-effective concentration being around 25 µg/ml. MTT results were confirmed by microscopic analysis. Western blotting and RT-PCR analyses showed that the expressions of caspases 3, 6, 7, and 9 were significantly decreased in the EuO-treated cells compared with the control (EuO- and H(2)O(2)-free) (P<0.05), with the half-effective concentration of EuO ranging from 12.5 to 25 µg/ml. We conclude that the ethanol-extracted EuO leaf extracts promoted the growth of MC3T3-E1 cells, and suppressed the H(2)O(2)-induced apoptosis in a rat MC3T3-E1 osteogenic cell model, likely due to the inhibition of caspases' activities. The results indicate that EuO is a potent antioxidant, which may contribute to its many cellular protective functions, including the promotion of bone growth. PMID:21194186

Lin, Jun; Fan, Yi-jing; Mehl, Christian; Zhu, Jia-jun; Chen, Hong; Jin, Ling-yan; Xu, Jing-hong; Wang, Hui-ming



Thyroid-Stimulating Hormone Induces the Secretion of Tumor Necrosis Factor-? from 3T3-L1 Adipocytes via a Protein Kinase A-Dependent Pathway.  


Numerous reports have suggested that thyroid-stimulating hormone (TSH) contributes to insulin resistance in adipocytes by directly stimulating the production of adipokines, such as tumor necrosis factor ? (TNF-?). The objective of this study was to clarify how TSH regulates TNF-? secretion in 3T3-L1 adipocytes and to determine which cell signaling pathways were involved.Mouse 3T3-L1 preadipocytes were differentiated into adipocytes and then exposed to 0.1 mIU/ml bovine TSH. The optimal treatment duration was determined by measuring the TNF-? concentration in the medium by ELISA. Thereafter, adipocytes were treated with 0.01, 0.1, and 1.0 mIU/ml bovine TSH, and the optimal TSH dose was determined. To decrease TSHR expression, TSHR shRNA was transfected into adipocytes, and the silencing was confirmed by Western blotting. The TSH signaling pathways responsible for regulating TNF-? secretion were studied. Phospho-NF-?Bp65 Ser276 was quantified by Western blotting, and co-immunopreci-pitations were performed to detect the formation of the I?B?/PKAc complex.TNF-? secretion from adipocytes peaked 4 h after TSH treatment. TSH induced TNF-? secretion in a dose-dependent manner, which was almost completely inhibited by TSHR shRNA. There was a significant positive correlation between phospho-NF-?Bp65 Ser276 levels and TNF-? secretion. H89, a cAMP-dependent protein kinase A inhibitor, significantly inhibited the effects of TSH. Bovine TSH and forskolin, which increases intracellular cAMP, simultaneously stimulated TNF-? secretion. The I?B?/PKAc complex could be detected in TSH-treated cells, but complex formation was inhibited by H89.TSH stimulated the cAMP-PKA pathway in 3T3-L1 adipocytes to increase TNF-? secretion. PMID:23864495

Zhang, Y-J; Zhao, W; Zhu, M-Y; Tang, S-S; Zhang, H



Mechanisms of Kirsten murine sarcoma virus transformation-induced changes in the collagen phenotype and synthetic rate of BALB 3T3 cells.  

PubMed Central

Specific viral transformation rather than cell selection can explain the previously observed increase in the proportion of type III procollagen compared to type I procollagen in BALB 3T3 cells transformed by Kirsten murine sarcoma virus (Ki-MSV). Two subclones of BALB 3T3 A31 were productively infected with with a temperature-sensitive Ki-MSV in the presence of helper murine leukemia virus (MLV), resulting in virtually complete transformation of cultures and eliminating selection of transformed foci. Analysis of radioactive collagen, derived from procollagen by pepsin treatment, showed that both of the tsKi-MSV/MLV-transformed subclones contained a 4-fold greater proportion of type III procollagen than did control MLV-infected cultures. A nonproducer derivative exhibited an even greater change (10-fold), indicating that viral replication was irrelevant. After 48 hr at a nonpermissive temperature, tsKi-MSV-transformed cells retained a high proportion of type III procollagen, suggesting that either this change is not induced by src protein or else there is a slowly reversible or irreversible step involved. Alternatively, type III procollagen mRNA may be long lived. In contrast, the relative rate of procollagen synthesis in transformed cells was clearly regulated by src protein. Translation of mRNA from cells preincubated at permissive or nonpermissive temperatures revealed that the decreased relative rate can be explained by a simultaneous small decrease in the level of procollagen mRNA and a large increase in mRNA for noncollagen proteins. Images

Bateman, J F; Peterkofsky, B



Expression of Caveolin-1 reduces cellular responses to TGF-{beta}1 through down-regulating the expression of TGF-{beta} type II receptor gene in NIH3T3 fibroblast cells  

SciTech Connect

Transcriptional repression of Transforming Growth Factor-{beta} type II receptor (T{beta}RII) gene has been proposed to be one of the major mechanisms leading to TGF-{beta} resistance. In this study, we demonstrate that expression of Caveolin-1 (Cav-1) gene in NIH3T3 fibroblast cells down-regulates the expression of T{beta}RII gene in the transcriptional level, eventually resulting in the decreased responses to TGF-{beta}. The reduced expression of T{beta}RII gene by Cav-1 appeared to be due to the changes of the sequence-specific DNA binding proteins to either Positive Regulatory Element 1 (PRE1) or PRE2 of the T{beta}RII promoter. In addition, Cav-1 expression inhibited TGF-{beta}-mediated cellular proliferation and Plasminogen Activator Inhibitor (PAI)-1 gene expression as well as TGF-{beta}-induced luciferase activity. Furthermore, the inhibition of endogeneous Cav-1 by small interfering RNA increased the expression of T{beta}RII gene. These findings strongly suggest that expression of Cav-1 leads to the decreased cellular responsiveness to TGF-{beta} through down-regulating T{beta}RII gene expression.

Lee, Eun Kyung [Inha University College of Medicine, Incheon 400-121 (Korea, Republic of); Lee, Youn Sook [Department of Biological Science, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Han, In-Oc [Inha University College of Medicine, Incheon 400-121 (Korea, Republic of); Park, Seok Hee [Inha University College of Medicine, Incheon 400-121 (Korea, Republic of) and Department of Biological Science, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of)]. E-mail:



Effects of paeoniflorin on tumor necrosis factor-?-induced insulin resistance and changes of adipokines in 3T3-L1 adipocytes.  


TNF? plays an important role in the adipocyte dysfunction, including lipolysis acceleration, insulin resistance and changes of adipokines. Recently, we showed that paeoniflorin attenuates adipocyte lipolysis and inhibits the phosphorylation of ERK, JNK, IKK stimulated by TNF?. However, the effects of paeoniflorin on adipocytes insulin resistance and changes of adipokines remain unknown. The aim of the current study was to investigate the role of paeoniflorin in preventing insulin resistance or inflammation in 3T3-L1 adipocytes treated with TNF?. Our results showed that paeoniflorin restored insulin-stimulated [(3)H]2-DOG uptake, which was reduced by TNF?, with concomitant restoration in serine phosphorylation of IRS-1 and insulin-stimulated phosphorylation of AKT in adipocytes. Paeoniflorin attenuated TNF?-mediated suppression of the expressions of PPAR? and PPAR? target genes, and the improvement of paeoniflorin on TNF?-induced insulin resistance was attenuated by GW9662, an antagonist of PPAR? activity. Moreover, paeoniflorin could inhibit the expressions and secretions of IL-6 and MCP-1 from adipocytes induced by TNF?. These results, together with our previous data, indicate that paeoniflorin exerts a beneficial effect on adipocytes to prevent TNF?-induced insulin resistance and inflammatory adipokine release. Our studies provide important evidence for an ability of paeoniflorin in amelioration of TNF?-induced adipocyte dysfunction, which would be helpful to clarify its potential role in the treatment of obesity. PMID:23978582

Kong, Poren; Chi, Rongxiang; Zhang, Linlin; Wang, Ningjian; Lu, Yingli



Correlations between radiation-induced double strand breaks, cell division delay, and cyclin-dependent signaling in x-irradiated NIH3T3 fibroblasts  

NASA Astrophysics Data System (ADS)

Molecular responses to radiation-induced DNA double strand breaks (DSB) are mediated by the phosphorylation of the histone variant H2AX which forms identifiable gamma-H2AX foci at the site of the DSB. This event is thought to be linked with the down-regulation of signaling proteins contributing to the checkpoints regulating cell cycle progression and, vis-a-vis , the induction of cell division delay. However, it is unclear whether this division delay is directly related to the number of DSB (gamma-H2AX foci) sustained by an irradiated cell and, if so, whether this number drives cells into cell cycle delay or apoptosis. For this reason, studies were conducted in the immortalized NIH/3T3 fibroblast cell in order to establish correlations between the temporal appearance of the gamma-H2AX foci (a DSB) and the expression of the cell cycle regulatory proteins, cyclin E, A, B1, and their cyclin kinase inhibitor, p21. Cell cycle kinetics and flow cytometry were used to establish radiation-induced division delay over a dose range of 1--6 Gy where a mitotic delay of 2.65 min/cGy was established. Correlations between the expression of cyclin E, A, B1, p21, and the generation of DSB were established in NIH/3T3 cells exposed to 2 or 4 Gy x-irradiation. The data suggest that the G1/S and S phase delay (cyclin E and cyclin A protein levels) are dependent on the dose of radiation while the G2/M (cyclin B1 protein levels) delay is dependent on the quantity of DSB sustained by the irradiated cell.

Cariveau, Mickael J.


The Novel Catenin p120 cas Binds Classical Cadherins and Induces an Unusual Morphological Phenotype in NIH3T3 Fibroblasts  

Microsoft Academic Search

p120cas(CAS) is a tyrosine kinase substrate whose phosphorylation has been implicated in cell transformation by Src and in ligand-induced signaling through the EGF, PDGF, and CSF-1 receptors. More recently, CAS has been shown to associate with E-cadherin and its cofactors (catenins), molecules that are involved in cell adhesion. Although both CAS and ?-catenin contain armadillo repeat domains (Arm domains), the

Albert B. Reynolds; Juliet M. Daniel; Yin-Yuan Mo; Jing Wu; Zhi Zhang



Nucleocytoplasmic transport is enhanced concomitant with nuclear accumulation of epidermal growth factor (EGF) binding activity in both 3T3-1 and EGF receptor reconstituted NR-6 fibroblasts  

PubMed Central

Measurements of nucleocytoplasmic transport of fluorescent-labeled macromolecules were performed in both an EGF-nonresponsive mutant fibroblast line (3T3-NR6) and in the same cell line reconstituted with active EGF receptors derived from rat hepatic membrane fraction. Immunolocalization studies of exogenously incorporated EGF receptors in reconstituted 3T3-NR6 fibroblasts demonstrated predominantly intracellular localization. The EGF receptor constructs also showed EGF- stimulated incorporation of [3H]thymidine, providing biochemical evidence for functional integration of the exogenously supplied EGF receptors into the reconstituted fibroblasts. Additional support for the functional incorporation of receptor may be inferred from the enhanced cellular accumulation of 125I-EGF in cells treated with chloroquine and leupeptin. 125I-EGF binding and transnuclear macromolecular transport measurements in mutant and reconstituted cells, in conjunction with such measurements on nuclei isolated from these cells, provide data consistent with a growth factor/nuclear signaling mechanism dependent on the nuclear acquisition of EGF binding activity from the plasma membrane.



Rosiglitazone Induces Mitochondrial Biogenesis in Differentiated Murine 3T3-L1 and C3H/10T1/2 Adipocytes  

PubMed Central

Growing evidence indicates that PPAR? agonists, including rosiglitazone (RSG), induce adipose mitochondrial biogenesis. By systematically analyzing mitochondrial gene expression in two common murine adipocyte models, the current study aimed to further establish the direct role of RSG and capture temporal changes in gene transcription. Microarray profiling revealed that in fully differentiated 3T3-L1 and C3H/10T1/2 adipocytes treated with RSG or DMSO vehicle for 1, 2, 4, 7, 24, and 48?hrs, RSG overwhelmingly increased mitochondrial gene transcripts time dependently. The timing of the increases was consistent with the cascade of organelle biogenesis, that is, initiated by induction of transcription factor(s), followed by increases in the biosynthesis machinery, and then by increases in functional components. The transcriptional increases were further validated by increased mitochondrial staining, citrate synthase activity, and O2 consumption, and were found to be associated with increased adiponectin secretion. The work provided further insight on the mechanism of PPAR?-induced mitochondrial biogenesis in differentiated adipocytes.

Rong, James X.; Klein, Jean-Louis D.; Qiu, Yang; Xie, Mi; Johnson, Jennifer H.; Waters, K. Michelle; Zhang, Vivian; Kashatus, Jennifer A.; Remlinger, Katja S.; Bing, Nan; Crosby, Renae M.; Jackson, Tymissha K.; Witherspoon, Sam M.; Moore, John T.; Ryan, Terence E.; Neill, Sue D.; Strum, Jay C.



Rosiglitazone Induces Mitochondrial Biogenesis in Differentiated Murine 3T3-L1 and C3H/10T1/2 Adipocytes.  


Growing evidence indicates that PPAR? agonists, including rosiglitazone (RSG), induce adipose mitochondrial biogenesis. By systematically analyzing mitochondrial gene expression in two common murine adipocyte models, the current study aimed to further establish the direct role of RSG and capture temporal changes in gene transcription. Microarray profiling revealed that in fully differentiated 3T3-L1 and C3H/10T1/2 adipocytes treated with RSG or DMSO vehicle for 1, 2, 4, 7, 24, and 48?hrs, RSG overwhelmingly increased mitochondrial gene transcripts time dependently. The timing of the increases was consistent with the cascade of organelle biogenesis, that is, initiated by induction of transcription factor(s), followed by increases in the biosynthesis machinery, and then by increases in functional components. The transcriptional increases were further validated by increased mitochondrial staining, citrate synthase activity, and O(2) consumption, and were found to be associated with increased adiponectin secretion. The work provided further insight on the mechanism of PPAR?-induced mitochondrial biogenesis in differentiated adipocytes. PMID:22013433

Rong, James X; Klein, Jean-Louis D; Qiu, Yang; Xie, Mi; Johnson, Jennifer H; Waters, K Michelle; Zhang, Vivian; Kashatus, Jennifer A; Remlinger, Katja S; Bing, Nan; Crosby, Renae M; Jackson, Tymissha K; Witherspoon, Sam M; Moore, John T; Ryan, Terence E; Neill, Sue D; Strum, Jay C



A short pulse of mechanical force induces gene expression and growth in MC3T3-E1 osteoblasts via an ERK 1/2 pathway.  


Physiological mechanical loading is crucial for maintenance of bone integrity and architecture. We have calculated the strain caused by gravity stress on osteoblasts and found that 4-30g corresponds to physiological levels of 40-300 microstrain. Short-term gravity loading (15 minutes) induced a 15-fold increase in expression of growth-related immediate early gene c-fos, a 5-fold increase in egr-1, and a 3-fold increase in autocrine bFGF. The non-growth-related genes EP-1, TGF-beta, and 18s were unaffected by gravity loading. Short-term physiological loading induced extracellular signal-regulated kinase (ERK 1/2) phosphorylation in a dose-dependent manner with maximum phosphorylation saturating at mechanical loading levels of 12g (p < 0.001) with no effect on total ERK. The phosphorylation of focal adhesion kinase (FAK) was unaffected by mechanical force. g-Loading did not activate P38 MAPK or c-jun N-terminal kinase (JNK). Additionally, a gravity pulse resulted in the localization of phosphorylated ERK 1/2 to the nucleus; this did not occur in unloaded cells. The induction of c-fos was inhibited 74% by the MEK1/2 inhibitor U0126 (p < 0.001) but was not affected by MEK1 or p38 MAPK-specific inhibitors. The long-term consequence of a single 15-minute gravity pulse was a 64% increase in cell growth (p < 0.001). U0126 significantly inhibited gravity-induced growth by 50% (p < 0.001). These studies suggest that short periods of physiological mechanical stress induce immediate early gene expression and growth in MC3T3-E1 osteoblasts primarily through an ERK 1/2-mediated pathway. PMID:12510806

Hatton, Jason P; Pooran, Milad; Li, Chai-Fei; Luzzio, Chris; Hughes-Fulford, Millie



Inhibitory effects of a tryptamine derivative on ultraviolet radiation-induced apoptosis in MC3T3-E1 mouse osteoblasts.  


MS-IPA1 is a new synthetic compound that is synthesized from tryptamine. Recently, our group demonstrated that SST-VED-I-1, which has a similar chemical structure to MS-IPA1, inhibits starvation-induced apoptosis in osteoblasts. However, the effects of MS-IPA1 on apoptosis in osteoblasts have not yet been examined. Therefore, this study examined the effects of this compound on apoptosis in osteoblasts. In this study, MC3T3-E1 mouse osteoblasts were used and apoptosis was induced by ultraviolet radiation (UV). We investigated the effect of MS-IPA1 on apoptosis by analyzing caspase3/7 activity, translocation of phosphatidylserine (PS), and mRNA expression levels of Bcl-2 and Bax. In addition, it was investigated whether MS-IPA1 affects cell proliferation and cell cycle progression. We found that MS-IPA1 had no effect on cell proliferation or cell cycle progression. However, MS-IPA1 suppressed UV-induced cell death in a dose-dependent manner, which was accompanied with the inhibition of caspase activation and translocation of PS. Furthermore, after UV exposure, Bcl-2 expression was increased in the MS-IPA1-treated cells as compared to that in the vehicle-treated cells. In contrast, Bax expression was decreased in the MS-IPA1-treated cell as compared to that in the vehicle-treated cells. These results suggest that MS-IPA1 has an inhibitory effect on apoptosis in osteoblasts through a Bcl-2 family-dependent signaling pathway. PMID:21282935

Mikami, Yoshikazu; Senoo, Motoki; Lee, Mio; Yamada, Kiyoshi; Ochiai, Kuniyasu; Honda, Masaki J; Watanabe, Eri; Watanabe, Nobukazu; Somei, Masanori; Takagi, Minoru



Insulin regulates hypoxia-inducible factor-1? transcription by reactive oxygen species sensitive activation of Sp1 in 3T3-L1 preadipocyte.  


Oxygen sensing transcription factor HIF-1 is activated due to accumulation of regulatory subunit HIF-1? by posttranslational stability mechanism during hypoxia or by several other stimuli even in normoxia. HIF-1? is also regulated by NF-kB mediated transcription mechanism. Reactive oxygen species (ROS) act as an important regulator of HIF-1 either by affecting prolyl hydroxylase activity, the critical determinant of HIF-1? stabilization or by activating NF-kB to promote HIF-1? transcription. Insulin is known to activate HIF-1 by a ROS dependent mechanism but the molecular mechanism of HIF-1? regulation is not known so far. Here we show that insulin regulates HIF-1? by a novel transcriptional mechanism by a ROS-sensitive activation of Sp1 in 3T3-L1 preadipocyte. Insulin shows little effect on HIF-1? protein stability, but increases HIF-1? promoter activity. Mutation analyses, electrophoretic mobility shift assay and chromatin immunoprecipitation assay confirm the role of Sp1 in HIF-1? transcription. We further demonstrate that insulin-induced ROS generation initiates signaling pathway involving phosphatidylinositol 3-kinase and protein kinase C for Sp1 mediated HIF-1? transcription. In summary, we reveal that insulin regulates HIF-1? by a novel transcriptional mechanism involving Sp1. PMID:23626778

Biswas, Sudipta; Mukherjee, Reshmi; Tapryal, Nisha; Singh, Amit K; Mukhopadhyay, Chinmay K



PPARgamma induces the insulin-dependent glucose transporter GLUT4 in the absence of C/EBPalpha during the conversion of 3T3 fibroblasts into adipocytes.  

PubMed Central

To define the molecular mechanisms that control GLUT4 expression during adipogenesis, NIH-3T3 fibroblasts ectopically expressing different adipogenic transcription factors (C/EBPbeta, C/EBPdelta, C/EBPalpha, and PPARgamma) under the control of a tetracycline-responsive inducible (C/EBPs) or a constitutive retroviral (PPARgamma) expression system were used. Enhanced production of C/EBPbeta (beta2 cell line), C/EBPbeta together with C/EBPdelta (beta/delta39 cell line), C/EBPalpha (alpha1 cell line), or PPARgamma (Pgamma2 cell line) in cells exposed to dexamethasone and the PPARgamma ligand ciglitazone (a thiazolidinedione) resulted in expression of GLUT4 mRNA as well as other members of the adipogenic gene program, including aP2 and adipsin. Focusing our studies on the beta/delta39 cells, we have demonstrated that C/EBPbeta along with C/EBPdelta in the presence of dexamethasone induces PPARgamma, adipsin, and aP2 mRNA production; however, GLUT4 mRNA is only expressed in cells exposed to ciglitazone. In addition, enhanced expression of a ligand-activated form of PPARgamma in the beta/delta39 fibroblasts stimulates synthesis of GLUT4 protein and gives rise to a population of adipocytic cells that take up glucose in direct response to insulin. C/EBPalpha is not expressed in the beta/delta39 cells under conditions that stimulate the adipogenic program. This observation suggests that PPARgamma alone or in combination with C/EBPbeta and C/EBPdelta is capable of activating GLUT4 gene expression.

Wu, Z; Xie, Y; Morrison, R F; Bucher, N L; Farmer, S R



I Lost Weight, but I Became Weak and Cannot Walk-A Case of Nutraceutical (T3)-Induced Thyrotoxic Periodic Paralysis.  


Thyrotoxic periodic paralysis (TPP) is a rare reversible cause of paralysis and cramping. TPP is usually precipitated by common causes of thyrotoxicosis such as Grave disease or multinodular goiter. TPP precipitated by exogenous triiodothyronine (T3) intake is an extremely rare occurrence with only 3 cases reported to date. We now report a 24-year-old healthy manual laborer who developed quadriparesis during a period of rest after heavy exertion and carbohydrate intake. He had severe hypokalemia (potassium level 1.9 mmole/L). Correction of his hypokalemia reversed the paralysis without rebound hyperkalemia. After a detailed history review, he reported that he had been consuming nutraceuticals containing T3 for 1 month to lose weight, and laboratory studies confirmed factitious T3 toxicosis. There was no evidence of renal or gastrointestinal potassium wasting. This episode of TPP was the first manifestation of thyrotoxicosis in this patient, and avoidance of T3 intake prevented more episodes. PMID:23567793

Panikkath, Ragesh; Nugent, Kenneth



Rapid Responses to Reverse T3 Hormone in Immature Rat Sertoli Cells: Calcium Uptake and Exocytosis Mediated by Integrin  

PubMed Central

There is increasing experimental evidence of the nongenomic action of thyroid hormones mediated by receptors located in the plasma membrane or inside cells. The aim of this work was to characterize the reverse T3 (rT3) action on calcium uptake and its involvement in immature rat Sertoli cell secretion. The results presented herein show that very low concentrations of rT3 are able to increase calcium uptake after 1 min of exposure. The implication of T-type voltage-dependent calcium channels and chloride channels in the effect of rT3 was evidenced using flunarizine and 9-anthracene, respectively. Also, the rT3-induced calcium uptake was blocked in the presence of the RGD peptide (an inhibitor of integrin-ligand interactions). Therefore, our findings suggest that calcium uptake stimulated by rT3 may be mediated by integrin ?v?3. In addition, it was demonstrated that calcium uptake stimulated by rT3 is PKC and ERK-dependent. Furthermore, the outcomes indicate that rT3 also stimulates cellular secretion since the cells manifested a loss of fluorescence after 4 min incubation, indicating an exocytic quinacrine release that seems to be mediated by the integrin receptor. These findings indicate that rT3 modulates the calcium entry and cellular secretion, which might play a role in the regulation of a plethora of intracellular processes involved in male reproductive physiology.

Zanatta, Ana Paula; Zanatta, Leila; Goncalves, Renata; Zamoner, Ariane; Silva, Fatima Regina Mena Barreto



Rapid responses to reverse t3 hormone in immature rat sertoli cells: calcium uptake and exocytosis mediated by integrin.  


There is increasing experimental evidence of the nongenomic action of thyroid hormones mediated by receptors located in the plasma membrane or inside cells. The aim of this work was to characterize the reverse T3 (rT3) action on calcium uptake and its involvement in immature rat Sertoli cell secretion. The results presented herein show that very low concentrations of rT3 are able to increase calcium uptake after 1 min of exposure. The implication of T-type voltage-dependent calcium channels and chloride channels in the effect of rT3 was evidenced using flunarizine and 9-anthracene, respectively. Also, the rT3-induced calcium uptake was blocked in the presence of the RGD peptide (an inhibitor of integrin-ligand interactions). Therefore, our findings suggest that calcium uptake stimulated by rT3 may be mediated by integrin ?v?3. In addition, it was demonstrated that calcium uptake stimulated by rT3 is PKC and ERK-dependent. Furthermore, the outcomes indicate that rT3 also stimulates cellular secretion since the cells manifested a loss of fluorescence after 4 min incubation, indicating an exocytic quinacrine release that seems to be mediated by the integrin receptor. These findings indicate that rT3 modulates the calcium entry and cellular secretion, which might play a role in the regulation of a plethora of intracellular processes involved in male reproductive physiology. PMID:24130850

Zanatta, Ana Paula; Zanatta, Leila; Gonçalves, Renata; Zamoner, Ariane; Silva, Fátima Regina Mena Barreto



Epigallocathechin-3 gallate selectively inhibits the PDGF-BB-induced intracellular signaling transduction pathway in vascular smooth muscle cells and inhibits transformation of sis-transfected NIH 3T3 fibroblasts and human glioblastoma cells (A172).  


Enhanced activity of receptor tyrosine kinases such as the PDGF beta-receptor and EGF receptor has been implicated as a contributing factor in the development of malignant and nonmalignant proliferative diseases such as cancer and atherosclerosis. Several epidemiological studies suggest that green tea may prevent the development of cancer and atherosclerosis. One of the major constituents of green tea is the polyphenol epigallocathechin-3 gallate (EGCG). In an attempt to offer a possible explanation for the anti-cancer and anti-atherosclerotic activity of EGCG, we examined the effect of EGCG on the PDGF-BB-, EGF-, angiotensin II-, and FCS-induced activation of the 44 kDa and 42 kDa mitogen-activated protein (MAP) kinase isoforms (p44(mapk)/p42(mapk)) in cultured vascular smooth muscle cells (VSMCs) from rat aorta. VSMCs were treated with EGCG (1-100 microM) for 24 h and stimulated with the above mentioned agonists for different time periods. Stimulation of the p44(mapk)/p42(mapk) was detected by the enhanced Western blotting method using phospho-specific MAP kinase antibodies that recognized the Tyr204-phosphorylated (active) isoforms. Treatment of VSMCs with 10 and 50 microM EGCG resulted in an 80% and a complete inhibition of the PDGF-BB-induced activation of MAP kinase isoforms, respectively. In striking contrast, EGCG (1-100 microM) did not influence MAP kinase activation by EGF, angiotensin II, and FCS. Similarly, the maximal effect of PDGF-BB on the c-fos and egr-1 mRNA expression as well as on intracellular free Ca2+ concentration was completely inhibited in EGCG-treated VSMCs, whereas the effect of EGF was not affected. Quantification of the immunoprecipitated tyrosine-phosphorylated PDGF-Rbeta, phosphatidylinositol 3'-kinase, and phospholipase C-gamma1 by the enhanced Western blotting method revealed that EGCG treatment effectively inhibits tyrosine phosphorylation of these kinases in VSMCs. Furthermore, we show that spheroid formation of human glioblastoma cells (A172) and colony formation of sis-transfected NIH 3T3 cells in semisolid agar are completely inhibited by 20-50 microM EGCG. Our findings demonstrate that EGCG is a selective inhibitor of the tyrosine phosphorylation of PDGF-Rbeta and its downstream signaling pathway. The present findings may partly explain the anti-cancer and anti-atherosclerotic activity of green tea. PMID:10198059

Ahn, H Y; Hadizadeh, K R; Seul, C; Yun, Y P; Vetter, H; Sachinidis, A



Regulation of Insulin Receptor Substrate 1 (IRS-1)/AKT Kinase-mediated Insulin Signaling by O-Linked ?-N-Acetylglucosamine in 3T3-L1 Adipocytes*  

PubMed Central

Increased O-linked ?-N-acetylglucosamine (O-GlcNAc) is associated with insulin resistance in muscle and adipocytes. Upon insulin treatment of insulin-responsive adipocytes, O-GlcNAcylation of several proteins is increased. Key insulin signaling proteins, including IRS-1, IRS-2, and PDK1, are substrates for OGT, suggesting potential O-GlcNAc control points within the pathway. To elucidate the roles of O-GlcNAc in dampening insulin signaling (Vosseller, K., Wells, L., Lane, M. D., and Hart, G. W. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 5313–5318), we focused on the pathway upstream of AKT. Increasing O-GlcNAc in 3T3-L1 adipocytes decreases phosphoinositide 3-kinase (PI3K) interactions with both IRS-1 and IRS-2. Elevated O-GlcNAc also reduces phosphorylation of the PI3K p85 binding motifs (YXXM) of IRS-1 and results in a concomitant reduction in tyrosine phosphorylation of Y608XXM in IRS-1, one of the two main PI3K p85 binding motifs. Additionally, insulin signaling stimulates the interaction of OGT with PDK1. We conclude that one of the steps at which O-GlcNAc contributes to insulin resistance is by inhibiting phosphorylation at the Y608XXM PI3K p85 binding motif in IRS-1 and possibly at PDK1 as well.

Whelan, Stephen A.; Dias, Wagner B.; Thiruneelakantapillai, Lakshmanan; Lane, M. Daniel; Hart, Gerald W.



PDGF-induces the glutathione-dependent enzyme PGH2/PGE2 isomerase in NIH3T3 and pEJ transformed fibroblasts.  


Exposure of NIH3T3 and pEJ serum-starved cells to platelet derived growth factor results in a 16 fold increase in the glutathione-dependent enzyme prostaglandin H2/prostaglandin E2 isomerase activity (EC The response is rapid as a detectable increase in NIH3T3 cells occurs after only 7 minutes of exposure to the growth factor. Only a mild increase in another microsomal glutathione-dependent enzyme, microsomal glutathione transferase (EC, was detected after a 2 hour exposure to the growth factor. PMID:8292033

Kelner, M J; Uglik, S F



Cinnamon extract and polyphenols affect the expression of tristetraprolin, insulin receptor, and glucose transporter 4 in mouse 3T3-L1 adipocytes  

Microsoft Academic Search

Cinnamon improves glucose and lipid profiles of people with type 2 diabetes. Water-soluble cinnamon extract (CE) and HPLC-purified cinnamon polyphenols (CP) with doubly linked procyanidin type-A polymers display insulin-like activity. The objective of this study was to investigate the effects of cinnamon on the protein and mRNA levels of insulin receptor (IR), glucose transporter 4 (GLUT4), and tristetraprolin (TTP\\/ZFP36) in

Heping Cao; Marilyn M. Polansky; Richard A. Anderson



Sodium Butyrate Induces NIH3T3 Cells to Senescence-like State and Enhances Promoter Activity of p21 WAF\\/CIP1in p53Independent Manner  

Microsoft Academic Search

Sodium butyrate, a histone deacetylase inhibitor, has been shown to induce differentiation of many cancer cells and senescence-like state of human fibroblasts. Here we show that sodium butyrate also induces senescence-like state of NIH3T3 cells. The treated cells were blocked at G1 phase and featured morphologically like senescent cells with enlarged cytoplasm and multiple nuclei. The expression of p21WAF\\/CIP1(p21) increased

Hengyi Xiao; Tadao Hasegawa; Osamu Miyaishi; Kozo Ohkusu; Ken-ichi Isobe



Inhibition of Calcium-Independent Phospholipases A2beta or A2gamma Inhibit Hormone-Induced Differentiation of 3T3-L1 Preadipocytes.  

National Technical Information Service (NTIS)

A method for identifying an agonist exhibiting molecular or pharmacologic inhibition which is effective against the activity of at least one of iPLA(sub 2)beta and iPLA(sub 2)gamma which comprises culturing 3T3-L1 cells and transfecting them with negative...

R. W. Gross



NIH3T3 cells overexpressing CD98 heavy chain resist early G1 arrest and apoptosis induced by serum starvation.  


CD98 is a heterodimeric glycoprotein of 125-kDa, which consists of a 90-kDa heavy chain (hc) subunit and 35-kDa to 55-kDa light chain (lc) subunits. It is strongly expressed on the surface of proliferating normal cells and almost all tumor cells. To investigate the participation of CD98 in cellular proliferation and malignant transformation, we analyzed cell-cycle progression of NIH3T3 clones transfected with cDNA of human CD98hc. Although NIH3T3 and control transfectant cells grown to the subconfluent state were arrested in the G0/G1 phase by serum starvation, considerable portions of CD98hc-transfected cells resided at S and G2/M phases. Under serum-starved and confluent conditions, significant fractions (20-25%) of NIH3T3 and control transfectant cells contained less than 2n content DNA, indicating occurrence of apoptosis, whereas no apoptotic cells were detected in CD98hc-transfectant cells. Under serum-starved conditions, a marked increase in the levels of cyclin D1 and cyclin E and a decrease in p16 were observed in CD98hc-transfectant cells. The reverse was true for NIH3T3 and control transfectant cells. Our results suggest that resistance to G1 arrest and apoptosis by CD98 overexpression are associated with high G1-cyclins and low p16 levels. PMID:22497681

Hara, Kaori; Ueda, Shiho; Ohno, Yoshiya; Tanaka, Toshiyuki; Yagi, Hideki; Okazaki, Shogo; Kawahara, Rieko; Masayuki, Takechi; Enomoto, Takemi; Hashimoto, Yoshiyuki; Masuko, Kazue; Masuko, Takashi



Genistein directly inhibits GLUT4-mediated glucose uptake in 3T3-L1 adipocytes  

Microsoft Academic Search

The isoflavone-derivative genistein is commonly applied as an inhibitor of tyrosine kinases. In this report we analyze the effect of genistein on insulin-stimulated glucose uptake in 3T3-L1 adipocytes. In these cells insulin-induced glucose uptake is primarily mediated by the GLUT4 glucose transporter. We observed that pre-treatment with genistein did not affect insulin-induced tyrosine kinase activity of the insulin receptor or

Merlijn Bazuine; Peter J. A. van den Broek; J. Antonie Maassen



Effect of quinupristin\\/dalfopristin on 3T3 and Eahy926 cells in vitro in comparison to other antimicrobial agents with the potential to induce infusion phlebitis  

Microsoft Academic Search

Infusion phlebitis is a common clinical problem that is observed with some antimicrobial agents, when being administered intravenously.\\u000a In this study, cultured murine fibroblasts and immortalised human endothelial cells were exposed to three antibiotics at clinically\\u000a relevant concentrations to assess their toxic potential in two established cytotoxicity assays. BALB\\/c 3T3 fibroblasts and\\u000a Eahy926 endothelial cells were exposed to quinupristin\\/dalfopristin (QD),

Matthias Kruse; Bülent Kilic; Burkhard Flick; Ralf Stahlmann



Cell volume regulation and signaling in 3T3-L1 pre-adipocytes and adipocytes: on the possible roles of caveolae, insulin receptors, FAK and ERK1/2.  


Caveolae have been implicated in sensing of cell volume perturbations, yet evidence is still limited and findings contradictory. Here, we investigated the possible role of caveolae in cell volume regulation and volume sensitive signaling in an adipocyte system with high (3T3-L1 adipocytes); intermediate (3T3-L1 pre-adipocytes); and low (cholesterol-depleted 3T3-L1 pre-adipocytes) caveolae levels. Using large-angle light scattering, we show that compared to pre-adipocytes, differentiated adipocytes exhibit several-fold increased rates of volume restoration following osmotic cell swelling (RVD) and osmotic cell shrinkage (RVI), accompanied by increased swelling-activated taurine efflux. However, caveolin-1 distribution was not detectably altered after osmotic swelling or shrinkage, and caveolae integrity, as studied by cholesterol depletion or expression of dominant negative Cav-1, was not required for either RVD or RVI in pre-adipocytes. The insulin receptor (InsR) localizes to caveolae and its expression dramatically increases upon adipocyte differentiation. In pre-adipocytes, InsR and its effectors focal adhesion kinase (FAK) and extracellular signal regulated kinase (ERK1/2) localized to focal adhesions and were activated by a 5 min exposure to insulin (100 nM). Osmotic shrinkage transiently inhibited InsR Y(146)-phosphorylation, followed by an increase at t=15 min; a similar pattern was seen for ERK1/2 and FAK, in a manner unaffected by cholesterol depletion. In contrast, cell swelling had no detectable effect on InsR, yet increased ERK1/2 phosphorylation. In conclusion, differentiated 3T3-L1 adipocytes exhibit greatly accelerated RVD and RVI responses and increased swelling-activated taurine efflux compared to pre-adipocytes. Furthermore, in pre-adipocytes, Cav-1/caveolae integrity is not required for volume regulation. Given the relationship between hyperosmotic stress and insulin signaling, the finding that cell volume regulation is dramatically altered upon adipocyte differentiation may be relevant for the understanding of insulin resistance and metabolic syndrome. PMID:22179011

Eduardsen, Kathrine; Larsen, Susanne L; Novak, Ivana; Lambert, Ian H; Hoffmann, Else K; Pedersen, Stine F



Insulin and IGF-1 Induce Different Patterns of Gene Expression in Mouse Fibroblast NIH-3T3 Cells: Identification by cDNA Microarray Analysis  

Microsoft Academic Search

The IGF-1 receptor and the related insulin receptor are sim- ilar in structure and activate many of the same postreceptor signaling pathways, yet they mediate distinct biological func- tions. It is still not understood how the specificity of insulin vs. IGF-1 signaling is controlled. In this study, we have used cDNA microarrays to monitor the gene expression patterns that are




Amaranth lunasin-like peptide internalizes into the cell nucleus and inhibits chemical carcinogen-induced transformation of NIH-3T3 cells.  


Because an unbalanced diet is an important risk factor for several illnesses, interest has increased in finding novel health-promoting foods. Amaranth produces seeds that not only have substantial nutritional properties but that also contain phytochemical compounds as rutin and nicotiflorin and peptides with antihypertensive and anticarcinogenic activities. We report that a cancer-preventive peptide in amaranth has activities similar to those of soybean lunasin. The amaranth lunasin-like peptide, however, requires less time than the soybean lunasin to internalize into the nucleus of NIH-3T3 cells, and inhibits histone acetylation (H(3) and H(4) in a 70 and 77%, respectively). The amaranth lunasin-like peptide inhibited the transformation of NIH-3T3 cells to cancerous foci. The open reading frame (ORF) of amaranth lunasin corresponds to a bifunctional inhibitor/lipid-transfer protein (LTP). LTPs are a family of proteins that in plants are implicated in different functions, albeit all linked to developmental processes and biotic and abiotic stress resistance. Our results open new intriguing questions about the function of lunasin in plants and support that amaranth is a food alternative containing natural peptides with health-promoting benefits. PMID:20599579

Maldonado-Cervantes, Enrique; Jeong, Hyung Jin; León-Galván, Fabiola; Barrera-Pacheco, Alberto; De León-Rodríguez, Antonio; González de Mejia, Elvira; de Lumen, Ben O; Barba de la Rosa, Ana P



Morphological transformation induced by multiwall carbon nanotubes on Balb/3T3 cell model as an in vitro end point of carcinogenic potential.  


In this work we investigated the toxicological effects of nude and chemically functionalised (-NH(2), -OH and -COOH groups) multiwall carbon nanotubes (mwCNTs) using immortalised mouse fibroblasts cell line (Balb/3T3) as in vitro model, alternative to the use of animals, to assess basal cytotoxicity, carcinogenic potential, genotoxicity and cell interaction of nanomaterials (NM). Combining in vitro tests such as cell transformation assay and micronucleus with physicochemical and topological analysis, we obtained results showing no cytotoxicity and genotoxicity. Carcinogenic potential and mwCNTs interaction with cells were instead evident. We stressed the importance that different toxicological end points have to be considered when studying NM, therefore, assays able to detect long-term effects, such as carcinogenicity, must be taken into account together with a panel of tests able to detect more immediate effects like basal cytotoxicity or genotoxicity. PMID:22279961

Ponti, Jessica; Broggi, Francesca; Mariani, Valentina; De Marzi, Laura; Colognato, Renato; Marmorato, Patrick; Gioria, Sabrina; Gilliland, Douglas; Pascual Garcěa, César; Meschini, Stefania; Stringaro, Annarita; Molinari, Agnese; Rauscher, Hubert; Rossi, François



Peroxisome Proliferator-Activated Receptor Gamma/Signal Transducers and Activators of Transcription 5A Pathway Plays a Key Factor in Adipogenesis of Human Bone Marrow-Derived Stromal Cells and 3T3-L1 Preadipocytes  

PubMed Central

Adipogenesis is largely dependent on the signal transducers and activators of transcription (STAT) pathway. However, the molecular mechanism of the STAT pathway in the adipogenesis of human bone marrow-derived stromal cells (hBMSCs) remains not well understood. The purpose of this research was to characterize the transcriptional regulation involved in expression of STAT5A and STAT5B during adipogenesis in hBMSCs and 3T3-L1 cells. The expression of STAT5A and STAT5B increases with the onset of adipogenesis in hBMSCs and 3T3-L1 cells. The PPAR response elements regulatory element of STAT5A exists at a promoter region ranging from ?346 to ?101, and the CCAAT/enhancer-binding protein (C/EBP) regulatory element is located at ?196 to ?118 of the STAT5B promoter. C/EBP? and C/EBP? bound to the STAT5B promoter region, whereas peroxisome proliferator-activated receptor ? (PPAR?) bound to STAT5A. RNA interference of STAT5A completely blocked differentiation, whereas the inhibition of STAT5B only partially blocked differentiation. We propose that C/EBP?, C/EBP?, and PPAR? control adipogenesis by regulating STAT5B and STAT5A and that STAT5A is necessary, whereas STAT5B plays a supplementary role during adipogenesis. Further, the regulation of PPAR?-STAT5 by C/EBP? signaling seems to be the crucial adipogenesis pathway-initiating cascade of the various adipogenic genes.

Jung, Ho Sun; Lee, Yoo Jeong; Kim, Yun Hee; Paik, Seungil; Kim, Jae Woo



Ribonuclease III Cleavage of Bacteriophage T3 RNA Polymerase Transcripts to Late T3 mRNAs  

Microsoft Academic Search

In vitro transcription of T3 DNA by T3 phage-induced RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase; EC yields eight discrete RNAs (designated I-VIII) with molecular weights of approximately 6.2, 4.7, 4, 2.8, 1.8, 0.9, 0.52, and 0.21 × 106, respectively. Comparison of the size of in vitro T3 RNA polymerase transcripts with in vivo late T3 mRNAs indicates that several late RNAs

Hemanta K. Majumder; Subal Bishayee; Prasanta R. Chakraborty; Umadas Maitra



Liganded Thyroid Hormone Receptor Induces Nucleosome Removal and Histone Modifications to Activate Transcription during Larval Intestinal Cell Death and Adult Stem Cell Development  

PubMed Central

Thyroid hormone (T3) plays an important role in regulating multiple cellular and metabolic processes, including cell proliferation, cell death, and energy metabolism, in vertebrates. Dysregulation of T3 signaling results in developmental abnormalities, metabolic defects, and even cancer. We used T3-dependent Xenopus metamorphosis as a model to study how T3 regulates transcription during vertebrate development. T3 exerts its metamorphic effects through T3 receptors (TR). TR recruits, in a T3-dependent manner, cofactor complexes that can carry out chromatin remodeling/histone modifications. Whether and how histone modifications change upon gene regulation by TR during vertebrate development is largely unknown. Here we analyzed histone modifications at T3 target genes during intestinal metamorphosis, a process that involves essentially total apoptotic degeneration of the simple larval epithelium and de novo development of the adult epithelial stem cells, followed by their proliferation and differentiation into the complex adult epithelium. We demonstrated for the first time in vivo during vertebrate development that TR induces the removal of core histones at the promoter region and the recruitment of RNA polymerase. Furthermore, a number of histone activation and repression marks have been defined based on correlations with mRNA levels in cell cultures. Most but not all correlate with gene expression induced by liganded TR during development, suggesting that tissue and developmental context influences the roles of histone modifications in gene regulation. Our findings provide important mechanistic insights on how chromatin remodeling affects developmental gene regulation in vivo.

Matsuura, Kazuo; Fujimoto, Kenta; Fu, Liezhen



Telmisartan-induced inhibition of vascular cell proliferation beyond angiotensin receptor blockade and peroxisome proliferator-activated receptor-gamma activation.  


We investigated the ability of angiotensin II type 1 (AT1) receptor blockers with peroxisome proliferator-activated receptor (PPAR)-gamma agonist activity (telmisartan and irbesartan) and AT1 receptor blockers devoid of PPARgamma agonist activity (eprosartan and valsartan) to inhibit vascular cell proliferation studied in the absence of angiotensin II stimulation. Telmisartan and, to a lesser extent, irbesartan inhibited proliferation of human aortic vascular smooth muscle cells in a dose-dependent fashion, whereas eprosartan and valsartan did not. To investigate the role of PPARgamma in the antiproliferative effects of telmisartan, we studied genetically engineered NIH3T3 cells that express PPARgamma. Pioglitazone inhibited proliferation of NIH3T3 cells expressing PPARgamma but had little effect on control NIH3T3 cells that lack PPARgamma. In contrast, telmisartan inhibited proliferation equally in NIH3T3 with and without PPARgamma. Valsartan failed to inhibit proliferation of either cell line. In addition, telmisartan inhibited proliferation equally in aortic smooth muscle cells derived from mice with targeted knockout of PPARgamma in the smooth muscle and from control mice, whereas valsartan had no effect on cell proliferation. Telmisartan, but not valsartan, reduced phosphorylation of AKT but not extracellular signal-regulated kinase otherwise induced by exposure to serum of quiescent human smooth muscle cells, quiescent mice smooth muscle cells lacking PPARgamma, or quiescent Chinese hamster ovary-K1 cells lacking the AT1 receptor. In summary, the antiproliferative effects of telmisartan in the absence of exogenously supplemented angiotensin II involve more than just AT1 receptor blockade and do not require activation of PPARgamma. It might be postulated that inhibition of AKT activation is a mechanism mediating the antiproliferative effects of telmisartan, including in cells lacking AT1 receptors or PPARgamma. PMID:19822796

Yamamoto, Koichi; Ohishi, Mitsuru; Ho, Christopher; Kurtz, Theodore W; Rakugi, Hiromi



Heat shock protein 90 suppresses tumor necrosis factor alpha induced apoptosis by preventing the cleavage of Bid in NIH3T3 fibroblasts  

Microsoft Academic Search

Two highly conserved mechanisms for maintaining cellular homeostasis are apoptosis and the cellular stress response. Hsp90 is one of the most abundant, highly conserved, and inducible Hsps in eukaryotes. Recently, Hsp90 has been shown to play important antiapoptotic roles through binding with Apaf-1, RIP and kinase domain of IKK?\\/?. Our present studies demonstrate that Hsp90 can suppress tumor necrosis factor

Chen Zhao; Enhua Wang



2-Methoxy-4-vinylphenol can induce cell cycle arrest by blocking the hyper-phosphorylation of retinoblastoma protein in benzo[a]pyrene-treated NIH3T3 cells.  


Benzo[a]pyrene (BaP) is an environment carcinogen that can enhance cell proliferation by disturbing the signal transduction pathways in cell cycle regulation. In this study, the effects of 2M4VP on cell proliferation, cell cycle and cell cycle regulatory proteins were studied in BaP-treated NIH 3T3 cells to establish the molecular mechanisms of 2M4VP as anti-proliferative agents. 2M4VP exerted a dose-dependent inhibitory effect on cell growth correlated with a G1 arrest. Analysis of G1 cell cycle regulators expression revealed 2M4VP increased expression of CDK inhibitor, p21Waf1/Cip1 and p15 INK4b, decreased expression of cyclin D1 and cyclin E, and inhibited kinase activities of CDK4 and CDK2. However, 2M4VP did not affect the expression of CDK4 and CDK2. Also, 2M4VP inhibited the hyper-phosphorylation of Rb induced by BaP. Our results suggest that 2M4VP induce growth arrest of BaP-treated NIH 3T3 cells by blocking the hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins. PMID:20816752

Jeong, Jin Boo; Jeong, Hyung Jin



Sucrose Ingestion Induces Rapid AMPA Receptor Trafficking  

PubMed Central

The mechanisms by which natural rewards such as sugar affect synaptic transmission and behavior are largely unexplored. Here, we investigate regulation of nucleus accumbens synapses by sucrose intake. Previous studies have shown that AMPA receptor trafficking is a major mechanism for regulating synaptic strength, and that in vitro, trafficking of AMPA receptors containing the GluA1 subunit takes place by a two-step mechanism involving extrasynaptic and then synaptic receptor transport. We report that in rat, repeated daily ingestion of a 25% sucrose solution transiently elevated spontaneous locomotion and potentiated accumbens core synapses through incorporation of Ca2+-permeable AMPA receptors (CPARs), which are GluA1-containing, GluA2-lacking AMPA receptors. Electrophysiological, biochemical and quantitative electron microscopy studies revealed that sucrose training (7 days) induced a stable (>24 hr) intraspinous GluA1 population, and that in these rats a single sucrose stimulus rapidly (5 min) but transiently (<24 hr) elevated GluA1 at extrasynaptic sites. CPARs and dopamine D1 receptors were required in vivo for elevated locomotion after sucrose ingestion. Significantly, a 7-day protocol of daily ingestion of a 3% solution of saccharin, a non-caloric sweetener, induced synaptic GluA1 similarly to 25% sucrose ingestion. These findings identify multi-step GluA1 trafficking, previously described in vitro, as a mechanism for acute regulation of synaptic transmission in vivo by a natural orosensory reward. Trafficking is stimulated by a chemosensory pathway that is not dependent on the caloric value of sucrose.

Tukey, David S.; Ferreira, Jainne M.; Antoine, Shannon O.; D'amour, James A.; Ninan, Ipe; de Vaca, Soledad Cabeza; Incontro, Salvatore; Wincott, Charlotte; Horwitz, Julian K.; Hartner, Diana T.; Guarini, Carlo B.; Khatri, Latika; Goffer, Yossef; Xu, Duo; Titcombe, Roseann F.; Khatri, Megna; Marzan, Dave S.; Mahajan, Shahana S.; Wang, Jing; Froemke, Robert C.; Carr, Kenneth D.; Aoki, Chiye; Ziff, Edward B.



Homologous Up-Regulation of the Gonadotropin-Releasing Hormone Receptor in alphaT3-1 Cells Is Associated with Unchanged Receptor Messenger RNA (mRNA) Levels and Altered mRNA Activity.  

National Technical Information Service (NTIS)

The role of altered biosynthesis was investigated in the mouse gonadotrope cell line, by studying the homologous up-regulation of GnRH receptor binding, mRNA levels, and mRNA activity. After GnRH exposure, ligand binding was performed on cell membranes. T...

M. Tsutsumi S. C. Laws S. C. Sealfon




EPA Science Inventory

Depending on the concentration and duration of agonist exposure, gonadotropin-releasing hormone (GnRH) receptor number either increases or decreases in response to GnRH. he molecular basis of this regulation could involve a combination of modulation of gene transcription, RNA pro...


Differential role for ERK2 in anoxia-induced activation of transcription and translation of Hsp70 in NIH 3T3 cells.  


Hsp70 has the ability to enhance the recovery of stressed cells by its ability to catalyze the reassembly of damaged proteins. Such a chaperoning function is essential for the Hsp70-mediated protection against anoxic stress that causes protein denaturation. We have studied induction of both transcription and translation of Hsp70 during recovery from chemical anoxia and the role of the extracellular signal regulated kinase ERK2 in this induction of Hsp70. 10 mM azide for 30 minutes (chemical anoxia) significantly inhibited the activity of ERK2 (measured as phospho-ERK) but the ERK-2 activity is rapidly increased in a MEK-independen manner, when azide is washed out of the cells. Chemical anoxia and overnight recovery induced Hsp70 expression (analyzed by Western blotting) and this was inhibited by actinomycin D as well as by cycloheximide showing that induction of both translation and transcription was involved. Inhibition of the MAP kinase p38, which was transiently activated during chemical anoxia, had no effect on the increase in Hsp70 expression whereas an inhibitor of reactive oxygen species and inhibition of the phosphatase PP1 and PP2a inhibited the increase in Hsp70 expression. Inhibition of ERK2 by the MEK inhibitor PD98059 resulted in strong inhibition of Hsp70 protein expression and simultaneous stimulation of hsp70 transcription. PMID:21325828

Ossum, Carlo G; Lauritsen, Anders N; Karottki, Dorina Gabriela; Hoffmann, Else K



Thyroid hormone inhibits ERK phosphorylation in pressure overload-induced hypertrophied mouse hearts through a receptor-mediated mechanism  

PubMed Central

Pressure overload-induced cardiac hypertrophy results in a pathological type of hypertrophy with activation of signaling cascades like the extracellular signal-regulated kinase (ERK) pathway, which promotes negative cardiac remodeling and decreased contractile function. In contrast, thyroid hormone mediates a physiological type of hypertrophy resulting in enhanced contractile function. In addition, thyroid hormone action is diminished in pressure overload-induced cardiac hypertrophy. We hypothesized that thyroid hormone status modulates ERK activity and that administration of thyroid hormone could alter the activity of this kinase in cardiac hypertrophy induced by pressure overload. ERK is activated by phosphorylation; accordingly, we investigated phosphorylation of ERK in hearts of control, hypothyroid, and hyperthyroid mice. In addition, the effect of T3 treatment on ERK phosphorylation in hypertrophied hearts from transverse aortic-constricted (TAC) mice was investigated. Results showed that phosphorylated ERK (p-ERK) was decreased by 25% in hyperthyroid mice. In contrast, hypothyroid mice presented increased p-ERK by 80%. TAC mice presented a greater than fourfold increase of p-ERK compared with control mice. Interestingly, T3 administration dramatically canceled TAC-induced ERK phosphorylation (36% lower compared with control). Raf-1 is upstream of the ERK pathway. TAC mice presented a 45% increase in phospho-Raf-1 (Ser338). T3 treatment inhibited this effect of pressure overload and further decreased p-Raf-1 (Ser338) by 37%, compared with control. Overexpression of thyroid hormone receptor-? in cultured cardiomyocytes potentiated the inhibitory effect of T3 on ERK phosphorylation. We concluded that thyroid hormone has an inhibitory effect on the Raf-1/ERK pathway. Furthermore, treatment of TAC mice with T3 inhibited Raf-1/ERK pathway by a thyroid hormone receptor-dependent mechanism.

Suarez, Jorge; Scott, Brian T.; Suarez-Ramirez, Jorge A.; Chavira, Citlalic V.



Close relationship between modulation of serum-induced stimulation of DNA synthesis and changes in gap-junctional intercellular communication in quiescent 3T3-L1 cells caused by cyclic AMP and the tumor-promoting phorbol ester TPA  

SciTech Connect

Involvement of gap-junctional intercellular communication in the stimulation of growth was investigated in quiescent 3T3-L1 cells. When the cells in monolayer were growth-arrested by culture in a low concentration of calf serum, addition of dibutyryl cyclic AMP enhanced dye-coupling and suppressed the enhancement of DNA synthesis, induced by calf serum, in quiescent cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA) suppressed dye-coupling in quiescent cells and enhanced DNA synthesis in both quiescent and serum-treated cells. When about 5000 cells were cultured in contact to form a colony, growth arrest of the cells was observed in the central region of such colonies rather than in the peripheral region, but addition of calf serum induced DNA synthesis in the cells in both the peripheral and central regions of the colonies. Addition of TPA enhanced serum-induced DNA synthesis in the cells in the central region of colonies rather than in the peripheral region. These results suggest that the ability of quiescent cells to escape from growth arrest is inversely correlated to the extent of gap-junctional intercellular communication.

Shiba, Yoshiki; Sasaki, Yasuto; Hirono, Chikara; Kanno, Yoshinobu (Hiroshima Univ. School of Dentistry (Japan))



Gas6-mediated survival in NIH3T3 cells activates stress signalling cascade and is independent of Ras  

Microsoft Academic Search

Gas6 is a growth factor membrane of the vitamin K-dependent family of proteins which is preferentially expressed in quiescent cells. Gas6 was identified as the ligand for Axl tyrosine kinase receptor family. Consistent with this, Gas6 was previously reported to induce cell cycle re-entry of serum-starved NIH3T3 cells and to prevent cell death after complete growth factor withdrawal, the survival

Sandro Goruppi; Elisabetta Ruaro; Brian Varnum; Claudio Schneider



Protective Effect of Phosphatidylinositol 4,5-Bisphosphate against Cortical Filamentous Actin Loss and Insulin Resistance Induced by Sustained Exposure of 3T3-L1 Adipocytes to Insulin*  

PubMed Central

Muscle and fat cells develop insulin resistance when cultured under hyperinsulinemic conditions for sustained periods. Recent data indicate that early insulin signaling defects do not fully account for the loss of insulin action. Given that cortical filamentous actin (F-actin) represents an essential aspect of insulin regulated glucose transport, we tested to see whether cortical F-actin structure was compromised during chronic insulin treatment. The acute effect of insulin on GLUT4 translocation and glucose uptake was diminished in 3T3-L1 adipocytes exposed to a physiological level of insulin (5 nM) for 12 h. This insulin-induced loss of insulin responsiveness was apparent under both low (5.5 mM) and high (25 mM) glucose concentrations. Microscopic and biochemical analyses revealed that the hyperinsulinemic state caused a marked loss of cortical F-actin. Since recent data link phosphatidylinositol 4,5-bisphosphate (PIP2) to actin cytoskeletal mechanics, we tested to see whether the insulin-resistant condition affected PIP2 and found a noticeable loss of this lipid from the plasma membrane. Using a PIP2 delivery system, we replenished plasma membrane PIP2 in cells following the sustained insulin treatment and observed a restoration in cortical F-actin and insulin responsiveness. These data reveal a novel molecular aspect of insulin-induced insulin resistance involving defects in PIP2/actin regulation.

Chen, Guoli; Raman, Priya; Bhonagiri, Padma; Strawbridge, Andrew B.; Pattar, Guruprasad R.; Elmendorf, Jeffrey S.



Mutations of CpG dinucleotides located in the triiodothyronine (T3)-binding domain of the thyroid hormone receptor (TR) beta gene that appears to be devoid of natural mutations may not be detected because they are unlikely to produce the clinical phenotype of resistance to thyroid hormone.  

PubMed Central

Thyroid hormone receptor (TR) beta gene mutations identified in patients with resistance to thyroid hormone (RTH) revealed two clusters ("hot" areas) of mutations (RTHmut) in the triiodothyronine (T3)-binding domain. Furthermore, 45% of RTHmuts and 90% of recurring mutations are located in CpG dinucleotides ("hot spots"). To investigate why the region between the two hot areas lacks RTHmuts, we produced 10 artificial mutant TR beta s (ARTmut) in this "cold" region according to the hot spot rule (C-->T or G-->A substitutions in CpGs). The properties of ARTmuts were compared with those of six RTHmuts. Among all RTHmuts, R320H manifesting a mild form of RTH showed the least impairment of T3-binding affinity (Ka). In contrast, Ka was normal in six ARTmuts (group A), reduced to a lesser extent than R320H in three (group B), and one that was truncated (R410X) did not bind T3. All RTHmuts had impaired ability to transactivate T3-responsive elements and exhibited a strong dominant negative effect on cotransfected wild-type TR beta. Group B and A ARTmuts had minimally impaired or normal transactivation and weak or no dominant negative effect, respectively. R410X showed neither transactivation nor dominant negative effect. Natural mutations expected to occur in the cold region of TR beta should fail to manifest as RTH (group A) or should escape detection (group B) since the serum thyroid hormone levels required to compensate for the reduced binding affinity should be inferior to those found in subjects with R320H. R410X would manifest RTH only in the homozygote state. The cold region of the putative T3-binding domain is relatively insensitive to amino acid changes and, thus, may not be involved in a direct interaction with T3. Images

Hayashi, Y; Sunthornthepvarakul, T; Refetoff, S



Soluble Receptor-Induced Retroviral Infection of Receptor-Deficient Cells  

Microsoft Academic Search

Current models of retroviral entry hypothesize that interactions between the host cell receptor(s) and viral envelope protein induce structural changes in the envelope protein that convert it to an active conformation, allowing it to mediate fusion with the membrane. Recent evidence supporting this hypothesis is the demon- stration that Tva, the receptor for subgroup A avian sarcoma and leukosis virus




Mediator subunit MED1 is a T3-dependent and T3-independent coactivator on the thyrotropin ? gene promoter.  


The MED1 subunit of the Mediator transcriptional coregulator complex is a nuclear receptor-specific coactivator. A negative feedback mechanism of thyroid-stimulating hormone (TSH, or thyrotropin) expression in the thyrotroph in the presence of triiodothyronine (T3) is employed by liganded thyroid hormone receptor ? (TR?) on the TSH? gene promoter, where conventional histone-modifying coactivators act as corepressors. We now provide evidence that MED1 is a ligand-dependent positive cofactor on this promoter. TSH? gene transcription was attenuated in MED1 mutant mice in which the nuclear receptor-binding ability of MED1 was specifically disrupted. MED1 stimulated GATA2- and Pit1-mediated TSH? gene promoter activity in a ligand-independent manner in cultured cells. MED1 also stimulated transcription from the TSH? gene promoter in a T3-dependent manner. The transcription was further enhanced when the T3-dependent corepressors SRC1, SRC2, and HDAC2 were downregulated. Hence, MED1 is a T3-dependent and -independent coactivator on the TSH? gene promoter. PMID:24055033

Matsui, Keiji; Oda, Kasumi; Mizuta, Shumpei; Ishino, Ruri; Urahama, Norinaga; Hasegawa, Natsumi; Roeder, Robert G; Ito, Mitsuhiro



Extract of Chaga mushroom (Inonotus obliquus) stimulates 3T3-L1 adipocyte differentiation.  


Chaga mushroom (Inonotus obliquus) has long been used as a folk medicine due to its numerous biological functions such as antibacterial, antiallergic, antiinflammatory and antioxidative activities. In the present study, it was found that the I. obliquus hot water extract (IOWE) activated adipogenesis of 3T3-L1 preadipocytes. Even in the absence of adipogenic stimuli by insulin, the IOWE strongly induced adipogenesis of 3T3-L1 preadipocytes. The major constituent of IOWE was glucose-rich polysaccharides with a molecular mass of 149? kDa. IOWE enhanced the differentiation of 3T3-L1 preadipocytes, increasing TG (triacylglycerol) accumulation that is critical for acquisition of the adipocyte phenotype, in a dose-dependent manner. IOWE stimulated gene expression of C/EBP? (CCAAT/enhancer-binding protein ?) and PPAR? (peroxisome proliferator-activated receptors ?) during adipocyte differentiation, and induced the expression of PPAR? target genes such as aP2 (adipocyte protein 2), LPL (lipoprotein lipase) and CD36 (fatty acid translocase). Immunoblot analysis revealed that IOWE increased the expression of adipogenic makers such as PPAR? and GLUT4 (glucose transporter 4). The luciferase reporter assay demonstrated that IOWE did not exhibit PPAR? ligand activity. Although these results require further investigation, the ability of natural mushroom product to increase PPAR? transcriptional activities may be expected to be therapeutic targets for dyslipidemia and type 2 diabetes. PMID:21031614

Joo, Jeong In; Kim, Dong Hyun; Yun, Jong Won



Resistin affects lipid metabolism during adipocyte maturation of 3T3-L1 cells.  


Resistin, an adipose-tissue-specific secretory factor, aggravates metabolic syndrome through impairment of glucose metabolism. Previously, we demonstrated that resistin expression was induced in both 3T3-L1 cells and primary pre-adipocytes derived from Zucker obese rats during the process of differentiation and maturation (Ikeda Y, Hama S, Kajimoto K, Okuno T, Tsuchiya H & Kogure K (2011) Biol Pharm Bull 34, 865-870). However, the biological function of resistin in adipocytes is poorly understood. In the present study, we examined the effects of resistin knockdown on the biological features of 3T3-L1 cells. We found that lipid content was significantly decreased in 3T3-L1 cells transfected with anti-resistin small interfering RNA (siRNA) after adipocyte differentiation. While expression of peroxisome proliferator activated receptor ? and CCAAT/enhancer-binding protein ? was not affected, protein expression and transcriptional activity levels of carbohydrate response element binding protein (ChREBP), which upregulates transcription of lipogenic genes, decreased after anti-resistin siRNA treatment. Moreover, gene expression of fatty acid synthase and acetyl-CoA carboxylase 2, which are known to be regulated by ChREBP, were also suppressed by resistin knockdown. In contrast, activity of the fatty acid ?-oxidation-regulating protein carnitine palmitoyltransferase 1 increased. These results suggest that resistin knockdown induces suppression of lipid production and activation of fatty acid ?-oxidation. Consequently, resistin may affect lipid metabolism during adipocyte maturation. PMID:24034627

Ikeda, Yoshito; Tsuchiya, Hiroyuki; Hama, Susumu; Kajimoto, Kazuaki; Kogure, Kentaro



The intracellular mechanism of alpha-fetoprotein promoting the proliferation of NIH 3T3 cells  

Microsoft Academic Search

AIM The existence and properties of alpha-fetoprotein (AFP) receptor on the surface of NIH 3T3 cells and the effects of AFP on cellular signal transduction pathway were investigated. METHODS The effect of AFP on the proliferation of NIH 3T3 cells was measured by incorporation of 3H-TdR. Receptor-binding assay of 125I-AFP was performed to detect the properties of AFP receptor in

Meng Sen LI; Ping Feng LI; Fei Yi YANG; Shi Peng HE; Guo Guang DU; Gang LI



Insulin receptor substrate-4 enhances insulin-like growth factor-I-induced cell proliferation.  


The insulin receptor substrates (IRSs)-1-4 play important roles in signal transduction emanating from the insulin and insulin-like growth factor (IGF)-I receptors. IRS-4 is the most recently characterized member, which has been found primarily in human cells and tissues. It interacts with SH2-containing proteins such as phosphatidylinositol 3'-kinase (PI3K), Grb2, Crk-II, and CrkL. In this study, we transfected IRS-4 in mouse NIH-3T3 cells that overexpress IGF-I receptors. Clones expressing IRS-4 showed enhanced cellular proliferation when cells were cultured in 1% fetal bovine serum without added IGF-I. Addition of IGF-I enhanced cellular proliferation in cells overexpressing the IGF-I receptor alone but had an even greater proliferative effect in cells overexpressing both the IGF-I receptors and IRS-4. When etoposide and methylmethane sulfonate (MMS), both DNA damaging agents, were added to the cells, they uniformly induced cell cycle arrest. Fluorescence-activated cell sorter analysis demonstrated that the arrest of the cell cycle occurred at the G(1) checkpoint, and furthermore no significant degree of apoptosis was demonstrated with the use of either agent. In cells, overexpressing IGF-I receptors alone, IGF-I addition enhanced cellular proliferation, even in the presence of etoposide and MMS. In cells overexpressing IGF-I receptors and IRS-4, the effect of IGF-I in overcoming the cell cycle arrest was even more pronounced. These results suggest that IRS-4 is implicated in the IGF-I receptor mitogenic signaling pathway. PMID:10531310

Qu, B H; Karas, M; Koval, A; LeRoith, D



Triiodothyronine (T3) supplementation maintains surfactant biochemical integrity during sepsis.  


Surfactant functional effectiveness is dependent on phospholipid compositional integrity: sepsis decreases this through an undefined mechanism. Sepsis-induced hypothyroidism is commensurate and may be related. This study examines the effect of triiodothyronine (T3) supplementation on surfactant function, metabolism, and composition during sepsis. Male Sprague-Dawley rats (n = 75) underwent sham laparotomy or cecal ligation and puncture (CLP) with or without T3 supplementation (CLP/T3; 3 ng/hr). Twenty-four hours later, surfactant was obtained by lavage. Total phospholipids were determined by chromatography. Choline phosphate cytidyltransferase (CT) activity was determined by the formation of cytidine diphosphate (CDP)-choline. In vivo lung compliance was determined by lung inflation; surfactant hysteresis plots were determined on a pulsating bubble surfactometer. Lung compliance and surfactant hysteresis plots were significantly affected by sepsis; T3 modulated this (dynamic compliance: sham = 0.66 +/- 0.02, CLP = 0.47 +/- 0.06, CLP/T3 = 0.56 +/- 0.02 mm Hg/mL; p < 0.05). Sepsis produced a decrease in phosphatidylglycerol, and phosphatidic acid, with an increase in lesser surface active lipids phosphatidylserine and phosphatidylinositol. Hormonal replacement prevented these alterations. Lung CT activity was increased by sepsis independent of T3 treatment. Thyroid hormone may have an active role in lung functional preservation during sepsis caused by maintenance of surfactant biophysical and compositional homeostasis. PMID:7636910

Dulchavsky, S A; Ksenzenko, S M; Saba, A A; Diebel, L N



Pharmacology of muscarinic acetylcholine receptor subtypes (m1–m5): high throughput assays in mammalian cells  

Microsoft Academic Search

Based on the ability of many receptors to amplify NIH 3T3 cells, we developed a high throughput assay of cloned receptor pharmacology. In this assay, receptors are transiently co-expressed with the marker enzyme ?-galactosidase. Receptors that induce cellular proliferation select and amplify the cells that also express the marker, thus the ability of ligands to alter receptor activity are reported

Hans Bräuner-Osborne; Mark R. Brann



Requirement of Phosphatidylinositol 3-Kinase-Dependent Pathway and Src for Gas6-Axl Mitogenic and Survival Activities in NIH 3T3 Fibroblasts  

Microsoft Academic Search

Gas6 is a secreted protein previously identified as the ligand of the Axl receptor tyrosine kinase. We have shown that Gas6 is able to induce cell cycle reentry of serum-starved NIH 3T3 cells and to efficiently prevent apoptosis after complete growth factor removal, a survival effect uncoupled from Gas6-induced mitogenesis. Here we report that the mitogenic effect of Gas6 requires




Regulation of CCAAT\\/Enhancer Binding Protein? (C\\/EBP?) Gene Expression by Thiazolidinediones in 3T3-L1 Adipocytes  

Microsoft Academic Search

Thiazolidinediones are a class of antidiabetic drugs that induce preadipocyte differentiation by binding and activating peroxisome proliferator-activated receptor ?2. Although thiazolidinediones are commonly thought of as insulin-sensitizing agents, these drugs have opposing and antagonistic effects to that of insulin on CCAAT\\/enhancer binding protein ? (C\\/EBP?) gene expression in fully differentiated 3T3-L1 adipocytes. Thiazolidinediones induce expression of C\\/EBP? mRNA and protein,

Nahid Hemati; Robin L. Erickson; Sarah E. Ross; Raymond Liu; Ormond A. MacDougald



A role of unliganded thyroid hormone receptor in postembryonic development in Xenopus laevis  

Microsoft Academic Search

A fascinating feature of thyroid hormone (T3) receptors (TR) is that they constitutively bind to promoter regions of T3-response genes, providing dual functions. In the presence of T3, TR activates T3-inducible genes, while unliganded TR represses these same genes. Although this dual function model is well demonstrated at the molecular level, few studies have addressed the presence or the role

Yukiyasu Sato; Daniel R. Buchholz; Bindu D. Paul; Yun-Bo Shi



Cytokines induce increased endothelin ET B receptor-mediated contraction  

Microsoft Academic Search

The effect of cytokines on the induction of contractile endothelin ETB receptors during organ culture was examined. Ring segments of rat superior mesenteric artery were used fresh or incubated for 24 h in Dulbecco's modified Eagle's medium alone, or with either interleukin-1?, tumor necrosis factor-? (TNF-?) or interleukin-2. In fresh arterial segments there was no endothelin ETB receptor-induced contraction. After

Erik Uddman; Sebastian Möller; Mikael Adner; Lars Edvinsson



Characterization of Ligand-Induced Endocytosis of EGF-Receptors.  

National Technical Information Service (NTIS)

Under the auspices of this training Fellowship (May 9, 1994-November 9, 1997), I have undertaken studies on the molecular mechanisms that govern ligand-induced endocytosis of epidermal growth factor receptors (EGFR) and the role of regulated endocytosis o...

S. L. Schmid



Involvement of glucocorticoid receptor on hyperpyrexia induced by methamphetamine administration.  


We have investigated the involvement of glucocorticoid on methamphetamine (MA) induced hyperpyrexia using a bio-telemetric system. A significant level of hyperpyrexia was observed in MA administered rats. In contrast, increase of body temperature was suppressed by adrenalectomy or by the administration of RU-486, an antagonist of the glucocorticoid receptor. These data suggest that the glucocorticoid receptor may be involved in hyperpyrexia induced by MA. Keywords: methamphetamine - hyperpyrexia - glucocorticoid - corticosterone. PMID:23121037

Yoshida, S; Kinoshita, H; Tatara, T; Tashiro, C; Nishiguchi, M; Ouchi, H; Minami, T; Hishida, S



T-3M Tokamak materials testing stand  

SciTech Connect

The T-3M stand, built on the basis of the T-3A widing, was simplified to the utmost in order to speed up its dismantling and assembly. The stand was started up in stages. The longitudinal magnetic field winding of the T-3M stand consists of 16 copper coils, connected in pairs into eight units. The plasma is excited inductively by an air-core inductor of a vortex electric field. The experimental complex of the T-3M stand incorporates the following component parts: a stand for high-power thermal action on materials of diaphragms; a stand of model experiments; devices for the study of plasma in the wall zone; and an IVK-2 information-computational complex based on an SM-4 computer.

Abagyan, A.A.; Alferov, A.A.; Babal'yants, V.F.; Baratov, D.G.; Dem'yanenko, V.N.; Leonov, S.B.



Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway.  


Ghrelin is a 28-amino-acid peptide that acts as a natural endogenous ligand of the growth hormone secretagogue receptor (GHSR) and strongly stimulates the release of growth hormone from the hypothalamus-pituitary axis. Previous studies have identified the important physiological effects of ghrelin on bone metabolism, such as regulating proliferation and differentiation of osteoblasts, independent of GH/IGF-1 axis. However, research on effects and mechanisms of ghrelin on osteoblast apoptosis is still rare. In this study, we identified expression of GHSR in MC3T3-E1 cells and determined the effects of ghrelin on the apoptosis of osteoblastic MC3T3-E1 cells and the mechanism involved. Our data demonstrated that ghrelin inhibited the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, as determined by terminal deoxynucleotidyl transferase-mediated deoxyribonucleotide triphosphate nick end-labeling (TUNEL) and ELISA assays. Moreover, ghrelin upregulated Bcl-2 expression and downregulated Bax expression in a dose-dependent manner. Our study also showed decreased activated caspase-3 activity under the treatment of ghrelin. Further study suggested that ghrelin stimulated the phosphorylation of ERK and AKT. Pretreatment of cells with the ERK inhibitor PD98059, PI3K inhibitor LY294002, and GHSR-siRNA blocked the ghrelin-induced activation of ERK and AKT, respectively; however, ghrelin did not stimulate the phosphorylation of p38 or JNK. PD90859, LY294002 and GHSR-siRNA attenuated the anti-apoptosis effect of ghrelin in MC3T3-E1 cells. In conclusion, ghrelin inhibits the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, which may be mediated by activating the GHSR/ERK and GHSR/PI3K/AKT signaling pathways. PMID:23921150

Liang, Qiu-Hua; Liu, Yuan; Wu, Shan-Shan; Cui, Rong-Rong; Yuan, Ling-Qing; Liao, Er-Yuan



Bombesin, vasopressin, and endothelin rapidly stimulate tyrosine phosphorylation in intact Swiss 3T3 cells  

SciTech Connect

The mitogenic neuropeptides bombesin and vasopressin markedly increased tyrosine and serine phosphorylation of multiple substrates in quiescent Swiss 3T3 fibroblasts, including two major bands of M{sub r} 90,000 and 115,000. Tyrosine phosphorylation of these proteins was increased as judged by immunoprecipitation of {sup 32}P{sub i}-labeled cells and immunoblotting of unlabeled cells with monoclonal antiphosphotyrosine antibodies, elution with phenyl phosphate, and phospho amino acid analysis. Phosphotyrosyl proteins generated by bombesin and vasopressin did not correspond either by apparent molecular weight or by immunological and biochemical criteria to several known tyrosine kinase substrates, including phospholipase C{sub {gamma}}, the microtubule-associated protein 2 kinase, GTPase-activating protein, or phosphatidylinositol kinase. The effect was rapid (within seconds), concentration dependent, and inhibited by specific receptor antagonists for both bombesin and vasopressin. The endothelin-related peptide, vasoactive intestinal contractor, also elicited a rapid and concentration-dependent tyrosine/serine phosphorylation of a similar set of substrates. These results demonstrate that neuropeptides, acting through receptors linked to GTP-binding proteins, stimulate tyrosine phosphorylation of a common set of substrates in quiescent Swiss 3T3 cells and suggest the existence of an additional signal transduction pathway in neuropeptide-induced mitogenesis.

Zachary, I.; Gil, J.; Lehmann, W.; Sinnett-Smith, J.; Rozengurt, E. (Imperial Cancer Research Fund, London (England))



A Phosphatidylinositol 3Kinase Docking Site in the Cytoplasmic Tail of the Jaagsiekte Sheep Retrovirus Transmembrane Protein Is Essential for Envelope-Induced Transformation of NIH 3T3 Cells  

Microsoft Academic Search

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a transmissible lung cancer of sheep known as ovine pulmonary carcinoma. Recently, we have found that the expression of the JSRV envelope (Env) is sufficient to transform mouse NIH 3T3 cells in classical transformation assays. To further investigate the mechanisms of JSRV oncogenesis, we generated a series of envelope chimeras between




Gsalpha signalling suppresses PPARgamma2 generation and inhibits 3T3L1 adipogenesis.  


Since TSH receptor (TSHR) expression increases during adipogenesis and signals via cAMP/phospho-cAMP-response element binding protein (CREB), reported to be necessary and sufficient for adipogenesis, we hypothesised that TSHR activation would induce preadipocyte differentiation. Retroviral vectors introduced constitutively active TSHR (TSHR*) into 3T3L1 preadipocytes; despite increased cAMP (RIA) and phospho-CREB (western blot) there was no spontaneous adipogenesis (assessed morphologically, using oil red O and QPCR measurement of adipogenesis markers). We speculated that Gbetagamma signalling may be inhibitory but failed to induce adipogenesis using activated Gsalpha (gsp*). Inhibition of phosphodiesterases did not promote adipogenesis in TSHR* or gsp* populations. Furthermore, differentiation induced by adipogenic medium with pioglitazone was reduced in TSHR* and abolished in gsp* expressing 3T3L1 cells. TSHR* and gsp* did not inactivate PPARgamma (PPARG as listed in the HUGO database) by phosphorylation but expression of PPARgamma1 was reduced and PPARgamma2 undetectable in gsp*. FOXO1 phosphorylation (required to inactivate this repressor of adipogenesis) was lowest in gsp* despite the activation of AKT by phosphorylation. PROF is a mediator that facilitates FOXO1 phosphorylation by phospho-Akt. Its transcript levels remained constantly low in the gsp* population. In most measurements, the TSHR* cells were between the gsp* and control 3T3L1 preadipocytes. The enhanced down-regulation of PREF1 (adipogenesis inhibitor) permits retention of some adipogenic potential in the TSHR* population. We conclude that Gsalpha signalling impedes FOXO1 phosphorylation and thus inhibits PPARgamma transcription and the alternative promoter usage required to generate PPARgamma2, the fat-specific transcription factor necessary for adipogenesis. PMID:19460852

Zhang, Lei; Paddon, Carol; Lewis, Mark D; Grennan-Jones, Fiona; Ludgate, Marian



Gs? signalling suppresses PPAR?2 generation and inhibits 3T3L1 adipogenesis  

PubMed Central

Since TSH receptor (TSHR) expression increases during adipogenesis and signals via cAMP/phospho-cAMP-response element binding protein (CREB), reported to be necessary and sufficient for adipogenesis, we hypothesised that TSHR activation would induce preadipocyte differentiation. Retroviral vectors introduced constitutively active TSHR (TSHR*) into 3T3L1 preadipocytes; despite increased cAMP (RIA) and phospho-CREB (western blot) there was no spontaneous adipogenesis (assessed morphologically, using oil red O and QPCR measurement of adipogenesis markers). We speculated that G?? signalling may be inhibitory but failed to induce adipogenesis using activated Gs? (gsp*). Inhibition of phosphodiesterases did not promote adipogenesis in TSHR* or gsp* populations. Furthermore, differentiation induced by adipogenic medium with pioglitazone was reduced in TSHR* and abolished in gsp* expressing 3T3L1 cells. TSHR* and gsp* did not inactivate PPAR? (PPARG as listed in the HUGO database) by phosphorylation but expression of PPAR?1 was reduced and PPAR?2 undetectable in gsp*. FOXO1 phosphorylation (required to inactivate this repressor of adipogenesis) was lowest in gsp* despite the activation of AKT by phosphorylation. PROF is a mediator that facilitates FOXO1 phosphorylation by phospho-Akt. Its transcript levels remained constantly low in the gsp* population. In most measurements, the TSHR* cells were between the gsp* and control 3T3L1 preadipocytes. The enhanced down-regulation of PREF1 (adipogenesis inhibitor) permits retention of some adipogenic potential in the TSHR* population. We conclude that Gs? signalling impedes FOXO1 phosphorylation and thus inhibits PPAR? transcription and the alternative promoter usage required to generate PPAR?2, the fat-specific transcription factor necessary for adipogenesis.

Zhang, Lei; Paddon, Carol; Lewis, Mark D; Grennan-Jones, Fiona; Ludgate, Marian



RACK1, a Receptor for Activated C Kinase and a Homolog of the b Subunit of G Proteins, Inhibits Activity of Src Tyrosine Kinases and Growth of NIH 3T3 Cells  

Microsoft Academic Search

To isolate and characterize proteins that interact with the unique domain and SH3 and SH2 domains of Src and potentially regulate Src activity, we used the yeast two-hybrid assay to screen a human lung fibroblast cDNA library. We identified RACK1, a receptor for activated C kinase and a homolog of the b subunit of G proteins, as a Src-binding protein.




Spurious t3 thyrotoxicosis unmasking multiple myeloma.  


Objective. To document a case of spurious T3 thyrotoxicosis in a 54-year-old woman. Methods. We present the diagnostic approach of a patient with euthyroid hypertri-iodothyronemia. Results. A 54-year-old, clinically euthyroid woman without personal or family history of thyroid disease referred to endocrinology for possible T3 thyrotoxicosis, after thyroid function tests revealed total T3 > 800?ng/dL (reference range 60-181), normal TSH, and T4. The laboratory data were not compatible with the clinical picture, so thyroid binding globulin abnormalities were suspected. Additional laboratory studies confirmed the diagnosis of multiple myeloma. Conclusion. Monoclonal gammopathy is characterized by the presence of a monoclonal immunoglobulin in the serum or urine, occurring in multiple myeloma, and can cause assay interference and spurious results. We identify a newly recognized cause of euthyroid hypertri-iodothyronemia, due to binding of T3 to monoclonal immunoglobulins in the setting of multiple myeloma. Our case is the only one to date suggesting that monoclonal immunoglobulins from multiple myeloma may exhibit binding to T3 only. PMID:23984117

Antonopoulou, Marianna; Silverberg, Arnold



G-protein receptor kinase 5 regulates the cannabinoid receptor 2-induced up-regulation of serotonin 2A receptors.  


We have recently reported that cannabinoid agonists can up-regulate and enhance the activity of serotonin 2A (5-HT2A) receptors in the prefrontal cortex (PFCx). Increased expression and activity of cortical 5-HT2A receptors has been associated with neuropsychiatric disorders, such as anxiety and schizophrenia. Here we report that repeated CP55940 exposure selectively up-regulates GRK5 proteins in rat PFCx and in a neuronal cell culture model. We sought to examine the mechanism underlying the regulation of GRK5 and to identify the role of GRK5 in the cannabinoid agonist-induced up-regulation and enhanced activity of 5-HT2A receptors. Interestingly, we found that cannabinoid agonist-induced up-regulation of GRK5 involves CB2 receptors, ?-arrestin 2, and ERK1/2 signaling because treatment with CB2 shRNA lentiviral particles, ?-arrestin 2 shRNA lentiviral particles, or ERK1/2 inhibitor prevented the cannabinoid agonist-induced up-regulation of GRK5. Most importantly, we found that GRK5 shRNA lentiviral particle treatment prevented the cannabinoid agonist-induced up-regulation and enhanced 5-HT2A receptor-mediated calcium release. Repeated cannabinoid exposure was also associated with enhanced phosphorylation of CB2 receptors and increased interaction between ?-arrestin 2 and ERK1/2. These latter phenomena were also significantly inhibited by GRK5 shRNA lentiviral treatment. Our results suggest that sustained activation of CB2 receptors, which up-regulates 5-HT2A receptor signaling, enhances GRK5 expression; the phosphorylation of CB2 receptors; and the ?-arrestin 2/ERK interactions. These data could provide a rationale for some of the adverse effects associated with repeated cannabinoid agonist exposure. PMID:23592773

Franklin, Jade M; Carrasco, Gonzalo A



Glucocorticoids inhibit proliferation and adhesion of the IL3-dependent mast cell line, MC\\/9, to NIH\\/3T3 fibroblasts, with an accompanying decrease in IL3 receptor expression  

Microsoft Academic Search

\\u000a Abstract We investigated the effects of glucocorticoids on IL-3-dependent proliferation and c-kit expression of cells of the mouse\\u000a mast cell line, MC\\/9. Glucocorticoids (dexamethasone, prednisolone, and hydrocortisone) inhibited IL-3-dependent MC\\/9 cell\\u000a proliferation, whereas sex steroids (progesterone, ?-estradiol, and testosterone) had no effect. Flow cytometric analysis\\u000a revealed that glucocorticoids reduced the expression of the IL-3 receptor on MC\\/9 cells. Immunoblot and

Hiroyuki Sakai; Noriaki Toyota; Fumihiko Ito; Hidetoshi Takahashi; Yoshio Hashimoto; Hajime Iizuka



[alpha ]-Lipoic acid prevents the development of glucose-induced insulin resistance in 3T3-L1 adipocytes and accelerates the decline in immunoreactive insulin during cell incubation  

Microsoft Academic Search

Oxidative stress has been implicated in glucose toxicity. We tested the hypothesis that certain antioxidants may prevent insulin-resistant glucose transport that develops in adipocytes after sustained exposure to high glucose, provided insulin is present. The antioxidant [alpha ]-lipoic acid has been proposed as an insulin sensitizer. 3T3-L1 adipocytes were preincubated 18 hours in media containing insulin (0.6 nmol\\/L) with low

Eddie L. Greene; Bryce A. Nelson; Katherine A. Robinson; Maria G. Buse



Anandamide induces overeating: mediation by central cannabinoid (CB1) receptors  

Microsoft Academic Search

Rationale: Central cannabinoid systems have been implicated in appetite regulation by the respective hyperphagic actions of exogenous\\u000a cannabinoids, such as ?9-THC, and hypophagic effects of selective cannabinoid receptor antagonists. Objective: This study examined whether an endogenous cannabinoid, anandamide, could induce overeating, via a specific action at central\\u000a (CB1) cannabinoid receptors. Methods: Pre-satiated male rats (n=18), received subcutaneous injections of anandamide

Claire M. Williams; Tim C. Kirkham



Effects of local anesthetics on cell morphology and membrane-associated cytoskeletal organization in BALB/3T3 cells  

PubMed Central

Tertiary amine local anesthetics (dibucaine, tetracaine, procaine) reversibly affect the morphology of untransformed BALB/3T3 cells and the organization of membrane-associated cytoskeletal elements. In the presence of these drugs cells contract and become rounded in shape with the appearance of numerous surface "blebs." Electron microscope examination of anesthetic-treated cells revealed significant reductions in plasma membrane-associated microtubules and microfilaments and/or their plasma membrane attachment. The relationship of the findings on local anesthetic-induced changes in cellular cytoskeletal systems is discussed in relation to previous proposals on plasma membrane organization and control of cell surface receptor topography and mobility.



A Cray T3D performance study  

SciTech Connect

We carry out a performance study using the Cray T3D parallel supercomputer to illustrate some important features of this machine. Timing experiments show the speed of various basic operations while more complicated operations give some measure of its parallel performance.

Nallana, A. [Interact, Inc., Austin, TX (United States); Kincaid, D.R. [Texas Univ., Austin, TX (United States)



Deletion of the thyroid hormone receptor 1 prevents the structural alterations of the cerebellum induced by hypothyroidism  

Microsoft Academic Search

Thyroid hormone (T3) controls critical aspects of cerebellar development, such as migration of postmitotic granule cells and terminal differentiation of Purkinje cells. T3 acts through nuclear receptors (TR) of two types, TR1 and TR, that either repress or activate gene expression. We have analyzed the cerebellar structure of developing mice lacking the TR1 isoform, which normally accounts for about 80%

Beatriz Morte; Jimena Manzano; Thomas Scanlan; Björn Vennström; Juan Bernal



PDGF receptor-? modulates metanephric mesenchyme chemotaxis induced by PDGF AA  

PubMed Central

PDGF B chain or PDGF receptor (PDGFR)-?-deficient (?/?) mice lack mesangial cells. To study responses of ?- and ?-receptor activation to PDGF ligands, metanephric mesenchymal cells (MMCs) were established from embryonic day E11.5 wild-type (+/+) and ?/? mouse embryos. PDGF BB stimulated cell migration in +/+ cells, whereas PDGF AA did not. Conversely, PDGF AA was chemotactic for ?/? MMCs. The mechanism by which PDGFR-? inhibited AA-induced migration was investigated. PDGF BB, but not PDGF AA, increased intracellular Ca2+ and the production of reactive oxygen species (ROS) in +/+ cells. Transfection of ?/? MMCs with the wild-type ?-receptor restored cell migration and ROS generation in response to PDGF BB and inhibited AA-induced migration. Inhibition of Ca2+ signaling facilitated PDGF AA-induced chemotaxis in the wild-type cells. The antioxidant N-acetyl-l-cysteine (NAC) or the NADPH oxidase inhibitor diphenyleneiodonium (DPI) abolished the BB-induced increase in intracellular Ca2+ concentration, suggesting that ROS act as upstream mediators of Ca2+ in suppressing PDGF AA-induced migration. These data indicate that ROS and Ca2+ generated by active PDGFR-? play an essential role in suppressing PDGF AA-induced migration in +/+ MMCs. During kidney development, PDGFR ?-mediated ROS generation and Ca2+ influx suppress PDGF AA-induced chemotaxis in metanephric mesenchyme.

Ricono, Jill M.; Wagner, Brent; Gorin, Yves; Arar, Mazen; Kazlauskas, Andrius; Choudhury, Goutam Ghosh; Abboud, Hanna E.



Anti-adipogenic diarylheptanoids from Alnus hirsuta f. sibirica on 3T3-L1 cells.  


A new diarylheptanoid, (5S)-hydroxy-1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl)-hepta-1E-en-3-one (1), was isolated along with seventeen known diarylheptanoids (2-18) from the methanol extract of Alnus hirsuta f. sibirica leaves using bioactivity-guided fractionation. Among the isolated compounds, compounds 1 and 2 and 4-12 reduced lipid accumulation dose-dependently in 3T3-L1 preadipocytes. Of the compounds active in the present assay system, the most potent compound 7, platyphyllonol-5-O-?-d-xylopyranoside, significantly suppressed the induction of peroxisome proliferator activated receptor ? (PPAR? and CCAAT/enhancer binding protein ? (C/EBP?) protein expression, and inhibited adipocyte differentiation induced by troglitazone, a PPAR? agonist. It was demonstrated that compound 7 has anti-adipogenic activity mediated by the regulation of PPAR? dependent pathways. PMID:23465614

Lee, Mina; Song, Ji Yeon; Chin, Young-Won; Sung, Sang Hyun



Chromatin architecture and the regulation of nuclear receptor inducible transcription.  


Epigenetic mechanisms alter the structure of local chromosome domains to dynamically regulate gene expression by signalling and propagating transcriptional states. Nuclear receptors, a stimulus-inducible class of transcription factors, interact with chromatin to regulate transcription. To promote transcription, nuclear receptors interact with genomic regulatory elements that are epigenetically marked by modified histone tails, DNA methylation status, histone variants, chromatin accessibility and long-range interactions. Advances in throughput have allowed the profiling of regulatory factor activity on a genome-wide scale, with recent evidence from genomic analyses highlighting novel aspects of DNA-binding factor actions on chromatin. In the present review, the current knowledge of the mechanisms regulating nuclear receptor occupancy at cis-regulatory elements is discussed, with particular emphasis on the glucocorticoid, oestrogen and androgen receptors. Epigenetic regulation of genomic elements direct cell-specific regulatory factor binding and contribute to human variation in factor occupancy. Through regulating nuclear receptor activity, the epigenome is a critical checkpoint in nuclear receptor induced gene expression in health and disease. PMID:21039975

Biddie, S C



Bisphenol A Diglycidyl Ether Induces Adipogenic Differentiation of Multipotent Stromal Stem Cells through a Peroxisome Proliferator-Activated Receptor Gamma-Independent Mechanism  

PubMed Central

Background: Bisphenol A (BPA) and bisphenol A diglycidyl ether (BADGE), used in manufacturing coatings and resins, leach from packaging materials into food. Numerous studies suggested that BPA and BADGE may have adverse effects on human health, including the possibility that exposure to such chemicals can be superimposed on traditional risk factors to initiate or exacerbate the development of obesity. BPA is a suspected obesogen, whereas BADGE, described as a peroxisome proliferator–activated receptor gamma (PPAR?) antagonist, could reduce weight gain. Objectives: We sought to test the adipogenic effects of BADGE in a biologically relevant cell culture model. Methods: We used multipotent mesenchymal stromal stem cells (MSCs) to study the adipogenic capacity of BADGE and BPA and evaluated their effects on adipogenesis, osteogenesis, gene expression, and nuclear receptor activation. Discussion: BADGE induced adipogenesis in human and mouse MSCs, as well as in mouse 3T3-L1 preadipocytes. In contrast, BPA failed to promote adipogenesis in MSCs, but induced adipogenesis in 3T3-L1 cells. BADGE exposure elicited an adipogenic gene expression profile, and its ability to induce adipogenesis and the expression of adipogenic genes was not blocked by known PPAR? antagonists. Neither BADGE nor BPA activated or antagonized retinoid “X” receptor (RXR) or PPAR? in transient transfection assays. Conclusions: BADGE can induce adipogenic differentiation in both MSCs and in preadipocytes at low nanomolar concentrations comparable to those that have been observed in limited human biomonitoring. BADGE probably acts through a mechanism that is downstream of, or parallel to, PPAR?.

Chamorro-Garcia, Raquel; Kirchner, Severine; Li, Xia; Janesick, Amanda; Casey, Stephanie C.; Chow, Connie



Characterization of Ligand-Induced Endocytosis of EGF Receptors.  

National Technical Information Service (NTIS)

Down-regulation of activated EGF-receptors (EGF-R) requires their tyrosine kinase (Y-kinase) activity. However, controversy existed as to whether ligand-induced activation of the EGF-R Y-kinase was required for internalization or for lysosomal targeting. ...

S. L. Schmid C. Lamaze



Soluble Receptor-Induced Retroviral Infection of Receptor-Deficient Cells  

PubMed Central

Current models of retroviral entry hypothesize that interactions between the host cell receptor(s) and viral envelope protein induce structural changes in the envelope protein that convert it to an active conformation, allowing it to mediate fusion with the membrane. Recent evidence supporting this hypothesis is the demonstration that Tva, the receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A), induces conformational changes in the viral envelope protein. These changes include conversion of the envelope protein to an active, membrane-binding state likely representing a fusogenic conformation. To determine whether binding of the soluble Tva (sTva) receptor was sufficient to activate fully the fusogenic potential of the ASLV-A envelope protein, we have evaluated the ability of ASLV-A to infect receptor-deficient cell lines in the presence of sTva. Soluble receptor efficiently mediated infection of cells devoid of endogenous Tva in a dose-dependent manner, and this infection was dependent absolutely on the addition of sTva. The infectivity of the virus was enhanced dramatically in the presence of the polycationic polymer Polybrene or when centrifugal forces were applied during inoculation, resulting in viral titers comparable to those achieved on cells expressing endogenous receptor. sTva functioned to mediate infection at low concentrations, approaching the estimated binding constant of the receptor and viral envelope protein. These results demonstrate that receptor binding can activate the ASLV-A envelope protein and convert it to a fusogenic conformation competent to mediate the fusion of the viral and cellular membranes.

Damico, Rachel; Bates, Paul



Epidermal Growth Factor Receptor Transactivation by the Cannabinoid Receptor (CB1) and Transient Receptor Potential Vanilloid 1 (TRPV1) Induces Differential Responses in Corneal Epithelial Cells.  

National Technical Information Service (NTIS)

Corneal epithelial injury induces release of endogenous metabolites that are cannabinoid receptor 1 (CB1) and transient receptor potential vanilloid 1 (TRPV1) agonists. We determined the functional contributions by CB1 and TRPV1 activation to eliciting re...

F. Zhang H. Yang J. E. Capo-Aponte Z. Pan Z. Wang



Involvement of matrix metalloproteinases in the adipose conversion of 3T3-L1 preadipocytes.  

PubMed Central

When mouse 3T3-L1 preadipocytes are induced to differentiate into adipocytes, they change from an extended fibroblast-like morphology to a rounded one. This change most likely occurs through extracellular matrix remodelling, a process known to be mediated in part by matrix metalloproteinases (MMPs). In this study, we have shown by semi-quantitative reverse transcriptase-PCR, zymographic and immunoblot analysis that MMP-2, MMP-9 and membrane type 1 (MT1)-MMP are regulated during adipose conversion. To assess the importance of MMPs for adipocytic differentiation we have used MMP-specific inhibitors as well as neutralizing antibodies. Treatment of 3T3-L1 preadipocytes with the broad MMP inhibitor Ilomastat or the more restricted MMP-2 Inhibitor I prevented their differentiation into adipocytes in a dose-dependent manner, as evidenced by absence of triglyceride accumulation. Inhibitor treatment prevented the fibronectin-network degradation, as well as the induction of the genes for peroxisome-proliferator-activated receptor gamma and adipsin, two adipocyte phenotype markers. Inhibitor treatment was effective when applied during the early stages of adipocytic conversion, whereas inhibitor treatment during later stages had little effect. Inhibitor treatment did not inhibit clonal mitotic expansion; nor did it affect the expression pattern of the adipogenic transcription factor CCAAT/enhancer-binding protein beta (C/EBPbeta) or its nuclear translocation. It did, however, markedly reduce C/EBPbeta DNA-binding capacity. Taken together, these results suggest that MMPs, and notably MMP-2 and MMP-9, may be necessary mediators of adipocytic differentiation of 3T3-L1 cells.

Croissandeau, Gilles; Chretien, Michel; Mbikay, Majambu



Forced swim-induced musculoskeletal hyperalgesia is mediated by CRF2 receptors but not by TRPV1 receptors.  


The exacerbation of musculoskeletal pain by stress in humans is modeled by the musculoskeletal hyperalgesia in rodents following a forced swim. We hypothesized that stress-sensitive corticotropin releasing factor (CRF) receptors and transient receptor vanilloid 1 (TRPV1) receptors are responsible for the swim stress-induced musculoskeletal hyperalgesia. We confirmed that a cold swim (26 °C) caused a transient, morphine-sensitive decrease in grip force responses reflecting musculoskeletal hyperalgesia in mice. Pretreatment with the CRF2 receptor antagonist astressin 2B, but not the CRF1 receptor antagonist NBI-35965, attenuated this hyperalgesia. Desensitizing the TRPV1 receptor centrally or peripherally using desensitizing doses of resiniferatoxin (RTX) failed to prevent the musculoskeletal hyperalgesia produced by cold swim. SB-366791, a TRPV1 antagonist, also failed to influence swim-induced hyperalgesia. Together these data indicate that swim stress-induced musculoskeletal hyperalgesia is mediated, in part, by CRF2 receptors but is independent of the TRPV1 receptor. PMID:23624287

Abdelhamid, Ramy E; Kovacs, Katalin J; Pasley, Jeffrey D; Nunez, Myra G; Larson, Alice A



Insulin-induced loss of the insulin receptor in IM-9 lymphocytes. A biological process mediated through the insulin receptor.  


Exposure of cultured lymphocytes of the IM-9 line to insulin results in a rapid, time-dependent reduction in the number of insulin receptors to a new steady state concentration. Both the rate of loss and the net loss of receptors were directly related to the ambient insulin concentration. The insulin-induced loss of receptors was mediated by binding of insulin to the receptor itself; insulins, which varied 200-fold in biopotency, produced receptor loss in direct proportion to the ability of each insulin to occupy the receptor. The residual insulin receptors were normal following insulin-mediated receptor loss by a variety of sensitive binding criteria. While insulin binding to its receptors was a necessary condition to induce receptor loss, it was not sufficient. Thus, reduction in the temperature of the preincubation from 37 degrees C to 20 degrees C (which enhanced the total amount of insulin bound to the receptor) abolished the loss of insulin receptors. Likewise, cycloheximide prevented the insulin-induced loss of receptors. Furthermore, turkey erythrocytes, which lack active macromolecular synthesis, had no change in the concentration of insulin receptors when exposed to insulin for similar periods. Interestingly, the turkey erythrocytes, when exposed to insulin or to proinsulin, showed a time- and concentration-dependent increase in the affinity of the insulin receptor over a restricted part of the insulin-binding isotherm, which was reversed over a period of several hours following removal of hormone. The insulin-mediated decrease in receptor number on IM-9 lymphocytes was reversible. Following removal of insulin from the growth medium, about one-half of the receptors were restored within 10 h and the full complement of insulin receptors was restored within 24 h. Cycloheximide prevented restoration of the insulin receptor. PMID:7000764

Kosmakos, F C; Roth, J



Interferon-gamma-induced regulation of peroxisome proliferator-activated receptor gamma and STATs in adipocytes.  


Interferon-gamma (IFN-gamma) is known primarily for its roles in immunological responses but also has been shown to affect fat metabolism and adipocyte gene expression. To further investigate the effects of IFN-gamma on fat cells, we examined the effects of this cytokine on the expression of adipocyte transcription factors in 3T3-L1 adipocytes. Although IFN-gamma regulated the expression of several adipocyte transcription factors, IFN-gamma treatment resulted in a rapid reduction of both peroxisome proliferator-activated receptor (PPAR) protein and mRNA. A 48-h exposure to IFN-gamma also resulted in a decrease of both CCAAT/enhancer-binding alpha and sterol regulatory element binding protein (SREBP-1) expression. The short half-life of both the PPARgamma mRNA and protein likely contributed to the rapid decline of both cytosolic and nuclear PPARgamma in the presence of IFN-gamma. Our studies clearly demonstrated that the IFN-gamma-induced loss of PPARgamma protein is partially inhibited in the presence of two distinct proteasome inhibitors. Moreover, IFN-gamma also inhibited the transcription of PPARgamma, which was accompanied by a decrease in PPARgamma mRNA accumulation. In addition, exposure to IFN-gamma resulted in a substantial increase in STAT 1 expression and a small increase in STAT 3 expression. IFN-gamma treatment of 3T3-L1 adipocytes (48-96 h) resulted in a substantial inhibition of insulin-sensitive glucose uptake. These data clearly demonstrate that IFN-gamma treatment results in the development of insulin resistance, which is accompanied by the regulation of various adipocyte transcription factors, in particular the synthesis and degradation of PPARgamma. PMID:11106650

Waite, K J; Floyd, Z E; Arbour-Reily, P; Stephens, J M



A primer on cytokines: sources, receptors, effects, and inducers.  

PubMed Central

Protection against pathogens is a prerequisite for survival of most organisms. To cope with this continuous challenge, complex defense mechanisms have evolved. The construction, adaptation, and maintenance of these mechanisms are under control of an extensive network of regulatory proteins called cytokines. A great number of cytokines have been described over the last 2 decades. This review consists of an overview of cytokines that are involved in immune responses and describes some historical and general aspects as well as prospective clinical applications. Major biological effects together with information on cytokine receptors, producers, inducers, and biochemical and molecular characteristics are listed in tables. In addition, some basic information is given on cytokine receptor signal transduction. Finally, the recent discoveries of cytokine receptors functioning as coreceptors in the pathogenesis of human immunodeficiency virus are summarized.

Curfs, J H; Meis, J F; Hoogkamp-Korstanje, J A



Mechanisms That Underlie ?-Opioid Receptor Agonist-Induced Constipation: Differential Involvement of ?-Opioid Receptor Sites and Responsible Regions.  


Reducing the side effects of pain treatment is one of the most important strategies for improving the quality of life of cancer patients. However, little is known about the mechanisms that underlie these side effects, especially constipation induced by opioid receptor agonists; i.e., do they involve naloxonazine-sensitive versus -insensitive sites or central-versus-peripheral ?-opioid receptors? The present study was designed to investigate the mechanisms of ?-opioid receptor agonist-induced constipation (i.e., the inhibition of gastrointestinal transit and colonic expulsion) that are antagonized by the peripherally restricted opioid receptor antagonist naloxone methiodide and naloxonazine in mice. Naloxonazine attenuated the fentanyl-induced inhibition of gastrointestinal transit more potently than the inhibition induced by morphine or oxycodone. Naloxone methiodide suppressed the oxycodone-induced inhibition of gastrointestinal transit more potently than the inhibition induced by morphine, indicating that ?-opioid receptor agonists induce the inhibition of gastrointestinal transit through different mechanisms. Furthermore, we found that the route of administration (intracerebroventricular, intrathecally, and/or intraperitoneally) of naloxone methiodide differentially influenced the suppressive effect on the inhibition of colorectal transit induced by morphine, oxycodone, and fentanyl. These results suggest that morphine, oxycodone, and fentanyl induce constipation through different mechanisms (naloxonazine-sensitive versus naloxonazine-insensitive sites and central versus peripheral opioid receptors), and these findings may help us to understand the characteristics of the constipation induced by each ?-opioid receptor agonist and improve the quality of life by reducing constipation in patients being treated for pain. PMID:23902939

Mori, Tomohisa; Shibasaki, Yumiko; Matsumoto, Kenjiro; Shibasaki, Masahiro; Hasegawa, Minami; Wang, Erika; Masukawa, Daiki; Yoshizawa, Kazumi; Horie, Syunji; Suzuki, Tsutomu



Dark chocolate receptors: epicatechin-induced cardiac protection is dependent on ?-opioid receptor stimulation  

PubMed Central

Epicatechin, a flavonoid, is a well-known antioxidant linked to a variety of protective effects in both humans and animals. In particular, its role in protection against cardiovascular disease has been demonstrated by epidemiologic studies. Low-dose epicatechin, which does not have significant antioxidant activity, is also protective; however, the mechanism by which low-dose epicatechin induces this effect is unknown. Our laboratory tested the hypothesis that low-dose epicatechin mediates cardiac protection via opioid receptor activation. C57BL/6 mice were randomly assigned to 1 of 10 groups: control, epicatechin, naloxone (nonselective opioid receptor antagonist), epicatechin + naloxone, naltrindole (?-specific opioid receptor antagonist), epicatechin + naltrindole, norbinaltorphimine (nor-BNI, ?-specific opioid receptor antagonist), epicatechin + nor-BNI, 5-hydroxydecanoic acid [5-HD, ATP-sensitive potassium channel antagonist], and epicatechin + 5-HD. Epicatechin (1 mg/kg) or other inhibitors (5 mg/kg) were administered by oral gavage or intraperitoneal injection, respectively, daily for 10 days. Mice were subjected to 30 min coronary artery occlusion followed by 2 h of reperfusion, and infarct size was determined via planimetry. Whole heart homogenates were assayed for downstream opioid receptor signaling targets. Infarct size was significantly reduced in epicatechin- and epicatechin + nor-BNI-treated mice compared with control mice. This protection was blocked by naloxone, naltrindole, and 5-HD. Epicatechin and epicatechin + nor-BNI increased the phosphorylation of Src, Akt, and I?B?, while simultaneously decreasing the expression of c-Jun NH2-terminal kinase and caspase-activated DNase. All signaling effects are consistent with opioid receptor stimulation and subsequent cardiac protection. Naloxone, naltrindole, and 5-HD attenuated these effects. In conclusion, epicatechin acts via opioid receptors and more specifically through the ?-opioid receptor to produce cardiac protection from ischemia-reperfusion injury.

Panneerselvam, Mathivadhani; Tsutsumi, Yasuo M.; Bonds, Jacqueline A.; Horikawa, Yousuke T.; Saldana, Michelle; Dalton, Nancy D.; Head, Brian P.; Patel, Piyush M.; Roth, David M.



Dark chocolate receptors: epicatechin-induced cardiac protection is dependent on delta-opioid receptor stimulation.  


Epicatechin, a flavonoid, is a well-known antioxidant linked to a variety of protective effects in both humans and animals. In particular, its role in protection against cardiovascular disease has been demonstrated by epidemiologic studies. Low-dose epicatechin, which does not have significant antioxidant activity, is also protective; however, the mechanism by which low-dose epicatechin induces this effect is unknown. Our laboratory tested the hypothesis that low-dose epicatechin mediates cardiac protection via opioid receptor activation. C57BL/6 mice were randomly assigned to 1 of 10 groups: control, epicatechin, naloxone (nonselective opioid receptor antagonist), epicatechin + naloxone, naltrindole (?-specific opioid receptor antagonist), epicatechin + naltrindole, norbinaltorphimine (nor-BNI, ?-specific opioid receptor antagonist), epicatechin + nor-BNI, 5-hydroxydecanoic acid [5-HD, ATP-sensitive potassium channel antagonist], and epicatechin + 5-HD. Epicatechin (1 mg/kg) or other inhibitors (5 mg/kg) were administered by oral gavage or intraperitoneal injection, respectively, daily for 10 days. Mice were subjected to 30 min coronary artery occlusion followed by 2 h of reperfusion, and infarct size was determined via planimetry. Whole heart homogenates were assayed for downstream opioid receptor signaling targets. Infarct size was significantly reduced in epicatechin- and epicatechin + nor-BNI-treated mice compared with control mice. This protection was blocked by naloxone, naltrindole, and 5-HD. Epicatechin and epicatechin + nor-BNI increased the phosphorylation of Src, Akt, and I?B?, while simultaneously decreasing the expression of c-Jun NH(2)-terminal kinase and caspase-activated DNase. All signaling effects are consistent with opioid receptor stimulation and subsequent cardiac protection. Naloxone, naltrindole, and 5-HD attenuated these effects. In conclusion, epicatechin acts via opioid receptors and more specifically through the ?-opioid receptor to produce cardiac protection from ischemia-reperfusion injury. PMID:20833967

Panneerselvam, Mathivadhani; Tsutsumi, Yasuo M; Bonds, Jacqueline A; Horikawa, Yousuke T; Saldana, Michelle; Dalton, Nancy D; Head, Brian P; Patel, Piyush M; Roth, David M; Patel, Hemal H



AMPA Receptor Potentiation can Prevent Ethanol-Induced Intoxication  

Microsoft Academic Search

We present a substantial series of behavioral and imaging experiments, which demonstrate, for the first time, that increasing AMPA receptor-mediated neurotransmission via administration of potent and selective biarylsulfonamide AMPA potentiators LY404187 and LY451395 reverses the central effects of an acutely intoxicating dose of ethanol in the rat. Using pharmacological magnetic resonance imaging (phMRI), we observed that LY404187 attenuated ethanol-induced reductions

Nicholas Jones; Marcus J Messenger; Michael J O'Neill; Anna Oldershaw; Gary Gilmour; Rosa M A Simmons; Smriti Iyengar; Vincenzo Libri; Mark Tricklebank; Steve C R Williams



Research report Glutamate receptor-induced toxicity in neostriatal cells  

Microsoft Academic Search

Infrared differential interference contrast (IR DIC) videomicroscopy was used to measure and characterize cell swelling induced by activation of glutamate receptors (GluR) in a neostriatal brain slice preparation. This swelling is, in many cases, a prelude to necrotic cell death. Activation of N-methyl-D-aspartate (NMDA) and non-NMDA ionotropic GluRs caused cell swelling. The concentration-response relationships and the time courses of the

Christopher S. Colwell; Michael S. Levine


Type beta transforming growth factor controls the adipogenic differentiation of 3T3 fibroblasts.  


Differentiating mouse 3T3-L1 preadipocytes have been used as a model system to study the ability of type beta transforming growth factor (TGF-beta) to modulate cell development. We find that TGF-beta inhibits potently (ID50 approximately equal to 25 pM) the adipogenic conversion of 3T3-L1 cells. Inhibition is observed only when cells are exposed to TGF-beta before they become committed to differentiation. Even a transient (4 hr) exposure to TGF-beta immediately before the commitment point is sufficient to prevent differentiation. This point coincides with the time point immediately preceding the onset of coordinate expression of differentiation-specific proteins in 3T3-L1 cells. TGF-beta interacts with cell surface receptors in 3T3-L1 cells that have structural and binding properties similar to TGF-beta receptors in other cell types in which TGF-beta acts as a growth activator or a growth inhibitor. However, TGF-beta does not markedly alter differentiation-related mitosis in 3T3-L1 cells. The action of TGF-beta on 3T3-L1 cells does not involve changes in cAMP or prostaglandin E levels. These results suggest that TGF-beta is a unique modulator of adipogenic differentiation of fibroblasts. PMID:3001708

Ignotz, R A; Massagué, J



Morphine induces ? opioid receptor endocytosis in guinea pig enteric neurons following prolonged receptor activation  

PubMed Central

Background & Aims The ? opioid receptor (?OR) undergoes rapid endocytosis following acute stimulation with opioids and most opiates, but not with morphine. We investigated whether prolonged activation of ?OR affects morphine’s ability to induce receptor endocytosis in enteric neurons. Methods We compared the effects of morphine, a poor ?OR-internalizing opiate, and [D-Ala2, MePhe4,Gly-ol5] enkephalin (DAMGO), a potent ?OR-internalizing agonist, on ?OR trafficking in enteric neurons and on the expression of dynamin and ?-arrestin immunoreactivity in the ileum of guinea pigs rendered tolerant by chronic administration of morphine. Results Morphine (100 µM) strongly induced endocytosis of ?OR in tolerant but not naďve neurons (55.7%±9.3% vs. 24.2%±7.3%, P<0.001) whereas DAMGO (10 µM) strongly induced internalization of ?OR in neurons from tolerant and naďve animals (63.6%±8.4% and 66.5%±3.6%). Morphine- or DAMGO-induced ?OR endocytosis resulted from direct interactions between the ligand and the ?OR, because endocytosis was not affected by tetrodotoxin, a blocker of endogenous neurotransmitter release. Ligand-induced ?OR internalization was inhibited by pretreatment with the dynamin inhibitor, dynasore. Chronic morphine administration resulted in a significant increase in dynamin and translocation of dynamin immunoreactivity from the intracellular pool to the plasma membrane, but did not affect ? arrestin immunoreactivity. Conclusion Chronic activation of ?ORs increases the ability of morphine to induce ?OR endocytosis in enteric neurons, which depends on the level and cellular localization of dynamin, a regulatory protein that has an important role in receptor-mediated signal transduction in cells.

Patierno, Simona; Anselmi, Laura; Jaramillo, Ingrid; Scott, David; Garcia, Rachel; Sternini, Catia



The Orphan Nuclear Receptor Estrogen Receptor-related Receptor ? Negatively Regulates BMP2-induced Osteoblast Differentiation and Bone Formation*  

PubMed Central

Estrogen receptor-related receptor ? (ERR?/ERR3/NR3B3) is a member of the orphan nuclear receptor with important functions in development and homeostasis. Recently it has been reported that ERR? is involved in osteoblast differentiation and bone formation. In the present study we examined the role of ERR? in osteoblast differentiation. Here, we showed that ERR? is expressed in osteoblast progenitors and primary osteoblasts, and its expression is increased temporarily by BMP2. Overexpression of ERR? reduced BMP2-induced alkaline phosphatase activity and osteocalcin production as well as calcified nodule formation, whereas inhibition of ERR? expression significantly enhanced BMP2-induced osteogenic differentiation and mineralization, suggesting that endogenous ERR? plays an important role in osteoblast differentiation. In addition, ERR? significantly repressed Runx2 transactivity on osteocalcin and bone sialoprotein promoters. We also observed that ERR? physically interacts with Runx2 in vitro and in vivo and competes with p300 to repress Runx2 transactivity. Notably, intramuscular injection of ERR? strongly inhibited BMP2-induced ectopic bone formation in a dose-dependent manner. Taken together, these results suggest that ERR? is a novel negative regulator of osteoblast differentiation and bone formation via its regulation of Runx2 transactivity.

Jeong, Byung-Chul; Lee, Yong-Soo; Park, Yun-Yong; Bae, In-Ho; Kim, Don-Kyu; Koo, Seung-Hoi; Choi, Hong-Ran; Kim, Sun-Hun; Franceschi, Renny T.; Koh, Jeong-Tae; Choi, Hueng-Sik



Bixin regulates mRNA expression involved in adipogenesis and enhances insulin sensitivity in 3T3-L1 adipocytes through PPARgamma activation.  


Insulin resistance is partly due to suppression of insulin-induced glucose uptake into adipocytes. The uptake is dependent on adipocyte differentiation, which is controlled at mRNA transcription level. The peroxisome proliferator-activated receptor (PPAR), a ligand-regulated nuclear receptor, is involved in the differentiation. Many food-derived compounds serve as ligands to activate or inactivate PPAR. In this study, we demonstrated that bixin and norbixin (annatto extracts) activate PPARgamma by luciferase reporter assay using GAL4-PPAR chimera proteins. To examine the effects of bixin on adipocytes, 3T3-L1 adipocytes were treated with bixin or norbixin. The treatment induced mRNA expression of PPARgamma target genes such as adipocyte-specific fatty acid-binding protein (aP2), lipoprotein lipase (LPL), and adiponectin in differentiated 3T3-L1 adipocytes and enhanced insulin-dependent glucose uptake. The observations indicate that bixin acts as an agonist of PPARgamma and enhances insulin sensitivity in 3T3-L1 adipocytes, suggesting that bixin is a valuable food-derived compound as a PPAR ligand to regulate lipid metabolism and to ameliorate metabolic syndrome. PMID:19891958

Takahashi, Nobuyuki; Goto, Tsuyoshi; Taimatsu, Aki; Egawa, Kahori; Katoh, Sota; Kusudo, Tatsuya; Sakamoto, Tomoya; Ohyane, Chie; Lee, Joo-Young; Kim, Young-Il; Uemura, Taku; Hirai, Shizuka; Kawada, Teruo



Dietary xenoestrogens differentially impair 3T3-L1 preadipocyte differentiation and persistently affect leptin synthesis.  


Recent observations have highlighted adipogenesis alterations under exposure to several xenoestrogens at critical stages, and pointed at their possible involvement in the pathogenesis of obesity. However, it remains unclear whether these effects are mediated by classical estrogen receptor (ER) binding and subsequent transcriptional modulation. The aim of this study was to determine the (anti-)adipogenic impact of apigenin, bisphenol A, genistein and 17beta-estradiol at the onset of adipose cell maturation, and to correlate it to their estrogenic potential. In steroid-free conditions, 3T3-L1 preadipocytes were induced to differentiate in the presence of xenoestrogens for 2 days. DNA and triglyceride levels, leptin secretion and expression of Pref-1, C/EBPbeta, PPARgamma2, FAS, leptin and ERs were measured on days 0, 3 and 8 of differentiation. Genistein potently blocked mitotic clonal expansion and all markers of maturation. Bisphenol A and estradiol did not modify triglyceride accumulation but increased the expression of differentiation genes. Apigenin caused a weak but reversible delay in adipogenesis although it unexpectedly enhanced leptin synthesis. However, the expression of steroid hormone receptors was not associated with these differential effects. In conclusion, we could not put a clear estrogen-dependent mechanism forward, but early exposure to xenoestrogens persistently disrupted adipocyte gene expression and leptin synthesis. PMID:18359623

Phrakonkham, Pascal; Viengchareun, Say; Belloir, Christine; Lombčs, Marc; Artur, Yves; Canivenc-Lavier, Marie-Chantal



Cyclic restricted feeding enhances lipid storage in 3 T3-L1 adipocytes  

PubMed Central

Background People who skip breakfast have more visceral fat than those who eat breakfast; however, the mechanism underlying this difference is unclear. In this study, we examined 3 T3-L1 adipocytes and assessed 1) whether restricted feeding (i.e., “breakfast skipping”) alters the cyclic expression of brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like protein 1 (BMAL1) and lipogenic proteins and 2) whether repeated exposure to growth media at the time-points with enhanced lipogenic regulatory signals increases de novo lipogenesis and lipid storage. Methods Differentiated adipocytes were divided into two groups: a control group and a restricted feeding group, for which incubation with growth medium from ZT 9 to ZT 12 was withheld. Results A bout of restricted feeding disrupted the cyclic expression of BMAL1 protein and increased the expression of lipogenic proteins, such as fatty acid synthase and peroxisome proliferator-activated receptor gamma in adipocytes. Furthermore, the repeated exposure to growth media at the time-points with enhanced lipogenic regulatory signals increased de novo lipogenesis and lipid storage. Conclusion These findings suggest that direct disruption of intracellular molecular clock systems by breakfast skipping and the concurrent changes in the daily cycle of lipogenic proteins in adipocytes, as a consequence of repeated nutrition at the time-points with enhanced lipogenic regulatory signals, would result in increased lipogenesis and lipid storage. These alterations are important molecular mechanisms underlying augmented adiposity induced by breakfast skipping.



Changes in serum triiodothyronine (T3) kinetics after prolonged Antarctic residence: the polar T3 syndrome.  


Humans who live in Antarctica for greater than 5 continuous months demonstrate alterations in the hypothalamic-pituitary-thyroid axis. These changes are characterized by 1) increased pituitary release of TSH in response to iv TRH, 2) increased serum clearance of orally administered T3, and 3) normal serum total, free T4, and unstimulated TSH levels. To clarify the mechanism responsible for these findings, serum kinetic studies of 125I-labeled T4 and T3 were carried out in a group of normal men, first in California, then after 20 and 42 weeks of continuous Antarctic residence. The kinetic parameters were calculated by noncompartmental analysis. The mean T4 residence time (MRT) was not different before and after 42 weeks (5.54 +/- 0.50 and 5.08 +/- 0.43 days). The total T4 volume of distribution (TVd) tended to fall over the same period (4.30 +/- 0.12, 3.56 +/- 0.27 L/m2), but was not significantly different (P = 0.075). In contrast to T4, there was an increase from control values for the T3 MRT from 0.83 +/- 0.03 to 1.10 +/- 0.03 days (P less than 0.002) and a more than doubling of the T3 TVd from 15.55 +/- 0.52 to 47.24 +/- 5.09 L/m2 (P less than 0.002) after 42 wk of Antarctic residence. Energy intake increased approximately 40% throughout the study without a change in body weight. The changes in T3 kinetic parameters may be accounted for by increased extravascular tissue binding. The marked increase in T3 TVd and the small increase in MRT are associated with increased T3 production and clearance and only minor changes in T4 kinetics. This is the first description of a mechanism for the change in thyroid hormone economy occurring with extended residence in Antarctica. PMID:2318952

Reed, H L; Silverman, E D; Shakir, K M; Dons, R; Burman, K D; O'Brian, J T



Apolipoprotein CIII Induces Monocyte Chemoattractant Protein-1 and Interleukin 6 Expression Via Toll-Like Receptor 2 Pathway in Mouse Adipocytes  

PubMed Central

Objective To examine the direct effect of apolipoprotein CIII (apoCIII) on adipokine expressions that are involved in obesity, insulin resistance, or metabolic syndrome. Methods and Results ApoCIII in triglyceride-rich lipoproteins is elevated in patients with obesity, insulin resistance, or metabolic syndrome. Its level is also associated with proinflammatory adipokines. Fully differentiated mouse 3T3L1 adipocytes were incubated with apoCIII. ApoCIII activated nuclear factor ?B of 3T3L1 adipocytes and induced the expression of monocyte chemoattractant protein (MCP) 1 and interleukin (IL) 6. ApoCIII also activated extracellular signal–regulated kinase and p38. Mitogen-activated protein kinase kinase (MEK)-1 inhibitor PD98059, but not p38 inhibitor SB203580, inhibited apoCIII-induced upregulation of MCP-1 and IL-6. Previously, it was shown that apoCIII activates proinflammatory signals through toll-like receptor (TLR) 2. TLR2-blocking antibody abolished activation of nuclear factor ?B and extracellular signal–regulated kinase induced by apoCIII and inhibited apoCIII-induced upregulation of MCP-1 and IL-6. ApoCIII also reduced adiponectin expression of 3T3L1 adipocytes, which was recovered by TLR2-blocking antibody. ApoCIII induced the expression of MCP-1 and IL-6 in TLR2-overexpressed human embryonic kidney 293 cells but not wild-type human embryonic kidney 293 cells without TLR2. ApoCIII induced the expression of MCP-1 and IL-6 and decreased adiponectin expression in white adipose tissue of wild-type mice but not of TLR2-deficient mice in vivo. Conclusion ApoCIII may activate extracellular signal–regulated kinase and nuclear factor kB through TLR2 and induce proinflammatory adipokine expression in vitro and in vivo. Thus, apoCIII links dyslipidemia to inflammation in adipocytes, which, in turn, may contribute to atherosclerosis.

Abe, Yasuko; Kawakami, Akio; Osaka, Mizuko; Uematsu, Satoshi; Akira, Shizuo; Shimokado, Kentaro; Sacks, Frank M.; Yoshida, Masayuki



Bombesin Stimulation of DNA Synthesis and Cell Division in Cultures of Swiss 3T3 Cells  

Microsoft Academic Search

Bombesin is shown to be a potent mitogen for Swiss 3T3 cells. At nanomolar concentrations the peptide markedly enhances the ability of fresh serum to stimulate DNA synthesis in confluent and quiescent cultures of these cells. In the presence of a low concentration (3.5%) of serum, bombesin stimulates 3T3 cell proliferation. In serum-free medium, bombesin induces DNA synthesis in the

Enrique Rozengurt; James Sinnett-Smith



Triiodothyronine (T3) stimulates food intake via enhanced hypothalamic AMP-activated kinase activity  

Microsoft Academic Search

Thyroid hormone regulates food intake. We previously reported that rats with triiodothyronine (T3)-induced thyrotoxicosis display hyperphagia associated with suppressed circulating leptin levels, increased hypothalamic neuropeptide Y (NPY) mRNA and decreased hypothalamic pro-opiomelanocortin (POMC) mRNA. AMP-activated kinase (AMPK) is a serine\\/threonine protein kinase that is activated when cellular energy is depleted. We hypothesized that T3 causes an increase in hypothalamic AMPK

Shinya Ishii; Jun Kamegai; Hideki Tamura; Takako Shimizu; Hitoshi Sugihara; Shinichi Oikawa



RIC-3 differentially modulates ?4?2 and ?7 nicotinic receptor assembly, expression, and nicotine-induced receptor upregulation  

PubMed Central

Background Recent work has shown that the chaperone resistant to inhibitors of acetylcholinesterase (RIC-3) is critical for the folding, maturation and functional expression of a variety of neuronal nicotinic acetylcholine receptors. ?7 nicotinic receptors can only assemble and functionally express in select lines of cells, provided that RIC-3 is present. In contrast, ?4?2 nicotinic receptors can functionally express in many cell lines even without the presence of RIC-3. Depending on the cell line, RIC-3 has differential effects on ?4?2 receptor function – enhancement in mammalian cells but inhibition in Xenopus oocytes. Other differences between the two receptor types include nicotine-induced upregulation. When expressed in cell lines, ?4?2 receptors readily and robustly upregulate with chronic nicotine exposure. However, ?7 nicotinic receptors appear more resistant and require higher concentrations of nicotine to induce upregulation. Could the coexpression of RIC-3 modulate the extent of nicotine-induced upregulation not only for ?7 receptors but also ?4?2 receptors? We compared and contrasted the effects of RIC-3 on assembly, trafficking, protein expression and nicotine-induced upregulation on both ?7 and ?4?2 receptors using fluorescent protein tagged nicotinic receptors and Förster resonance energy transfer (FRET) microscopy imaging. Results RIC-3 increases assembly and cell surface trafficking of ?7 receptors but does not alter ?7 protein expression in transfected HEK293T cells. In contrast, RIC-3 does not affect assembly of ?4?2 receptors but increases ?4 and ?2 subunit protein expression. Acute nicotine (30 min exposure) was sufficient to upregulate FRET between ?4 and ?2 subunits. Surprisingly, when RIC-3 was coexpressed with ?4?2 receptors nicotine-induced upregulation was prevented. ?7 receptors did not upregulate with acute nicotine in the presence or absence of RIC-3. Conclusions These results provide interesting novel data that RIC-3 differentially regulates assembly and expression of different nicotinic receptor subunits. These results also show that nicotine-mediated upregulation of ?4?2 receptors can be dynamically regulated by the presence of the chaperone, RIC-3. This could explain a novel mechanism why high affinity ?4?2 receptors are upregulated in specific neuronal subtypes in the brain and not others.



Endothelin1 and endothelin B type receptor are induced in mesangial proliferative nephritis in the rat  

Microsoft Academic Search

Endothelin-1 and endothelin B type receptor are induced in mesangial proliferative nephritis in the rat. We studied whether endothelin-1 (ET-1) and its receptor subtypes (ETaR, endothelin A type receptor; and ETbR, B type receptor) were up-regulated in the glomerulus of a rat model of mesangial proliferative glomerulonephritis induced by anti-thymocyte serum (anti-Thy-1 GN). A marked increase in preproET-1 mRNA could

Ashio Yoshimura; Shigeki Iwasaki; Kiyoko Inui; Terukuni Ideura; Shozo Koshikawa; Masashi Yanagisawa; Tomoh Masaki



5HT 2 receptor regulation of acetylcholine release induced by dopaminergic stimulation in rat striatal slices  

Microsoft Academic Search

The role of 5-hydroxytryptamine (5-HT) receptor subtypes in acetylcholine (ACh) release induced by dopamine or neurokinin receptor stimulation was studied in rat striatal slices. The dopamine D1 receptor agonist SKF 38393 potentiated in a tetrodotoxin-sensitive manner the K+-evoked [3H]ACh release while SCH 23390, a dopamine D1 receptor antagonist, had no effect. [3H]ACh release was decreased by the dopamine D2 receptor

M. J Ram??rez; E Cenarruzabeitia; B Lasheras; J Del Rio



The role of adrenomedullin and receptors in glomerular hyperfiltration in streptozotocin-induced diabetic rats  

Microsoft Academic Search

The role of adrenomedullin and receptors in glomerular hyperfiltration in streptozotocin-induced diabetic rats.BackgroundSince adrenomedullin (AM) elicits vasodilatation by binding to specific AM receptors consisted of calcitonin-receptor-like receptor (CRLR)\\/receptor-activity-modifying protein 2 (RAMP2) or CRLR\\/receptor-activity-modifying protein 3 (RAMP3) on endothelial cells and stimulating nitric oxide production, AM possibly involves in glomerular capillary dilatation in early phase of diabetic nephropathy.MethodsStreptozotocin (STZ)-induced diabetic Sprague-Dawley




Glucagon-like Peptide-1 Upregulates Visfatin Expression in 3T3-L1 Adipocytes.  


The incretin hormone glucagon-like peptide-1 (GLP-1) exerts important functions in controlling glucose homeostasis. Many studies have revealed molecular targets of GLP-1, but its influence on adipokines has not been determined. Visfatin, a recently discovered adipokine, has been shown to attenuate insulin resistance by binding to insulin receptor. Our study shows that GLP-1 induced secretion of visfatin into the culture medium of 3T3-L1 adipocytes due to increased visfatin mRNA expression. Furthermore, the effect of GLP-1 on visfatin was dose- and time-dependent. H89, a protein kinase A inhibitor, prevented the induction of visfatin expression by GLP-1. Moreover, inhibition of NF-?B by PDTC reduced the basal visfatin release while having no effect on the transcription regulation by GLP-1. In addition, GLP-1 alleviated the decrease of visfatin mRNA expression under endoplasmic reticulum stress induced by thapsigargin. Taken together, our study suggests that GLP-1 promotes the novel insulin-mimetic adipocytokine visfatin expression via the PKA pathway and might influence glucose metabolism. PMID:23670349

Liu, R; Ding, X; Wang, Y; Wang, M; Peng, Y



Androgen-Induced Cell Migration: Role of Androgen Receptor\\/Filamin A Association  

Microsoft Academic Search

BackgroundAndrogen receptor (AR) controls male morphogenesis, gametogenesis and prostate growth as well as development of prostate cancer. These findings support a role for AR in cell migration and invasiveness. However, the molecular mechanism involved in AR-mediated cell migration still remains elusive.Methodology\\/Principal FindingsMouse embryo NIH3T3 fibroblasts and highly metastatic human fibrosarcoma HT1080 cells harbor low levels of transcriptionally incompetent AR. We

Gabriella Castoria; Loredana D'Amato; Alessandra Ciociola; Pia Giovannelli; Tiziana Giraldi; Leandra Sepe; Giovanni Paolella; Maria Vittoria Barone; Antimo Migliaccio; Ferdinando Auricchio; Irina Agoulnik



Requirement of phosphatidylinositol 3-kinase-dependent pathway and Src for Gas6-Axl mitogenic and survival activities in NIH 3T3 fibroblasts.  

PubMed Central

Gas6 is a secreted protein previously identified as the ligand of the Axl receptor tyrosine kinase. We have shown that Gas6 is able to induce cell cycle reentry of serum-starved NIH 3T3 cells and to efficiently prevent apoptosis after complete growth factor removal, a survival effect uncoupled from Gas6-induced mitogenesis. Here we report that the mitogenic effect of Gas6 requires phosphatidylinositol 3-kinase (PI3K) activity since it is abrogated both by the specific inhibitor wortmannin and by overexpression of the dominant negative P13K p85 subunit. Consistently, Gas6 activates the P13K downstream targets S6K and Akt, whose activation is abrogated by addition of wortmannin. Moreover, rapamycin treatment blocks Gas6-induced entry into the S phase of serum-starved NIH 3T3 cells. We also demonstrate the requirement of Src tyrosine kinase for Gas6 signalling since stable or transient expression of a catalytically inactive form of Src significantly inhibited Gas6-stimulated entry into the S phase. Accordingly, Gas6 addition to serum-starved NIH 3T3 cells causes activation of the intrinsic Src kinase activity. When specifically analyzed in a survival assay, these elements were found to be required for the survival effect of Gas6. Taken together, the evidence presented here identifies elements involved in the Gas6 transduction pathway that are responsible for its antiapoptotic effect and suggests that Src is involved in the events regulating cell survival.

Goruppi, S; Ruaro, E; Varnum, B; Schneider, C



T3 and Triac inhibit leptin secretion and expression in brown and white rat adipocytes.  


Leptin regulates appetite, inhibits food intake, and seems to increase energy expenditure. We investigated the effect of triiodothyroacetic acid (Triac), a metabolite of T3, which seems to be more thermogenic than T3, on leptin secretion and mRNA expression. Rat primary cultures of white and brown adipocytes were treated with increasing concentrations of Triac and T3. The effect of different types of serum and insulin concentrations was also tested. Serum inhibited leptin secretion and mRNA expression. Leptin secretion was also clearly inhibited by Triac and T3 in a dose-dependent manner and with similar potency. In the presence of norepinephrine (NE), Triac and T3 had a similar inhibitory effect, but the inhibition was almost complete in white adipocytes. Parallel results were found at the mRNA level, where Triac and T3 had similar inhibitory potency, both alone and with NE. We also show that insulin induced dose- and time-dependent increases in leptin secretion, reaching maximum levels at 0.5 and 3 nM insulin for white and brown adipocytes, respectively. Leptin secretion was higher in white than in brown adipocytes. The increases in leptin secretion were preceded by increases in leptin mRNA. In conclusion, these data demonstrate for the first time that Triac, like T3 and serum, inhibits leptin secretion and expression in white and brown adipocytes, whereas insulin has the opposite effect. PMID:15158754

Medina-Gomez, Gema; Calvo, Rosa-Maria; Obregón, Maria-Jesus




EPA Science Inventory

ZINC-INDUCED EGF RECEPTOR SIGNALING REQUIRES Src-MEDIATED PHOSPHORYLATION OF THE EGF RECEPTOR ON TYROSINE 845 (Y845) Weidong Wu1, Lee M. Graves2, Gordon N. Gill3 and James M. Samet4 1Center for Environmental Medicine and Lung Biology; 2Department of Pharmacology, University o...


Drosophila D1 dopamine receptor mediates caffeine-induced arousal  

PubMed Central

The arousing and motor-activating effects of psychostimulants are mediated by multiple systems. In Drosophila, dopaminergic transmission is involved in mediating the arousing effects of methamphetamine, although the neuronal mechanisms of caffeine (CAFF)-induced wakefulness remain unexplored. Here, we show that in Drosophila, as in mammals, the wake-promoting effect of CAFF involves both the adenosinergic and dopaminergic systems. By measuring behavioral responses in mutant and transgenic flies exposed to different drug-feeding regimens, we show that CAFF-induced wakefulness requires the Drosophila D1 dopamine receptor (dDA1) in the mushroom bodies. In WT flies, CAFF exposure leads to downregulation of dDA1 expression, whereas the transgenic overexpression of dDA1 leads to CAFF resistance. The wake-promoting effects of methamphetamine require a functional dopamine transporter as well as the dDA1, and they engage brain areas in addition to the mushroom bodies.

Andretic, Rozi; Kim, Young-Cho; Jones, Frederick S.; Han, Kyung-An; Greenspan, Ralph J.



Role of hypothalamic angiotensin type 1 receptors in pressure overload-induced mineralocorticoid receptor activation and salt-induced sympathoexcitation.  


Pressure overload enhances salt-induced sympathoexcitation through hypothalamic mineralocorticoid receptor (MR)-epithelial Na channel activation. Pressure overload also increases hypothalamic angiotensin type 1 receptors (AT1R). However, the role of AT1R in pressure overload-induced MR activation and salt-induced sympathoexcitation remains unknown. Therefore, the aim of the present study was to address this question. We performed aortic banding (AB) on mice from the Institute of Cancer Research. The expression of hypothalamic MR, serum/glucocorticoid-induced protein kinase-1 (SGK-1) and AT1R increased independently of plasma renin activity at 2 or 4 weeks after AB. Next, we performed AB in AT1aR-knockout (KO) mice and c57BL6/J wild-type (WT) mice. Sham-operated (Sham) mice were used as a control. Four weeks after AB (AB-KO or AB-WT), the expression of hypothalamic MR and SGK-1 increased in both AB-WT and AB-KO compared with Sham-WT and Sham-KO, respectively. The expression of AT1R was also greater in AB-WT than in Sham-WT. In addition, mice were fed a high-salt (8%) diet for an additional 4 weeks (ABH-KO and ABH-WT). High salt loading increased the urinary excretion of norepinephrine, a marker of sympathetic activity in ABH-WT, concomitant with hypothalamic MR activation, but not in ABH-KO. These results indicate that pressure overload activated hypothalamic MR independently of AT1R. After salt intake, however, AT1R was necessary to maintain hypothalamic MR activation and salt-induced sympathoexcitation. PMID:23364339

Ito, Koji; Hirooka, Yoshitaka; Nakano, Masatsugu; Honda, Nobuhiro; Matsukawa, Ryuichi; Sunagawa, Kenji



Protein Kinase C? Mediates ?-Opioid Receptor-induced Cross-desensitization of Chemokine Receptor CCR5*  

PubMed Central

We have previously shown that the ?-opioid receptor (MOR) is capable of mediating cross-desensitization of several chemokine receptors including CCR5, but the biochemical mechanism of this process has not been fully elucidated. We have carried out a series of functional and biochemical studies and found that the mechanism of MOR-induced cross-desensitization of CCR5 involves the activation of PKC?. Inhibition of PKC? by its pseudosubstrate inhibitor, or its siRNA, or dominant negative mutants suppresses the cross-desensitization of CCR5. Our results further indicate that the activation of PKC? is mediated through a pathway involving phosphoinositol-dependent kinase-1 (PDK1). In addition, activation of MOR elevates the phosphorylation level and kinase activity of PKC?. The phosphorylation of PKC? can be suppressed by a dominant negative mutant of PDK1. We observed that following MOR activation, the interaction between PKC? and PDK1 is immediately increased based on the analysis of fluorescent resonance energy transfer in cells with the expression of PKC?-YFP and PDK1-CFP. In addition, cells expressing PKC? kinase motif mutants (Lys-281, Thr-410, Thr-560) fail to exhibit full MOR-induced desensitization of CCR5 activity. Taken together, we propose that upon DAMGO treatment, MOR activates PKC? through a PDK1-dependent signaling pathway to induce CCR5 phosphorylation and desensitization. Because CCR5 is a highly proinflammatory receptor, and a critical coreceptor for HIV-1, these results may provide a novel approach for the development of specific therapeutic agents to treat patients with certain inflammatory diseases or AIDS.

Song, Changcheng; Rahim, Rahil T.; Davey, Penelope C.; Bednar, Filip; Bardi, Giuseppe; Zhang, Lily; Zhang, Ning; Oppenheim, Joost J.; Rogers, Thomas J.



Activation of type 5 metabotropic glutamate receptors attenuates deficits in cognitive flexibility induced by NMDA receptor blockade  

PubMed Central

Metabotropic glutamate (mGlu) receptors provide a mechanism by which the function of NMDA glutamate receptors can be modulated. As NMDA receptor hypofunction is implicated in the etiology of psychiatric disorders, including schizophrenia, the pharmacological regulation of mGlu receptor activity represents a promising therapeutic approach. We examined the effects of the positive allosteric mGlu5 receptor modulator 3- cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB), alone and in combination with the NMDA receptor antagonist MK-801, on a task measuring cognitive set-shifting ability. This task measures NMDA receptor-dependent cognitive abilities analogous to those impaired in schizophrenia. Systemic administration of CDPPB (10 & 30 mg/kg i.p) blocked MK-801 (0.1 mg/kg, i.p.)-induced impairments in set-shifting ability. The effect on learning was dose-dependent, with the 30 mg/kg dose having a greater effect than the 10 mg/kg dose across all trials. This ameliorative effect of CDPPB reflected a reduction in MK-801-induced perseverative responding. These results add to the evidence that mGlu5 receptors interact functionally with NMDA receptors to regulate behavior, and suggest that positive modulators of mGlu5 receptors may have therapeutic potential in the treatment of disorders, like schizophrenia, characterized by impairments in cognitive flexibility and memory.

Stefani, Mark R.; Moghaddam, Bita



Angiotensin (1-7) induces MAS receptor internalization.  


Angiotensin (Ang) (1-7) is the endogenous ligand for the G protein-coupled receptor Mas, a receptor associated with cardiac, renal, and cerebral protective responses. Physiological evidence suggests that Mas receptor (MasR) undergoes agonist-dependent desensitization, but the underlying molecular mechanism regulating receptor activity is unknown. We investigated the hypothesis that MasR desensitizes and internalizes on stimulation with Ang-(1-7). For this purpose, we generated a chimera between the MasR and the yellow fluorescent protein (YFP; MasR-YFP). MasR-YFP-transfected HEK 293T cells were incubated with Ang-(1-7), and the relative cellular distribution of MasR-YFP was observed by confocal microscopy. In resting cells, MasR-YFP was mostly localized to the cell membrane. Ang-(1-7) induced a redistribution of MasR-YFP to intracellular vesicles of various sizes after 5 minutes. Following the time course of [(125)I]Ang-(1-7) endocytosis, we observed that half of MasR-YFP underwent endocytosis after 10 minutes, and this was blocked by a MasR antagonist. MasR-YFP colocalized with Rab5, the early endosome antigen 1, and the adaptor protein complex 2, indicating that the R is internalized through a clathrin-mediated pathway and targeted to early endosomes after Ang-(1-7) stimulation. A fraction of MasR-YFP also colocalized with caveolin 1, suggesting that at some point MasR-YFP traverses caveolin 1-positive compartments. In conclusion, MasR undergoes endocytosis on stimulation with Ang-(1-7), and this event may explain the desensitization of MasR responsiveness. In this way, MasR activity and density may be tightly controlled by the cell. PMID:21670420

Gironacci, Mariela M; Adamo, Hugo P; Corradi, Gerardo; Santos, Robson A; Ortiz, Pablo; Carretero, Oscar A



Identification of the target proteins of rosiglitazone in 3T3-L1 adipocytes through proteomic analysis of cytosolic and secreted proteins  

Microsoft Academic Search

Rosiglitazone, one of the thiazolidinedione (TZD), is an oral antidiabetic drug that activates a gamma isoform of peroxisome\\u000a proliferator-activated receptor (PPAR?). To identify target proteins induced by rosiglitazone in adipocytes, we first performed\\u000a simultaneous in-depth proteomic profiling of cytosolic proteins and secreted proteins (secretome) from 3T3-L1 adipocytes using\\u000a a label-free quantification method with nano-UPLC MS\\/MS. In total, we identified 646

Hyun-Ho Hwang; Pyong-Gon Moon; Jeong-Eun Lee; Jung-Guk Kim; Wan Lee; Sung-Ho Ryu; Moon-Chang Baek



Common molecular mechanisms in field- and agrin-induced acetylcholine receptor clustering.  


1. The aggregation of acetylcholine receptors at the developing neuromuscular junction is critical to the development and function of this synapse. In vitro studies have shown that receptor aggregation can be induced by the finding of agrin to the muscle cell surface and by the electric field-induced concentration of a (nonreceptor) molecule at the cathodal cell pole. 2. We report here on the interaction between agrin binding and electric fields with respect to the distribution of receptors and agrin binding sites. 3. (a) Pretreatment of cells with agrin completely blocks the development of field-induced receptor clusters. (b) Field-induced aggregation of receptors precedes the field-induced aggregation of agrin binding sites by approximately 30 min. (c) Electric fields prevent agrin-induced receptor clustering despite the presence of agrin binding sites and freely diffusing receptors. 4. These results indicate that another membrane component-but not the agrin binding site and not the receptor-is required for agrin-induced receptor clustering. They also suggest that electric fields and agrin cause receptor clustering via common molecular mechanisms. PMID:9140698

Sabrina, F; Stollberg, J



Opiate receptor knockout mice define mu receptor roles in endogenous nociceptive responses and morphine-induced analgesia.  


Morphine produces analgesia at opiate receptors expressed in nociceptive circuits. mu, delta, and kappa opiate receptor subtypes are expressed in circuits that can modulate nociception and receive inputs from endogenous opioid neuropeptide ligands. The roles played by each receptor subtype in nociceptive processing in drug-free and morphine-treated states have not been clear, however. We produced homologous, recombinant mu, opiate receptor, heterozygous and homozygous knockout animals that displayed approximately 54% and 0% of wild-type levels of mu receptor expression, respectively. These mice expressed kappa receptors and delta receptors at near wild-type levels. Untreated knockout mice displayed shorter latencies on tail flick and hot plate tests for spinal and supraspinal nociceptive responses than wild-type mice. These findings support a significant role for endogenous opioid-peptide interactions with mu opiate receptors in normal nociceptive processing. Morphine failed to significantly reduce nociceptive responses in hot plate or tail flick tests of homozygous mu receptor knockout mice, and heterozygote mice displayed right and downward shifts in morphine analgesia dose-effect relationships. These results implicate endogenous opioid-peptide actions at mu opiate receptors in several tests of nociceptive responsiveness and support mu receptor mediation of morphine-induced analgesia in tests of spinal and supraspinal analgesia. PMID:9037090

Sora, I; Takahashi, N; Funada, M; Ujike, H; Revay, R S; Donovan, D M; Miner, L L; Uhl, G R



[Adipocyte differentiation and nuclear receptor].  


The roles of nuclear receptors in differentiation and function of adipocytes were reviewed and discussed. Expression of peroxisome proliferator-activated receptor (PPAR) gamma have been reported to be strongly induced during adipocyte differentiation and maintained in matured adipocytes. Forced expression of PPAR gamma converted NIH3T3 fibroblasts to adipocytes, indicating PPAR gamma regulates essential genes to obtain the adipocyte phenotype. Newly developed antidiabetic thiazolidinediones known as high affinity ligands for PPAR gamma improved insulin resistance. This finding suggests that PPAR gamma contributes regulation of insulin action. Several genes regulated by troglitazone, one of the most potent thiazolidinediones, in matured 3T3-L1 adipocytes-were obtained by differential display PCR method. Orphan receptors ROR alpha/gamma and Rev-ErbA which bind to the same response element are also induced during adipocyte differentiation but their function is still to be investigated. PMID:9200953

Hirose, T; Kurebayashi, S



Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) Induces Death Receptor 5 Networks That Are Highly Organized*  

PubMed Central

Recent evidence suggests that TNF-related apoptosis-inducing ligand (TRAIL), a death-inducing cytokine with anti-tumor potential, initiates apoptosis by re-organizing TRAIL receptors into large clusters, although the structure of these clusters and the mechanism by which they assemble are unknown. Here, we demonstrate that TRAIL receptor 2 (DR5) forms receptor dimers in a ligand-dependent manner at endogenous receptor levels, and these receptor dimers exist within high molecular weight networks. Using mutational analysis, FRET, fluorescence microscopy, synthetic biochemistry, and molecular modeling, we find that receptor dimerization relies upon covalent and noncovalent interactions between membrane-proximal residues. Additionally, by using FRET, we show that the oligomeric structure of two functional isoforms of DR5 is indistinguishable. The resulting model of DR5 activation should revise the accepted architecture of the functioning units of DR5 and the structurally homologous TNF receptor superfamily members.

Valley, Christopher C.; Lewis, Andrew K.; Mudaliar, Deepti J.; Perlmutter, Jason D.; Braun, Anthony R.; Karim, Christine B.; Thomas, David D.; Brody, Jonathan R.; Sachs, Jonathan N.



Liquiritigenin isolated from Glycyrrhiza uralensis stimulates osteoblast function in osteoblastic MC3T3-E1 cells.  


Liquiritigenin is one of the flavonoids present in Glycyrrhizae radix. In the present study, the effects of liquiritigenin on the function of osteoblastic MC3T3-E1 cells were studied. Liquiritigenin caused a significant elevation of cell growth, alkaline phosphatase activity, collagen synthesis, mineralization, and glutathione content in the cells (P<0.05). Moreover, liquiritigenin significantly decreased the production of reactive oxygen species (ROS) and osteoclast differentiation inducing factors such as tumor necrosis factor ? (TNF-?), interleukin-6 (IL-6), and receptor activator of nuclear factor-?B ligand (RANKL) in the presence of antimycin A, which inhibits mitochondrial electron transport and has been used as a ROS generator. These results demonstrate that liquiritigenin may have positive effects on skeletal structure. PMID:22116056

Choi, Eun Mi



Collagen-Induced Arthritis in TNF Receptor1Deficient Mice: TNF Receptor2 Can Modulate Arthritis in the Absence of TNF Receptor1  

Microsoft Academic Search

TNF is a potent proinflammatory cytokine important for the development of arthritis in human and animals. We have investigated the roles of TNF receptor-1 (TNFR1) and TNF receptor-2 (TNFR2) in collagen-induced arthritis (CIA) by inducing CIA in mice genetically deficient in TNFR1. TNFR1?\\/? mice developed arthritis with similar incidence and severity as TNFR1+\\/? littermates, indicating that TNFR1 is redundant for

Yoshifumi Tada; Alexandra Ho; Syuichi Koarada; Fumitaka Morito; Osamu Ushiyama; Noriaki Suzuki; Yuji Kikuchi; Akihide Ohta; Tak W. Mak; Kohei Nagasawa



Serum thyroid hormone levels in patients with fulminant hepatitis: Usefulness of rT3 and the rT3\\/T3 ratio as prognostic indices  

Microsoft Academic Search

Summary  To evaluate thyroid function in 19 patients with fulminant hepatitis (FH), we have measured total and free 3,5,3?-triiodothyronine\\u000a (T3) and thyroxine (T4), 3,3?,5?-triiodothyronine (reverse T3, rT3), thyroidstimulating hormone (TSH) and thyroxin-binding\\u000a globulin (TBG) in patients with FH, compared with those of 80 patients with other various liver diseases and of 10 healthy\\u000a controls. Patients with FH showed the lowest values

Takashi Kano; Takao Kojima; Takeshi Takahashi; Yasutoshi Muto



Ethanol inhibits neuritogenesis induced by astrocyte muscarinic receptors  

PubMed Central

In utero alcohol exposure can lead to fetal alcohol spectrum disorders, characterized by cognitive and behavioral deficits. In vivo and in vitro studies have shown that ethanol alters neuronal development. We have recently shown that stimulation of M3 muscarinic receptors in astrocytes increases the synthesis and release of fibronectin, laminin, and plasminogen activator inhibitor-1, causing neurite outgrowth in hippocampal neurons. As M3 muscarinic receptor signaling in astroglial cells is strongly inhibited by ethanol, we hypothesized that ethanol may also inhibit neuritogenesis in hippocampal neurons induced by carbachol-stimulated astrocytes. In the present study we report that the effect of carbachol-stimulated astrocytes on hippocampal neuron neurite outgrowth was inhibited in a concentration-dependent manner (25–100 mM) by ethanol. This effect was due to the inhibition of the release of fibronectin, laminin, and plasminogen activator inhibitor-1. Similar effects on neuritogenesis and on the release of astrocyte extracellular proteins were observed after the incubation of astrocytes with carbachol in the presence of 1-butanol, another short-chain alcohol that, like ethanol, is a competitive substrate for phospholipase D, but not by tert-butanol, its analogue that is not a substrate for this enzyme. This study identifies a potential novel mechanism involved in the developmental effects of ethanol mediated by the interaction of ethanol with cell signaling in astrocytes, leading to an impairment in neuron-astrocyte communication.

Guizzetti, Marina; Moore, Nadia H.; Giordano, Gennaro; VanDeMark, Kathryn L.; Costa, Lucio G.



Blockade of 5HT2 Receptor Selectively Prevents MDMA-Induced Verbal Memory Impairment  

Microsoft Academic Search

3,4-Methylenedioxymethamphetamine (MDMA) or ‘ecstasy’ has been associated with memory deficits during abstinence and intoxication. The human neuropharmacology of MDMA-induced memory impairment is unknown. This study investigated the role of 5-HT2A and 5-HT1A receptors in MDMA-induced memory impairment. Ketanserin is a 5-HT2A receptor blocker and pindolol a 5-HT1A receptor blocker. It was hypothesized that pretreatment with ketanserin and pindolol would protect

J H P van Wel; K P C Kuypers; E L Theunissen; W M Bosker; K Bakker; J G Ramaekers; JHP van Wel



Attenuation of D-1 antagonist-induced D-1 receptor upregulation by conccomitant D-2 receptor blockade  

SciTech Connect

The effect of chronic selective D-1 and/or D-2 dopamine receptor blockade on regional D-1 receptor binding was studied in rat brain following chronic treatment with the specific D-1 antagonist SCH 23390 and/or the predominantly D-2 antagonist haloperidol. D-1 receptor density and affinity were evaluated by quantitative autoradiography using /sup 125/I-SCH 23982. Chronic SCH 23390 treatment increased D-1 receptor density by 30 to 40% in the striatum, accumbens and tuberculum olfactorium; receptor affinity remained unchanged. Haloperidol had no effect on D-1 receptor Bmax or Kd values, although, when administered with SCH 23390, reduced the D-1 receptor upregulation induced by the D-1 antagonist in striatum and tuberculum olfactorium, but not in nucleus accumbens, These results may be attributable to D-1/D-2 dopamine receptor interactions occurring in the striatum and tuberculum olfactorium and may have implications for the prevention and treatment of drug-induced extrapyramidal disorders. 34 references, 1 figure, 2 tables.

Parashos, S.A.; Barone, P.; Tucci, I.; Chase, T.N.



Interaction of NT4 and BDNF with gp145 trkb receptor: effect on cellular metabolism  

Microsoft Academic Search

In the present study a silicon microphysiometer (Cytosensor®) was applied in investigating interactions of gp145trkb, a member of the tyrosine kinase receptor family, with different neurotrophic factors. NIH-3T3 cells transfected with gp145 trkb receptors (NIH3T3\\/trkB cells) were utilized in the studies. Treatment with brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4) and neurotrophin-3 (NT-3) induced changes in the metabolic rate of NIH3T3\\/trkB

Meghan A Hopkins; Mary P Rosser; Prabhavathi B Fernandes; Isia Bursuker



Effect of TAT-obestatin on proliferation, differentiation, apoptosis and lipolysis in 3T3-L1 preadipocytes.  


It has been reported that obestatin regulates adipocyte metabolism via receptors on the cell surface. We wondered whether obestatin can interact with intracellular components that activated signalling pathways in adipocytes. Because obestatin (human) only presents one lysine (at position 10), which cannot penetrate the cell membrane, therefore, we used a cell-permeable peptide TAT (49-57) as a vector to carry obestatin across the cell membrane. The goal of this study was to further understand the function of obestatin after penetrating the cell membrane. Our results showed that TAT-obestatin could cross the 3T3-L1 cell membrane in the absence of cytotoxicity. TAT-obestatin showed no effect on the proliferation of 3T3-L1 preadipocytes. In contrast, obestatin significantly stimulated proliferation at a dose of 10(-11) ?M and 10(-13) ?M. In addition, TAT-obestatin demonstrated a more potent inhibitory effect on cell apoptosis induced by serum starvation than that of obestatin. During the progress of adipocyte differentiation, TAT-obestatin and obestatin had no effect on adipogenesis. In the lipolysis assay, TAT-obestatin significantly increased glycerol and free fatty acid release from 3T3-L1 adipocytes after 3?h treatment but showed no significant effect on lipolysis after 24?h and 48?h of treatment. In contrast, obestatin (10(-7) ?M) had no effect on glycerol release after 3, 24 and 48?h of treatment. The difference between the effect of TAT-obestatin and obestatin on adipocytes metabolism indicated that TAT-obestatin may trigger intracellular signalling as well as signalling at the cell membrane. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. PMID:24106000

Ren, Guangcai; He, Zuyong; Cong, Peiqing; Yu, Jingwei; Qin, Yufeng; Chen, Yaosheng; Liu, Xiaohong



Endothelin-A receptor antagonists prevent amyloid-?-induced increase in ETA receptor expression, oxidative stress, and cognitive impairment.  


Alzheimer's disease is a neurodegenerative disorder associated with abnormal accumulation of amyloid-? (A?) which can release endothelin (ET). The present study was conducted to investigate the effect of ET antagonists on A?-induced changes in ETA and ETB receptor expression, oxidative stress, and cognitive impairment. Male Sprague-Dawley rats were treated with A?1-40 in the lateral cerebral ventricles and were administered vehicle or ET antagonists for 14 days. A? treatment produced an increase in ETA receptor expression in the cerebral cortex, hippocampus, and brain stem by 72%, 85%, and 90%, respectively. No change in ETB receptor expression was observed. There was an increase in malondialdehyde (MDA) and decrease in reduced glutathione (GSH) and superoxide dismutase (SOD) levels in A?-treated rats. In the Morris swim task, A? treated rats showed a significant impairment in spatial memory. ET receptor antagonists, BQ123, BMS182874, and TAK-044, significantly decreased A?-induced increase in ETA expression in the cortex, hippocampus, and brain stem. Rats treated with ET antagonists showed significant attenuation of A?-induced changes in the brain MDA, GSH, and SOD levels. Rats treated with specific ETA receptor antagonists, BQ123 and BMS182874, significantly reduced the cognitive impairment induced by A?. However, nonspecific ETA/ETB receptor antagonist TAK-044 did not show any improvement in the learning and memory parameter. This study demonstrates that ETA receptor antagonists are effective in preventing cognitive impairment, changes in ETA expression and oxidative stress induced by A?. It is concluded that ETA receptor antagonists may be useful in improving cognitive impairment due to Alzheimer's disease. PMID:21116051

Briyal, Seema; Philip, Tina; Gulati, Anil



TRPM7 Channels Regulate Proliferation and Adipogenesis in 3T3-L1 Preadipocytes.  


Transient receptor potential melastatin-7 (TRPM7) channels are involved in many cellular physiological and pathological processes. The present study was designed to investigate the expression of TRPM7 channels and the potential role in regulating cell proliferation and adipogenesis in 3T3-L1 preadipocytes with approaches of whole-cell patch voltage-clamp, molecular biology, cell proliferation, adipogenesis, etc. We found that a TRPM7-like current was recorded with Mg(2+) -free pipette solution in 3T3-L1 preadipocytes, and the current was inhibited by intercellular free Mg(2+) . The TRPM7-like current was potentiated by acidic pH and inhibited by 2-aminoethoxydiphenyl borate (2-APB). RT-PCR, Western blot and immunocytochemistry revealed that gene and protein of TRPM7 channels were abundant in 3T3-L1 preadipocytes. Blockade of TRPM7 channels with 2-APB inhibited cell proliferation in 3T3-L1 cells. In addition, knockdown of TRPM7 with specific siRNA inhibited both proliferation and adipogenesis. The present study demonstrates for the first time that TRPM7 channels regulate cell cycle and adipogenesis of 3T3-L1 preadipocytes. J. Cell. Physiol. 229: 60-67, 2014. © 2013 Wiley Periodicals, Inc. PMID:23765921

Chen, Kui-Hao; Xu, Xiao-Hui; Liu, Yi; Hu, Yan; Jin, Man-Wen; Li, Gui-Rong



Low Number of Insulin Receptors but High Receptor Protein Content in Adipose Tissue of Rats with Monosodium Glutamate-Induced Obesity  

Microsoft Academic Search

In order to better understand the mechanisms leading to insulin resis- tance, the number of fat tissue insulin receptors, their anity and insulin receptor protein in rats with monosodium glutamate-induced obesity were studied. Obese rats displayed signicantly lower number of insulin receptors with high anity. Sur- prisingly, the amount of insulin receptor protein was signicantly elevated in these animals. The

S. Zora; D. Jezova; L. Szabova; L. Macho; K. Tybitanclova



?-cryptoxanthin suppresses the adipogenesis of 3T3-L1 cells via RAR activation.  


We recently reported that the oral intake of ?-cryptoxanthin exerted anti-obesity effects by lowering visceral fat levels. In the present study, we characterized the molecular mechanisms underlying the lipid-lowering effects of ?-cryptoxanthin on 3T3-L1 cells. Consistent with our previous findings, ?-cryptoxanthin rapidly reduced the level of intracellular lipids in 3T3-L1 cells as assessed by Oil red O staining. Using an in vitro nuclear receptor binding assay, we demonstrated the ability of ?-cryptoxanthin to bind to and activate members of the retinoic acid receptor (RAR) family. Accordingly, treatment of cells with LE540, an RAR antagonist, abolished the ?-cryptoxanthin-dependent suppression of 3T3-L1 adipogenesis, suggesting that ?-cryptoxanthin mediates its effects on 3T3-L1 cells via RAR activation. In addition, real-time RT-PCR analysis revealed that ?-cryptoxanthin down-regulates mRNA expression of PPAR?, a key regulator of adipocyte differentiation, and that this inhibition was blocked by LE540 treatment. Taken together, these data indicate that RAR activation contributes to the molecular mechanism by which ?-cryptoxanthin prevents obesity. PMID:22472285

Shirakura, Yoshiyuki; Takayanagi, Katsuhiko; Mukai, Katsuyuki; Tanabe, Hiroki; Inoue, Makoto



Agarwood induced laxative effects via acetylcholine receptors on loperamide-induced constipation in mice.  


Agarwood (Aquilaria sinensis, Aquilaria crasna) is well known as an incense in the oriental region such as Thailand, Taiwan, and Cambodia, and is used as a digestive in traditional medicine. We investigated the laxative effects and mechanism of agarwood leaves extracted with ethanol (EEA-1, Aquilaria sinensis; EEA-2, Aquilaria crasna). EEA-1, EEA-2, the main constituents of EEAs (mangiferin, and genkwanin-5-O-primeveroside), and senna increased the frequency and weight of stools in loperamide-induced constipation model mice. EEA-1 and EEA-2 did not induce diarrhea as a side effect, but senna induced severe diarrhea. EEA-1 and senna increased gastro-intestinal (GI) transit in the model mice. EEA-1, but not senna, also increased the intestinal tension of isolated jejunum and ileum in guinea pigs, and the tension increase was blocked by atropine, a muscarinic receptor antagonist, but not by other inhibitors (granicetron, pyrilamine, or bradykinin-antagonist peptide). Furthermore, the increase in frequency and weight of stools induced by EEA-1 were blocked by pre-administration of atropine in the model mice. These findings indicate that EEAs exerted a laxative effect via acetylcholine receptors in the mouse constipation model. PMID:20699592

Kakino, Mamoru; Izuta, Hiroshi; Ito, Tetsuro; Tsuruma, Kazuhiro; Araki, Yoko; Shimazawa, Masamitsu; Oyama, Masayoshi; Iinuma, Munekazu; Hara, Hideaki



P2X7 receptor as sensitive flow sensor for ERK activation in osteoblasts  

Microsoft Academic Search

The involvement of the P2 receptor in the activation of ERK induced by a short transient fluid flow stimulation in MC3T3-E1 osteoblasts was examined in the current study. The ERK activation induced by this transient fluid flow stimulation was followed by an increase in c-fos mRNA expression. Suramin, a non-selective P2 receptor antagonist, and two different P2X7 receptor (P2X7R) antagonists,

Hisashi Okumura; Dai Shiba; Toshikazu Kubo; Takahiko Yokoyama



B Lymphocytes Are Resistant to Death Receptor 5-induced Apoptosis  

PubMed Central

Death Receptor 5 (DR5) induces apoptosis in various types of cells and is a potential therapeutic target. We have investigated whether targeting DR5 could be used to eliminate pathogenic B lymphocytes from systemic lupus erythematosus (SLE) patients. We examined DR5 expression and function on B lymphocytes from healthy controls subjects, SLE patients, and human tonsil. DR5 was expressed similarly on all B cell subpopulations, including resting and activated B cells. Expression of DR5 was equivalent on B cells from SLE patients and healthy subjects. Additionally, DR5 expression was unchanged after B lymphocyte stimulation. However, B cells were resistant to DR5-induced apoptosis, including after in vitro activation. No changes in subsets of B cells were observed in subjects of a trial of CS-1008, an agonist anti-DR5. While DR5 shows promise as a way to selectively eliminate tumor cells and activated synoviocytes, these data suggest DR5 alone cannot be used as a target to remove pathogenic SLE B cells.

Crowder, Roslyn N.; Zhao, Hong; Chatham, W. Winn; Zhou, Tong; Carter, Robert H.



Signal transduction induced in endothelial cells by growth factor receptors involved in angiogenesis  

PubMed Central

Summary New vessel formation during development and in the adult is triggered by concerted signals of largely endothelial-specific receptors for ligands of the VEGF, angiopoietin and ephrin families. The signals and genes induced by these receptors operate in the context of additional signals transduced by non-endothelial specific growth factor receptors, inflammatory cytokine receptors as well as adhesion molecules. We summarize here available data on characteristic signaling of the VEGF receptor-2 and the current state of knowledge regarding the additional different receptor tyrosine kinases of the VEGF, Tie and Ephrin receptor families. Furthermore, the potential cross-talk with signals induced by other growth factors and inflammatory cytokines as well as the modulation by VE-cadherin is discussed.

Hofer, Erhard; Schweighofer, Bernhard



The Selective mGlu5 Receptor Antagonist MTEP, Similar to NMDA Receptor Antagonists, Induces Social Isolation in Rats  

Microsoft Academic Search

It has repeatedly been shown that uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonists can mimic certain aspects of positive and negative symptoms of schizophrenia in human volunteers and laboratory animals. The purpose of the present study was to expand these findings and to determine whether the selective metabotropic glutamate receptor subtype 5 (mGluR5) antagonist, MTEP (3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine), could induce similar effects in Wistar

Eliza Koros; Holger Rosenbrock; Gerald Birk; Carmen Weiss; Frank Sams-Dodd



Human Platelet Aggregation and Degranulation Is Induced In Vitro by L-Thyroxine, but Not by 3,5,3?-Triiodo-L-Thyronine or Diiodothyropropionic Acid (DITPA)  

Microsoft Academic Search

The endogenous thyroid hormones L-thyroxine (T4) and 3,5,3?-triiodo-L-thyronine (T3) induce angiogenesis via an endothelial cell iodothyronine receptor on integrin ?V?3. This receptor also exists on platelets. Diiodothyropropionic acid (DITPA) and GC-1, a noniodinated thyroid hormone analog, also induce angiogenesis. Here we examined the effects of iodothyronines (L-T4 vs L-T3) and analogs DITPA and GC-1 on human platelet function. Subthreshold aggregation

Shaymaa S. Mousa; Faith B. Davis; Paul J. Davis; Shaker A. Mousa



Cannabinoid Receptor Type I Modulates Alcohol-Induced Liver Fibrosis  

PubMed Central

The cannabinoid system (CS) is implicated in the regulation of hepatic fibrosis, steatosis and inflammation, with cannabinoid receptors 1 and 2 (CB1 and CB2) being involved in regulation of pro- and antifibrogenic effects. Daily cannabis smoking is an independent risk factor for the progression of fibrosis in chronic hepatitis C and a mediator of experimental alcoholic steatosis. However, the role and function of CS in alcoholic liver fibrosis (ALF) is unknown so far. Thus, human liver samples from patients with alcoholic liver disease (ALD) were collected for analysis of CB1 expression. In vitro, hepatic stellate cells (HSC) underwent treatment with acetaldehyde, ?9-tetrahydrocannabinol H2O2, endo- and exocannabinoids (2-arachidonoylglycerol (2-AG) and [THC]), and CB1 antagonist SR141716 (rimonabant). In vivo, CB1 knockout (KO) mice received thioacetamide (TAA)/ethanol (EtOH) to induce fibrosis. As a result, in human ALD, CB1 expression was restricted to areas with advanced fibrosis only. In vitro, acetaldehyde, H2O2, as well as 2-AG and THC, alone or in combination with acetaldehyde, induced CB1 mRNA expression, whereas CB1 blockage with SR141716 dose-dependently inhibited HSC proliferation and downregulated mRNA expression of fibrosis-mediated genes PC?1(I), TIMP-1 and MMP-13. This was paralleled by marked cytotoxicity of SR141716 at high doses (5–10 ?mol/L). In vivo, CB1 knockout mice showed marked resistance to alcoholic liver fibrosis. In conclusion, CB1 expression is upregulated in human ALF, which is at least in part triggered by acetaldehyde (AA) and oxidative stress. Inhibition of CB1 by SR141716, or via genetic knock-out protects against alcoholic-induced fibrosis in vitro and in vivo.

Patsenker, Eleonora; Stoll, Matthias; Millonig, Gunda; Agaimy, Abbas; Wissniowski, Till; Schneider, Vreni; Mueller, Sebastian; Brenneisen, Rudolf; Seitz, Helmut K; Ocker, Matthias; Stickel, Felix



Histrionicotoxin enhances agonist-induced desensitization of acetylcholine receptor.  


Dihydroisohistrionicotoxin inhibits acetylcholine receptor-dependent 22Na+ uptake of cultured chick muscle cells with a KI of 0.2 micrometer. The inhibition is noncompetitive with respect to agonists. The toxin enhances desensitization of the receptor by agonists which is accompanied by a 10-fold increase in receptor affinity for agonists. Dihydroisohistrionicotoxin increases the affinity of the desensitized form of the receptor for agonists but not antagonists. The results suggest that dihydroisohistrionicotoxin inhibits the acetylcholine receptor by causing an increase in the affinity of the desensitized form of the receptor for agonists and thereby stabilizing the desensitized state. PMID:272000

Burgermeister, W; Catterall, W A; Witkop, B



Nuclear localization and signalling activity of phosphoinositidase Cbeta in Swiss 3T3 cells  

Microsoft Academic Search

THE hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) is a widespread receptor-coupled signalling system at the plasma membrane of most eukaryotic cells. The existence of an entirely separate nuclear phosphoinositide signalling system is suggested from evidence that purified nuclei synthesize PtdInsP2and phosphatidylinositol 4-phosphate (PtdlnsP) in vitro1and that a transient decrease in the mass of these lipids occurs when Swiss 3T3 cells are

Alberto M. Martelli; R. Stewart Gilmour; Valeria Bertagnolo; Luca M. Neri; Lucia Manzoli; Lucio Cocco



Metformin induces osteoblast differentiation via orphan nuclear receptor SHP-mediated transactivation of Runx2  

Microsoft Academic Search

Metformin is an oral anti-diabetic drug of the biguanide class that is commonly used to treat type 2 diabetes mellitus. This study examined the molecular mechanism for the action of metformin on osteoblast differentiation. Metformin-induced mRNA expression of the osteogenic genes and small heterodimer partner (SHP) in MC3T3E1 cells were determined by RT-PCR and real-time PCR. Metformin increased significantly the

Won Gu Jang; Eun Jung Kim; In-Ho Bae; Kkot-Nim Lee; Yong Deuk Kim; Don-Kyu Kim; Sun-Hun Kim; Chul-Ho Lee; Renny T Franceschi; Hueng-Sik Choi; Jeong-Tae Koh



Influence of surface condition on the fatigue of an aluminum-lithium alloy (2090-T3)  

Microsoft Academic Search

This report examines the axial fatigue life data of the shot peened and unpeened 2090-T3 aluminum-lithium alloy. The effect of shot peening on life was detrimental and insignificant at higher and lower stresses respectively. This life variation is discussed in terms of crack initiation and, subsequently, such shot peened induced surface characteristics as the surface roughness and the surface compressive

A. Eftekhari; J. E. Talia; P. K. Mazumdar



Angiotensin-(1-7) Induces Mas Receptor Internalization  

PubMed Central

Angiotensin (Ang) (1-7) is the endogenous ligand for the G protein-coupled receptor Mas, a receptor (R) associated with cardiac, renal and cerebral protective responses. Physiological evidence suggests that Mas R undergoes agonist-dependent desensitization, but the underlying molecular mechanism regulating R activity is unknown. We investigated the hypothesis that Mas R desensitizes and internalizes upon stimulation with Ang-(1-7). For this purpose, we generated a chimera between the Mas R and the fluorescent protein YFP (MasR-YFP). MasR-YFP transfected HEK 293T cells were incubated with Ang-(1-7) and the relative cellular distribution of MasR-YFP was observed by confocal microscopy. In resting cells, MasR-YFP was mostly localized to the cell membrane. Ang-(1-7) induced a redistribution of MasR-YFP to intracellular vesicles of various sizes after 5 min. Following the time course of [125I]Ang-(1-7) endocytosis we observed that half of MasR-YFP underwent endocytosis after 10 min and this was blocked by a Mas R antagonist. MasR-YFP colocalized with Rab5, the early endosome antigen 1 and the adaptor protein complex 2, indicating that the R is internalized through a clathrin-mediated pathway and targeted to early endosomes after Ang-(1-7) stimulation. A fraction of MasR-YFP also colocalized with caveolin-1 suggesting that at some point MasR-YFP traverses caveolin-1 positive compartments. In conclusion, Mas R undergoes endocytosis upon stimulation with Ang-(1-7) and this event may explain the desensitization of Mas R responsiveness. In this way, Mas R activity and density may be tightly controlled by the cell.

Gironacci, Mariela M.; Adamo, Hugo P.; Corradi, Gerardo; Santos, Robson A.; Ortiz, Pablo; Carretero, Oscar A.



?2-Adrenergic receptor supports prolonged theta tetanus-induced LTP  

PubMed Central

The widespread noradrenergic innervation in the brain promotes arousal and learning by molecular mechanisms that remain largely undefined. Recent work shows that the ?2-adrenergic receptor (?2AR) is linked to the AMPA-type glutamate receptor subunit GluA1 via stargazin and PSD-95 (Joiner ML, Lise MF, Yuen EY, Kam AY, Zhang M, Hall DD, Malik ZA, Qian H, Chen Y, Ulrich JD, Burette AC, Weinberg RJ, Law PY, El-Husseini A, Yan Z, Hell JW. EMBO J 29: 482–495, 2010). We now demonstrate that the ?2AR plays a prominent role in long-term potentiation (LTP) induced by a train of 900 stimuli at 5 Hz (prolonged theta-tetanus-LTP, or PTT-LTP) in the hippocampal CA1 region in mice, which requires simultaneous ?-adrenergic stimulation. Although PTT-LTP was impaired in hippocampal slices from ?1AR and ?2AR knockout (KO) mice, only ?2AR-selective stimulation with salbutamol supported this PTT-LTP in wild-type (WT) slices, whereas ?1AR-selective stimulation with dobutamine (+ prazosin) did not. Furthermore, only the ?2AR-selective antagonist ICI-118551 and not the ?1AR-selective antagonist CGP-20712 inhibited PTT-LTP and phosphorylation of GluA1 on its PKA site S845 in WT slices. Our analysis of S845A knockin (KI) mice indicates that this phosphorylation is relevant for PTT-LTP. These results identify the ?2AR-S845 signaling pathway as a prominent regulator of synaptic plasticity.

Qian, Hai; Matt, Lucas; Zhang, Mingxu; Nguyen, Minh; Patriarchi, Tommaso; Koval, Olha M.; Anderson, Mark E.; He, Kaiwen; Lee, Hey-Kyoung



Differential effects of selective adenosine A 1 and A 2A receptor agonists on dopamine receptor agonist-induced behavioural responses in rats  

Microsoft Academic Search

The effects of the systemic (i.p.) administration of the selective adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) and the selective adenosine A2A receptor agonist sodium 2-p-carboxyethyl)phenylamino-5?-N-carboxamidoadenosine (CGS 21680) on different dopamine receptor agonist-induced behaviours were studied in the male rat. CGS 21680 (1 ?mol\\/kg), but not CPA, was found to counteract the stereotypies induced by the non-selective dopamine receptor agonist apomorphine

Roberto Rimondini; Sergi Ferré; Lydia Giménez-Llort; Sven Ove Ögren; Kjell Fuxe



Protective effect of ?-lipoic acid on islet cells co-cultured with 3T3L1 adipocytes  

PubMed Central

Obesity and ?-cell dysfunction due to oxidative stress impact the pathogenesis of type 2 diabetes mellitus. We co-cultured 3T3L1 adipocytes and islet cells in the presence or absence of the antioxidant ?-lipoic acid (LA) and assayed the effects of the adipocytes and LA on the secretion of insulin by the islet cells and on the activities of factors involved in secretion and oxidative stress. At low glucose concentrations (2.8 mmol/l), the presence of adipocytes (co-culture) increased insulin secretion compared with islet cells cultured alone (control) and this increase was diminished by LA (co-culture plus LA). At high glucose concentrations (22 mmol/l), insulin secretion levels were similar for all islet groups, resulting in a restoration of the stimulation index in the presence of LA. The mRNA levels of the glucose-stimulated insulin secretion (GSIS) genes glucokinase, glucose transporter 2 and Kir6.2 were downregulated under co-culture and co-culture plus LA conditions. Protein and tyrosine phosphorylation levels of insulin receptor-? and insulin receptor substrate-1 were decreased under co-culture conditions and were restored by LA treatment. Cellular malondialdehyde levels increased in the co-cultured islets and this increase was blocked by LA. The mRNA levels of superoxide dismutase and catalase were reduced under co-culture conditions and these reductions were eliminated by the addition of LA. In conclusion, 3T3L1 adipocytes disturb insulin secretion and induce islet dysfunction. The effects may be mediated by multiple pathways, which include downregulation of GSIS gene expression, suppression of islet cell insulin signaling and the induction of oxidative stress. LA may protect islet cells via activation of islet cell insulin signaling and the mRNA expression of antioxidant enzymes.




Methotrexate enhances 3T3-L1 adipocytes hypertrophy.  


Methotrexate (MTX) is broadly used in the treatment of chronic inflammatory diseases such as rheumatoid arthritis (RA). The prevalence of metabolic syndrome (MeS) in patients with this condition is relatively high. Given the importance of adipose tissue in the development of obesity metabolic complications, this study aimed to investigate the effect of methotrexate on preadipocyte proliferation, adipogenesis, and glucose uptake by adipocytes. 3T3-L1 preadipocytes proliferation was evaluated by sulforhodamine B staining and (3)H-thymidine incorporation, after 24 or 48 h of treatment with MTX (0.1 and 10 ?M). Preadipocytes were induced to differentiate with an appropriate adipogenic cocktail in the presence or absence of MTX. Adipogenesis was determined by measuring lipid accumulation after staining with oil red O. (3)H-Deoxyglucose ((3)H-DG) uptake was determined by liquid scintillation counting. MTX treatment reduced culture protein content in a concentration-dependent manner and (3)H-thymidine incorporation (P?

Marques, Cláudia; Teixeira, Diana; Cunha, Ana; Meireles, Manuela; Pestana, Diogo; Keating, Elisa; Calhau, Conceiçăo; Monteiro, Rosário; Faria, Ana



Low number of insulin receptors but high receptor protein content in adipose tissue of rats with monosodium glutamate-induced obesity.  


In order to better understand the mechanisms leading to insulin resistance, the number of fat tissue insulin receptors, their affinity and insulin receptor protein in rats with monosodium glutamate-induced obesity were studied. Obese rats displayed significantly lower number of insulin receptors with high affinity. Surprisingly, the amount of insulin receptor protein was significantly elevated in these animals. The same relations have been already reported for angiotensin II binding and AT1 receptor protein in the same model of obesity. Therefore we suggest an existence of general defect of adipocyte cell membrane in monosodium glutamate-induced obesity characterized by the presence of high quantity of impaired receptor protein. PMID:15113127

Zorad, S; Jezova, D; Szabova, L; Macho, L; Tybitanclova, K



HIV-induced kidney cell injury: role of ROS-induced downregulated vitamin D receptor.  


Reactive oxygen species (ROS) have been demonstrated to contribute to HIV-induced tubular cell injury. We hypothesized that HIV-induced ROS generation may be causing tubular cell injury through downregulation of vitamin D receptor (VDR) and associated downstream effects. In the present study, HIV not only downregulated tubular cell VDR expression but also inflicted DNA injury. On the other hand, EB-1089, a VDR agonist (VD), inhibited both downregulation of VDR and tubular cell DNA injury in the HIV milieu. H(2)O(2) (an O(-) donor) directly downregulated tubular cell VDR, whereas catalase, a free radical scavenger, inhibited HIV-induced downregulation of tubular cell VDR expression. HIV also stimulated the tubular cell renin-angiotensin system (RAS) through downregulation of VDR. Because losartan (an ANG II blolcker) partially inhibited HIV-induced tubular cell ROS generation while ANG II directly stimulated tubular cell ROS generation, it appears that HIV-induced ROS production was partly contributed by the RAS activation. VD not only inhibited HIV-induced RAS activation but also attenuated tubular cell ROS generation. Tubular cells displayed double jeopardy in the HIV milieu induction of double-strand breaks and attenuated DNA repair; additionally, in the HIV milieu, tubular cells exhibited enhanced expression of phospho-p53 and associated downstream signaling. A VDR agonist and an ANG II blocker not only preserved expression of tubular cell DNA repair proteins but also inhibited induction of double-strand breaks. In in vivo studies, renal cortical sections of Tg26 mice displayed attenuated expression of VDR both in podocytes and tubular cells. In addition, renal cortical sections of Tg26 mice displayed enhanced oxidative stress-induced kidney cell DNA damage. These findings indicated that HIV-induced tubular cell downregulation of VDR contributed to the RAS activation and associated tubular cell DNA damage. However, both VD and RAS blockade provided protection against these effects of HIV. PMID:22647636

Salhan, Divya; Husain, Mohammad; Subrati, Ashaan; Goyal, Rohan; Singh, Tejinder; Rai, Partab; Malhotra, Ashwani; Singhal, Pravin C



HIV-induced kidney cell injury: role of ROS-induced downregulated vitamin D receptor  

PubMed Central

Reactive oxygen species (ROS) have been demonstrated to contribute to HIV-induced tubular cell injury. We hypothesized that HIV-induced ROS generation may be causing tubular cell injury through downregulation of vitamin D receptor (VDR) and associated downstream effects. In the present study, HIV not only downregulated tubular cell VDR expression but also inflicted DNA injury. On the other hand, EB-1089, a VDR agonist (VD), inhibited both downregulation of VDR and tubular cell DNA injury in the HIV milieu. H2O2 (an O? donor) directly downregulated tubular cell VDR, whereas catalase, a free radical scavenger, inhibited HIV-induced downregulation of tubular cell VDR expression. HIV also stimulated the tubular cell renin-angiotensin system (RAS) through downregulation of VDR. Because losartan (an ANG II blolcker) partially inhibited HIV-induced tubular cell ROS generation while ANG II directly stimulated tubular cell ROS generation, it appears that HIV-induced ROS production was partly contributed by the RAS activation. VD not only inhibited HIV-induced RAS activation but also attenuated tubular cell ROS generation. Tubular cells displayed double jeopardy in the HIV milieu induction of double-strand breaks and attenuated DNA repair; additionally, in the HIV milieu, tubular cells exhibited enhanced expression of phospho-p53 and associated downstream signaling. A VDR agonist and an ANG II blocker not only preserved expression of tubular cell DNA repair proteins but also inhibited induction of double-strand breaks. In in vivo studies, renal cortical sections of Tg26 mice displayed attenuated expression of VDR both in podocytes and tubular cells. In addition, renal cortical sections of Tg26 mice displayed enhanced oxidative stress-induced kidney cell DNA damage. These findings indicated that HIV-induced tubular cell downregulation of VDR contributed to the RAS activation and associated tubular cell DNA damage. However, both VD and RAS blockade provided protection against these effects of HIV.

Salhan, Divya; Husain, Mohammad; Subrati, Ashaan; Goyal, Rohan; Singh, Tejinder; Rai, Partab; Malhotra, Ashwani



Functional selectivity induced by mGlu? receptor positive allosteric modulation and concomitant activation of Gq coupled receptors.  


Metabotropic glutamate receptors (mGlus) are a group of Family C Seven Transmembrane Spanning Receptors (7TMRs) that play important roles in modulating signaling transduction, particularly within the central nervous system. mGlu(4) belongs to a subfamily of mGlus that is predominantly coupled to G(i/o) G proteins. We now report that the ubiquitous autacoid and neuromodulator, histamine, induces substantial glutamate-activated calcium mobilization in mGlu(4)-expressing cells, an effect which is observed in the absence of co-expressed chimeric G proteins. This strong induction of calcium signaling downstream of glutamate activation of mGlu(4) depends upon the presence of H(1) histamine receptors. Interestingly, the potentiating effect of histamine activation does not extend to other mGlu(4)-mediated signaling events downstream of G(i/o) G proteins, such as cAMP inhibition, suggesting that the presence of G(q) coupled receptors such as H(1) may bias normal mGlu(4)-mediated G(i/o) signaling events. When the activity induced by small molecule positive allosteric modulators of mGlu(4) is assessed, the potentiated signaling of mGlu(4) is further biased by histamine toward calcium-dependent pathways. These results suggest that G(i/o)-coupled mGlus may induce substantial, and potentially unexpected, calcium-mediated signaling events if stimulation occurs concomitantly with activation of G(q) receptors. Additionally, our results suggest that signaling induced by small molecule positive allosteric modulators may be substantially biased when G(q) receptors are co-activated. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'. PMID:22426233

Yin, Shen; Zamorano, Rocio; Conn, P Jeffrey; Niswender, Colleen M



Adenosine A2A receptor induces protein kinase A-dependent functional modulation of human ?3?4 nicotinic receptor  

PubMed Central

Abstract Adenosine modulates the function of nicotinic ACh receptors (nAChRs) in a variety of preparations, possibly through pathways involving protein kinase A (PKA), but these phenomena have not yet been investigated in detail. In this work we studied, using the patch clamp technique, the functional modulation of recombinant human ?3?4 nAChR by the A2A adenosine receptor, co-expressed in HEK cells. Tonic activation of A2A receptor slowed current decay during prolonged applications of nicotine and accelerated receptor recovery from desensitization. Together, these changes resulted into a more sustained current response upon multiple nicotine or ACh applications. These findings were confirmed in cultured mouse superior cervical ganglion neurones, which express nAChR containing the ?3 subunit together with ?2 and/or ?4 and A2A receptor. Expression of the A2A receptor in HEK cells also increased the apparent potency of nAChR for nicotine, further supporting a general A2A-induced gain of function for nAChR. These effects were dependent on PKA since the direct activation of PKA mimicked, and its inhibition prevented almost completely, the effects of the A2A receptor. Mutations of R385 and S388 in the cytoplasmic loop of the ?3 subunit abolished the functional modulation of nAChR induced by activation of A2A receptor, PKA and other Ser/Thr kinases, suggesting that this region constitutes a putative consensus site for these kinases. These data provide conclusive evidence that activation of the A2A receptor determines functional changes of human ?3?4 nAChR, through pathways involving PKA.

Di Angelantonio, Silvia; Piccioni, Alessio; Moriconi, Claudia; Trettel, Flavia; Cristalli, Gloria; Grassi, Francesca; Limatola, Cristina



Inhibitor of DNA Binding 2 Is a Small Molecule-Inducible Modulator of Peroxisome Proliferator-Activated Receptor-? Expression and Adipocyte Differentiation  

PubMed Central

We previously identified the small molecule harmine as a regulator of peroxisome proliferator activated-receptor ? (PPAR?) and adipocyte differentiation. In an effort to identify signaling pathways mediating harmine’s effects, we performed transcriptional profiling of 3T3-F442A preadipocytes. Inhibitor of DNA biding 2 (Id2) was identified as a gene rapidly induced by harmine but not by PPAR? agonists. Id2 is also induced in 3T3-L1 preadipocytes treated with dexamethasone, 3-isobutyl-1-methylxanthine, and insulin, suggesting that Id2 regulation is a common feature of the adipogenic program. Stable overexpression of Id2 in preadipocytes promotes expression of PPAR? and enhances morphological differentiation and lipid accumulation. Conversely, small interfering RNA-mediated knockdown of Id2 antagonizes adipocyte differentiation. Mice lacking Id2 expression display reduced adiposity, and embryonic fibroblasts derived from these mice exhibit reduced PPAR? expression and a diminished capacity for adipocyte differentiation. Finally, Id2 expression is elevated in adipose tissues of obese mice and humans. These results outline a role for Id2 in the modulation of PPAR? expression and adipogenesis and underscore the utility of adipogenic small molecules as tools to dissect adipocyte biology.

Won Park, Kye; Waki, Hironori; Villanueva, Claudio J.; Monticelli, Laurel A.; Hong, Cynthia; Kang, Sona; MacDougald, Ormond A.; Goldrath, Ananda W.; Tontonoz, Peter



Behavior and Cellular Evidence for Propofol-Induced Hypnosis Involving Brain Glycine Receptors  

PubMed Central

Background It is well documented that several general anesthetics, including propofol, potentiate glycine receptor function. Furthermore, glycine receptors exist throughout the central nervous system, including areas of the brain thought to be involved in sleep. However, the role of glycine receptors in anesthetic-induced hypnosis has not been determined. Methods Experiments were conducted in rats, where the loss of righting reflex (LORR) was used as a marker of the hypnotic state. Propofol-induced LORR was examined in the presence and the absence of strychnine (a glycine receptor antagonist), GABAzine (a ?-aminobutyric acid A receptor antagonist), as well as ketamine (an antagonist of N-methyl-D-aspartic acid subtype of glutamate receptors). Furthermore, the effects of propofol on the currents elicited by glycine and ?-aminobutyric acid were analyzed in neurons isolated from the posterior hypothalamus of rats. The effects of strychnine and GABAzine on propofol-induced currents were also evaluated. Results Strychnine and GABAzine dose-dependently reduced the percentage of rats exhibiting LORR induced by propofol. Furthermore, strychnine significantly increased the onset time and reduced the duration of LORR induced by propofol. In contrast, strychnine did not affect the LORR induced by ketamine. Additionally, propofol markedly increased the currents elicited by glycine and GABA of hypothalamic neurons. Conversely, strychnine and GABAzine both profoundly attenuated the current induced by propofol. Conclusion Strychnine, the glycine receptor antagonist dose-dependently reduced propofol-induced loss of righting reflex in rats and propofol-induced current of rat hypothalamic neurons. These results suggest that neuronal glycine receptors partially contribute to propofol-induced hypnosis.

Nguyen, Hai T; Li, Ke-yong; da Graca, Ralph L; Delphin, Ellise; Xiong, Ming; Ye, Jiang H



Ligand-induced interaction between. alpha. - and. beta. -type platelet-derived growth factor (PDGF) receptors: Role of receptor heterodimers in kinase activation  

SciTech Connect

Two types of PDGF receptors have been cloned and sequenced. Both receptors are transmembrane glycoproteins with a ligand-stimulatable tyrosine kinase site. The authors have shown earlier that ligand-induced activation of the {beta}-type PDGF receptor is due to the conversion of the monomeric form of the receptor to the dimeric form. In the present studies, they have established the ligand-binding specificity of two receptor types and extended it further to investigate the ligand-induced association state of the {alpha}-receptor and the role of {alpha}-receptor in the activation of {beta}-receptor. These studies were conducted with cells that express one or the other type of PDGF receptor as well as with cells that express both types of receptors. Moreover, ligand-binding characteristics of the receptor were confirmed by immunoprecipitation of the receptor-{sup 125}I-PDGF covalent complex with type-specific anti-PDGF receptor antibodies. These studies revealed that all three isoforms of PDGF bind to {alpha}-receptor, and such binding leads to dimerization as well as activation of the receptor. In contrast, {beta}-receptor can be activated only by PDGF BB and not by PDGF AB or PDGF AA. However, by using antipeptide antibodies that are specific for {alpha}- or {beta}-type PDGF receptor, they demonstrated that in the presence of {alpha}-receptor, {beta}-receptor kinase can be activated by PDGF AB. They present here direct evidence that strongly suggests that such PDGF AB induced activation of {beta}-receptor is due to the formation of a noncovalently linked {alpha}-{beta} receptor heterodimer.

Kanakaraj, P.; Raj, S.; Bishayee, S. (Coriell Institute for Medical Research, Camden, NJ (USA)); Khan, S.A. (Wistar Institute, Philadelphia, PA (USA))



Glyphosate induces human breast cancer cells growth via estrogen receptors.  


Glyphosate is an active ingredient of the most widely used herbicide and it is believed to be less toxic than other pesticides. However, several recent studies showed its potential adverse health effects to humans as it may be an endocrine disruptor. This study focuses on the effects of pure glyphosate on estrogen receptors (ERs) mediated transcriptional activity and their expressions. Glyphosate exerted proliferative effects only in human hormone-dependent breast cancer, T47D cells, but not in hormone-independent breast cancer, MDA-MB231 cells, at 10(-12) to 10(-6)M in estrogen withdrawal condition. The proliferative concentrations of glyphosate that induced the activation of estrogen response element (ERE) transcription activity were 5-13 fold of control in T47D-KBluc cells and this activation was inhibited by an estrogen antagonist, ICI 182780, indicating that the estrogenic activity of glyphosate was mediated via ERs. Furthermore, glyphosate also altered both ER? and ? expression. These results indicated that low and environmentally relevant concentrations of glyphosate possessed estrogenic activity. Glyphosate-based herbicides are widely used for soybean cultivation, and our results also found that there was an additive estrogenic effect between glyphosate and genistein, a phytoestrogen in soybeans. However, these additive effects of glyphosate contamination in soybeans need further animal study. PMID:23756170

Thongprakaisang, Siriporn; Thiantanawat, Apinya; Rangkadilok, Nuchanart; Suriyo, Tawit; Satayavivad, Jutamaad



Acetylcholine receptor-inducing factor from chicken brain increases the level of mRNA encoding the receptor. alpha. subunit  

SciTech Connect

A 42-kDa glycoprotein isolated from chicken brain, referred to as acetylcholine receptor-inducing activity (ARIA), that stimulates the rate of incorporation of acetylcholine receptors into the surface of chicken myotubes may play a role in the nerve-induced accumulation of receptors at developing neuromuscular synapses. Using nuclease-protection assays, the authors have found that ARIA causes a 2- to 16-fold increase in the level of mRNA encoding the {alpha} subunit of the receptor, with little or no change in the levels of {gamma}- and {delta}-subunit messengers. ARIA also increases the amount of a putative nuclear precursor of {alpha}-subunit mRNA, consistent with an activation of gene transcription. These results suggest that the concentration of {alpha} subunit may limit the rate of biosynthesis of the acetylcholine receptors in chicken myotubes. They also indicate that neuronal factors can regulate the expression of receptor subunit genes in a selective manner. Tetrodotoxin, 8-bromo-cAMP, and forskolin also increase the amount of {alpha}-subunit mRNA, with little change in the amount of {gamma}- and {delta}-subunit mRNAs. Unlike ARIA, however, these agents have little effect on the concentration of the {alpha}-subunit nuclear precursor.

Harris, D.A.; Falls, D.L.; Dill-Devor, R.M.; Fischbach, G.D. (Washington Univ. School of Medicine, St. Louis, MO (USA))



A1 and A3 adenosine receptors inhibit LPS-induced hypoxia-inducible factor-1 accumulation in murine astrocytes.  


Adenosine (Ado) exerts neuroprotective and anti-inflammatory functions by acting through four receptor subtypes A1, A2A, A2B and A3. Astrocytes are one of its targets in the central nervous system. Hypoxia-inducible factor-1 (HIF-1), a master regulator of oxygen homeostasis, is induced after hypoxia, ischemia and inflammation and plays an important role in brain injury. HIF-1 is expressed by astrocytes, however the regulatory role played by Ado on HIF-1? modulation induced by inflammatory and hypoxic conditions has not been investigated. Primary murine astrocytes were activated with lipopolysaccharide (LPS) with or without Ado, Ado receptor agonists, antagonists and receptor silencing, before exposure to normoxia or hypoxia. HIF-1? accumulation and downstream genes regulation were determined. Ado inhibited LPS-increased HIF-1? accumulation under both normoxic and hypoxic conditions, through activation of A1 and A3 receptors. In cells incubated with the blockers of p44/42 MAPK and Akt, LPS-induced HIF-1? accumulation was significantly decreased in normoxia and hypoxia, suggesting the involvement of p44/42 MAPK and Akt in this effect and Ado inhibited kinases phosphorylation. A series of angiogenesis and metabolism related genes were modulated by hypoxia in an HIF-1 dependent way, but not further increased by LPS, with the exception of GLUT-1 and hexochinase II that were elevated by LPS only in normoxia and inhibited by Ado receptors. Instead, genes involved in inflammation, like inducible nitric-oxide synthase (iNOS) and A2B receptors, were increased by LPS in normoxia, strongly stimulated by LPS in concert with hypoxia and inhibited by Ado, through A1 and A3 receptor subtypes. In conclusion A1 and A3 receptors reduce the LPS-mediated HIF-1? accumulation in murine astrocytes, resulting in a downregulation of genes involved in inflammation and hypoxic injury, like iNOS and A2B receptors, in both normoxic and hypoxic conditions. PMID:23969284

Gessi, Stefania; Merighi, Stefania; Stefanelli, Angela; Fazzi, Debora; Varani, Katia; Borea, Pier Andrea



Differential subcellular distribution of rat brain dopamine receptors and subtype-specific redistribution induced by cocaine  

PubMed Central

We investigated the subcellular distribution of dopamine D1, D2 and D5 receptor subtypes in rat frontal cortex, and examined whether psychostimulant-induced elevation of synaptic dopamine could alter the receptor distribution. Differential detergent solubilization and density gradient centrifugation were used to separate various subcellular fractions, followed by semi-quantitative determination of the relative abundance of specific receptor proteins in each fraction. D1 receptors were predominantly localized to detergent-resistant membranes, and a portion of these receptors also floated on sucrose gradients. These properties are characteristic of proteins found in lipid rafts and caveolae. D2 receptors exhibited variable distribution between cytoplasmic, detergent-soluble and detergent-resistant membrane fractions, yet were not present in buoyant membranes. Most D5 receptor immunoreactivity was distributed into the cytoplasmic fraction, failing to sediment at forces up to 300,000g, while the remainder was localized to detergent-soluble membranes in cortex. D5 receptors were undetectable in detergent-resistant fractions or raft-like subdomains. Following daily cocaine administration for seven days, a significant portion of D1 receptors translocated from detergent-resistant membranes to detergent-soluble membranes and the cytoplasmic fraction. The distributions of D5 and D2 receptor subtypes were not significantly altered by cocaine treatment. These data imply that D5 receptors are predominantly cytoplasmic, D2 receptors are diffusely distributed within the cell, whereas D1 receptors are mostly localized to lipid rafts within the rat frontal cortex. Dopamine receptor subtype localization is susceptible to modulation by pharmacological manipulations that elevate synaptic dopamine, however the functional implications of such drug-induced receptor warrant further investigation.

Voulalas, Pamela J.; Schetz, John; Undieh, Ashiwel S.



5-HT1A receptor blockade reverses GABAA receptor ?3 subunit-mediated anxiolytic effects on stress-induced hyperthermia  

PubMed Central

Rationale Stress-related disorders are associated with dysfunction of both serotonergic and GABAergic pathways, and clinically effective anxiolytics act via both neurotransmitter systems. As there is evidence that the GABAA and the serotonin receptor system interact, a serotonergic component in the anxiolytic actions of benzodiazepines could be present. Objectives The main aim of the present study was to investigate whether the anxiolytic effects of (non-)selective ? subunit GABAA receptor agonists could be reversed with 5-HT1A receptor blockade using the stress-induced hyperthermia (SIH) paradigm. Results The 5-HT1A receptor antagonist WAY-100635 (0.1–1 mg/kg) reversed the SIH-reducing effects of the non-?-subunit selective GABAA receptor agonist diazepam (1–4 mg/kg) and the GABAA receptor ?3-subunit selective agonist TP003 (1 mg/kg), whereas WAY-100635 alone was without effect on the SIH response or basal body temperature. At the same time, co-administration of WAY-100635 with diazepam or TP003 reduced basal body temperature. WAY-100635 did not affect the SIH response when combined with the preferential ?1-subunit GABAA receptor agonist zolpidem (10 mg/kg), although zolpidem markedly reduced basal body temperature. Conclusions The present study suggests an interaction between GABAA receptor ?-subunits and 5-HT1A receptor activation in the SIH response. Specifically, our data indicate that benzodiazepines affect serotonergic signaling via GABAA receptor ?3-subunits. Further understanding of the interactions between the GABAA and serotonin system in reaction to stress may be valuable in the search for novel anxiolytic drugs.

van Oorschot, Ruud; Korte, S. Mechiel; Olivier, Berend; Groenink, Lucianne



A transgenic mouse model of neuroepithelial cell specific inducible overexpression of dopamine D1-receptor  

PubMed Central

Dopamine and its receptors appear in the brain during early embryonic period suggesting a role for dopamine in brain development. In fact, dopamine receptor imbalance resulting from impaired physiological balance between D1- and D2-receptor activities can perturb brain development and lead to persisting changes in brain structure and function. Dopamine receptor imbalance can be produced experimentally using pharmacological or genetic methods. Pharmacological methods tend to activate or antagonize the receptors in all cell types. In the traditional gene knockout models the receptor imbalance occurs during development and also at maturity. Therefore, assaying the effects of dopamine imbalance on specific cell types (e.g. precursor versus postmitotic cells) or at specific periods of brain development (e.g. pre- or postnatal periods) is not feasible in these models. We describe a novel transgenic mouse model based on the tetracycline dependent inducible gene expression system in which dopamine D1-receptor transgene expression is induced selectively in neuroepithelial cells of the embryonic brain at experimenter-chosen intervals of brain development. In this model, doxycycline-induced expression of the transgene causes significant overexpression of the D1-receptor and significant reductions in the incorporation of the S-phase marker bromodeoxyuridine into neuroepithelial cells of the basal and dorsal telencephalon indicating marked effects on telencephalic neurogenesis. The D1-receptor overexpression occurs at higher levels in the medial ganglionic eminence than the lateral ganglionic eminence or cerebral wall. Moreover, although the transgene is induced selectively in the neuroepithelium, D1-receptor protein overexpression appears to persist in postmitotic cells. The mouse model can be modified for neuroepithelial cell-specific inducible expression of other transgenes or induction of the D1-receptor transgene in other cells in specific brain regions by crossbreeding the mice with transgenic mouse lines available already.

Fujimoto, Kumiko; Araki, Kiyomi; McCarthy, Deirdre M.; Sims, John R.; Ren, Jia-Qian; Zhang, Xuan; Bhide, Pradeep G.



Effect of Cannabinoid Receptor Agonists on Streptozotocin-Induced Hyperalgesia in Diabetic Neuropathy  

Microsoft Academic Search

The effect of CB-1 and CB-2 receptor agonists, as well as an influence of a non-selective inhibitor of nitric oxide synthase (NOS), L-NOArg, and an inhibitor acting preferentially on cyclooxygenase-1 (COX-1), indomethacin, on the action of cannabinoid receptor agonists in a streptozotocin (STZ)-induced neuropathic model was investigated. When administered alone, a non-selective cannabinoid receptor agonist, WIN 55,212-2, a potentially selective

Magdalena Bujalska



Proteomic analysis of rosiglitazone and guggulsterone treated 3T3-L1 preadipocytes.  


Adipogenesis is the differentiation of preadipocytes to adipocytes which is marked by the accumulation of lipid droplets. Adipogenic differentiation of 3T3-L1 cells is achieved by exposing the cells to Insulin, Dexamethasone and IBMX for 5-7 days. Thiazolidinedione drugs, like rosiglitazone are potent insulin sensitizing agents and have been shown to enhance lipid droplet formation in 3T3-L1 cells, a model cell line for preadipocyte differentiation. Guggulsterone is a natural drug extracted from the gum resin of tree Commiphora mukul. Guggulsterone has been shown to inhibit adipogenesis and induce apoptosis in 3T3-L1 cells. In this study we treated the 3T3-L1 preadipocytes with rosiglitazone and guggulsterone and assessed the protein expression profile using 2D gel electrophoresis-based proteomics to find out differential target proteins of these drugs. The proteins that were identified upon rosiglitazone treatment generally regulate cell proliferation and/or exhibit anti-inflammatory effect which strengthens its differentiation-inducing property. Guggulsterone treatment resulted in the identification of the apoptosis-inducing proteins to be up regulated which rightly is in agreement with the apoptosis-inducing property of guggulsterone in 3T3-L1 cells. Some of the proteins identified in our proteomic screen such as Galectin1, AnnexinA2 & TCTP were further confirmed by Real Time qPCR. Thus, the present study provides a better outlook of proteins being differentially regulated/expressed upon treatment with rosiglitazone and guggulsterone. The detailed study of the differentially expressed proteins identified in this proteomic screen may further provide the better molecular insight into the mode of action of these anti-diabetic drugs rosiglitazone and guggulsterone. PMID:23275126

Pal, Pooja; Kanaujiya, Jitendra K; Lochab, Savita; Tripathi, Shashi B; Sanyal, Sabyasachi; Behre, Gerhard; Trivedi, Arun K



The role of hypothalamic H1 receptor antagonism in antipsychotic-induced weight gain.  


Treatment with second generation antipsychotics (SGAs), notably olanzapine and clozapine, causes severe obesity side effects. Antagonism of histamine H1 receptors has been identified as a main cause of SGA-induced obesity, but the molecular mechanisms associated with this antagonism in different stages of SGA-induced weight gain remain unclear. This review aims to explore the potential role of hypothalamic histamine H1 receptors in different stages of SGA-induced weight gain/obesity and the molecular pathways related to SGA-induced antagonism of these receptors. Initial data have demonstrated the importance of hypothalamic H1 receptors in both short- and long-term SGA-induced obesity. Blocking hypothalamic H1 receptors by SGAs activates AMP-activated protein kinase (AMPK), a well-known feeding regulator. During short-term treatment, hypothalamic H1 receptor antagonism by SGAs may activate the AMPK-carnitine palmitoyltransferase 1 signaling to rapidly increase caloric intake and result in weight gain. During long-term SGA treatment, hypothalamic H1 receptor antagonism can reduce thermogenesis, possibly by inhibiting the sympathetic outflows to the brainstem rostral raphe pallidus and rostral ventrolateral medulla, therefore decreasing brown adipose tissue thermogenesis. Additionally, blocking of hypothalamic H1 receptors by SGAs may also contribute to fat accumulation by decreasing lipolysis but increasing lipogenesis in white adipose tissue. In summary, antagonism of hypothalamic H1 receptors by SGAs may time-dependently affect the hypothalamus-brainstem circuits to cause weight gain by stimulating appetite and fat accumulation but reducing energy expenditure. The H1 receptor and its downstream signaling molecules could be valuable targets for the design of new compounds for treating SGA-induced weight gain/obesity. PMID:23640535

He, Meng; Deng, Chao; Huang, Xu-Feng



Role of spinal 5-HT receptors in cutaneous hypersensitivity induced by REM sleep deprivation.  


Previous studies indicate that rapid eye movement (REM) sleep deprivation facilitates pain sensitivity. Since serotoninergic raphe neurons are involved both in regulation of sleep and descending pain modulation, we studied whether spinal 5-HT receptors have a role in sleep deprivation-induced facilitation of pain-related behavior. REM sleep deprivation of 48h was induced by the flower pot method in the rat. The pain modulatory influence of various serotoninergic compounds administered intrathecally was assessed by determining limb withdrawal response to monofilaments. REM sleep deprivation produced a marked hypersensitivity. Sleep deprivation-induced hypersensitivity and normal sensitivity in controls were reduced both by a 5-HT(1A) receptor antagonist (WAY-100635) and a 5-HT(2C) receptor antagonist (RS-102221). An antagonist of the 5-HT(3) receptor (LY-278584) failed to modulate hypersensitivity in sleep-deprived or control animals. Paradoxically, sensitivity in sleep-deprived and control animals was reduced not only by a 5-HT(1A) receptor antagonist but also by a 5-HT(1A) receptor agonist (8-OHDPAT). The results indicate that serotoninergic receptors in the spinal cord have a complex role in the control of sleep-deprivation induced cutaneous hypersensitivity as well as baseline sensitivity in control conditions. While endogenous serotonin acting on 5-HT(1A) and 5-HT(2C) receptors may facilitate mechanical sensitivity in animals with a sleep deprivation-induced hypersensitivity as well as in controls, increased activation of spinal 5-HT(1A) receptors by an exogenous agonist leads to suppression of mechanical sensitivity in both conditions. Spinal 5-HT(3) receptors do not contribute to cutaneous hypersensitivity induced by sleep deprivation. PMID:18602835

Wei, Hong; Ma, Ainiu; Wang, Yong-Xiang; Pertovaara, Antti



Smurf1 interacts with transforming growth factor-beta type I receptor through Smad7 and induces receptor degradation.  


Smad7 is an inhibitory Smad that acts as a negative regulator of signaling by the transforming growth factor-beta (TGF-beta) superfamily proteins. Smad7 is induced by TGF-beta, stably interacts with activated TGF-beta type I receptor (TbetaR-I), and interferes with the phosphorylation of receptor-regulated Smads. Here we show that Smurf1, an E3 ubiquitin ligase for bone morphogenetic protein-specific Smads, also interacts with Smad7 and induces Smad7 ubiquitination and translocation into the cytoplasm. In addition, Smurf1 associates with TbetaR-I via Smad7, with subsequent enhancement of turnover of TbetaR-I and Smad7. These results thus reveal a novel function of Smad7, i.e. induction of degradation of TbetaR-I through recruitment of an E3 ligase to the receptor. PMID:11278251

Ebisawa, T; Fukuchi, M; Murakami, G; Chiba, T; Tanaka, K; Imamura, T; Miyazono, K



Inhibition by chondroitin sulfate E can specify functional Wnt/?-catenin signaling thresholds in NIH3T3 fibroblasts.  


Aberrant activation of the Wnt/?-catenin signaling pathway is frequently associated with human disease, including cancer, and thus represents a key therapeutic target. However, Wnt/?-catenin signaling also plays critical roles in many aspects of normal adult tissue homeostasis. The identification of mechanisms and strategies to selectively inhibit the disease-related functions of Wnt signaling, while preserving normal physiological functions, is in its infancy. Here, we report the identification of exogenous chondroitin sulfate-E (CS-E) as an inhibitor of specific molecular and biological outcomes of Wnt3a signaling in NIH3T3 fibroblasts. We demonstrate that CS-E can decrease Wnt3a signaling through the negative regulation of LRP6 receptor activation. However, this inhibitory effect of CS-E only affected Wnt3a-mediated induction, but not repression, of target gene expression. We went on to identify a critical Wnt3a signaling threshold that differentially affects target gene induction versus repression. This signaling threshold also controlled the effects of Wnt3a on proliferation and serum starvation-induced apoptosis. Limiting Wnt3a signaling to this critical threshold, either by CS-E treatment or by ligand dilution, interfered with Wnt3a-mediated stimulation of proliferation but did not impair Wnt3a-mediated reduction of serum starvation-induced apoptosis. Treatment with pharmacological inhibitors demonstrated that both induction and repression of Wnt3a target genes in NIH3T3 cells require the canonical Wnt/?-catenin signaling cascade. Our data establish the feasibility of selective inhibition of Wnt/?-catenin transcriptional programs and biological outcomes through the exploitation of intrinsic signaling thresholds. PMID:22915582

Willis, Catherine M; Klüppel, Michael




Technology Transfer Automated Retrieval System (TEKTRAN)

We have identified the xenobiotic receptor CAR (constitutive androstane receptor) as a key regulator of acetaminophen metabolism and hepatotoxicity. Known CAR activators as well as high doses of acetaminophen induced expression of three acetaminophen-metabolizing enzymes in wild-type but not in CAR-...


Dansyl cadaverine regulates ligand induced endocytosis of interleukin-8 receptor in human polymorphonuclear neutrophils  

Microsoft Academic Search

Interleukin-8 (IL-8), a neutrophil chemotactic agent, acts as a key mediator in a large number of acute and chronic inflammatory diseases. At 37°C, the receptor for IL-8 is rapidly internalized with its ligand. But no specific inhibitor of this ligand induced internalization of the receptor has been reported so far. We have found that monodansyl cadaverine (MDC) inhibited about 70%

Ena Ray; Ajoy Kumar Samanta



Blockade of Mineralocorticoid and Glucocorticoid Receptors Reverses Stress-Induced Motor Impairments  

Microsoft Academic Search

Aim: Stress and glucocorticoids can influence movement performance and pathologies of the motor system. The classic notion assumes that the glucocorticoid receptor (GR) mediates the majority of stress-induced behavioral changes. Nevertheless, recent findings have attributed a more prominent role to the mineralocorticoid receptor (MR) in modulating behavior. The purpose of this study was to dissociate the impact of MR versus

Nafisa M. Jadavji; Rebecca D. Supina; Gerlinde A. Metz



Diet-Induced Hypercholesterolemia in Mice: Prevention by Overexpression of LDL Receptors  

Microsoft Academic Search

The current studies were designed to determine whether chronic overexpression of low density lipoprotein (LDL) receptors in the liver would protect mice from the increase in plasma LDL-cholesterol that is induced by high-fat diets. A line of transgenic mice was studied that express the human LDL receptor gene in the liver under control of the transferrin promoter. When fed a

Masayuki Yokode; Robert E. Hammer; Shun Ishibashi; Michael S. Brown; Joseph L. Goldstein



Effects of AT1 receptor antagonist, losartan, on rat hepatic fibrosis induced by CCl4  

Microsoft Academic Search

AIM To investigate effect o f losartan, an AT1 receptor antagonist, on hepatic fibrosis induced by CCl4; and to determine whether or not AT1 receptors are expressed on hepatic stellate cells. METHODS AND RESULTS Fifty male Sprague- Dawley rats, weighing (180 ± 20) g, were randomized into five groups (control group, model group, and three los artan treated groups), in

Hong Shan Wei; Ding Guo Li; Han Ming Lu; Yu Tao Zhan; Zhi Rong Wang; Xin Huang; Jing Zhang; Lin Cheng; Qin Fang Xu



Toward a Consensus on the Operation of Receptor-Induced Calcium Entry Signals  

NSDL National Science Digital Library

Receptor-induced Ca2+ signals involve both Ca2+ release from intracellular stores and extracellular Ca2+ entry across the plasma membrane. The channels mediating Ca2+ entry and the mechanisms controlling their function remain largely a mystery. Here we critically assess current views on the Ca2+ entry process and consider certain modifications to the widely held hypothesis that Ca2+ store emptying is the fundamental trigger for receptor-induced Ca2+ entry channels. Under physiological conditions, receptor-induced store depletion may be quite limited. A number of distinct channel activities appear to mediate receptor-induced Ca2+ entry, and their activation is observed to occur through quite diverse coupling processes.

Donald L. Gill (University of Maryland School of Medicine;Department of Biochemistry and Molecular Biology REV); Randen L. Patterson (Pennsylvania State University;Mueller Laboratory REV)



Resistin promotes 3T3-L1 preadipocyte differentiation  

Microsoft Academic Search

Objective: To investigate the relationship between resistin (a potential link between obesity and type 2 diabetes) and preadipocyte differentiation. Design: A rat resistin expression vector was transfected into 3T3-L1 preadipocytes and differentiation was compared between normal 3T3-L1 cells, rat resistin-transfected cells and non-transfected cells grown in conditioned medium taken from resistin-expressing cultures. Methods: The rat resistin gene was inserted into

Haixia Gong; Yuhui Ni; Xirong Guo; L i Fei; Xiaoqin Pan; Mei Guo; Ronghua Chen



Activity-induced and developmental downregulation of the Nogo receptor  

Microsoft Academic Search

The three axon growth inhibitory proteins, myelin associated glycoprotein, oligodendrocyte-myelin glycoprotein and Nogo-A, can all bind to the Nogo-66 receptor (NgR). This receptor is expressed by neurons with high amounts in regions of high plasticity where Nogo expression is also high. We hypothesized that simultaneous presence of high levels of Nogo and its receptor in neurons confers a locked state

Anna Josephson; Alexandra Trifunovski; Camilla Schéele; Johan Widenfalk; Claes Wahlestedt; Stefan Brené; Lars Olson; Christian Spenger



Suppressive effects of the extracts of Japanese edible seaweeds on mutagen-induced umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002) and tumor promotor-dependent ornithine decarboxylase induction in BALB/c 3T3 fibroblast cells.  


Some of epidemiological data indicated that ubiquitous consumption of seaweeds in Japan may be a possible protective factor against some types of tumor. To analyse this problem, the authors studied the antimutagenic and antitumor promotion activities in methanol-soluble extracts of typical edible seaweeds which showed suppressive effects on 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indol (Trp-P-1)-induced umu C gene expression in SOS response of Salmonella typhimurium (TA 1535/pSK 1002) and 12-O-tetradecanoylphorbol-13-acetate (TPA)-dependent ornithine decarboxylase induction in BALB/c 3T3 fibroblast cells. Although eight varieties of edible seaweeds including chlorophyta, Phaenophyta and Rhodophyta showed significant antimutagenic and antipromotion activities, they expressed the activities different from each other. Among these seaweeds, Enteromorpha prolifera ('Sujiaonori' in Japanese) and Porphyra tenera ('Asakusanori') showed relatively strong suppressive activities in both antimutagenic and antipromotion assays compared with other seaweeds. These seaweeds contained considerable amounts of beta-carotene as a possible active principle with anticarcinogenic activity. This compound was partially associated with the antimutagenic activity in the seaweed extract, but did not contribute to the antipromotion activity of seaweed extract under our experimental conditions. These results strongly suggest that Japanese edible seaweeds have possible antimutagenic and antipromotion activities probably associated with antitumor activity. PMID:7954366

Okai, Y; Higashi-Okai, K; Nakamura, S; Yano, Y; Otani, S



Mast cell desensitization to IgE fails to induce a parallel adenosine receptor desensitization  

Microsoft Academic Search

Desensitization induced by challenge of mast cells with antigen is specific for IgE-dependent signals. During the secretory process mast cells release adenosine, which can induce a desensitization of adenosine receptors. To determine whether adenosine receptors may de desensitized from a previous antigen challenge, mast cells were sensitized with anti-DNP IgE antibody, challenged with DNP-BSA antigen, returned to culture overnight, resensitized,

D. L. Marquardt; A. Lwin; L. L. Walker



Angiotensin protects cortical neurons from hypoxic-induced apoptosis via the angiotensin type 2 receptor  

Microsoft Academic Search

The effects of angiotensin on mouse cortical neuronal cultures exposed to chemical-induced hypoxia was investigated. Cultures exposed to 10 mM sodium azide for 5 min showed a 17% increase in apoptosis when assayed 24 h postinsult. The N-methyl-d-aspartate (NMDA) receptor antagonist MK-801 blocked sodium azide-induced cell death suggesting that the NMDA receptor contributes to the mediated cell death. Pretreatment of

Tom Grammatopoulos; Katherine Morris; Paul Ferguson; James Weyhenmeyer



Peroxisome Proliferator-Activated Receptor g-Dependent Repression of the Inducible Nitric Oxide Synthase Gene  

Microsoft Academic Search

The peroxisome proliferator-activated receptor g (PPARg) is a member of the nuclear receptor superfamily that activates target gene transcription in a ligand-dependent manner. In addition, liganded PPARg can inhib- it transcription of genes induced by gamma interferon (IFN-g) and\\/or lipopolysaccharides (LPSs), including the inducible nitric oxide synthase (iNOS) gene. Inhibition of the iNOS promoter is achieved partially through an- tagonizing




Activation of GABA-A Receptor Ameliorates Homocysteine-Induced MMP-9 Activation by ERK Pathway  

PubMed Central

Hyperhomocysteinemia (HHcy) is a risk factor for neuroinflammatory and neurodegenerative diseases. Homocysteine (Hcy) induces redox stress, in part, by activating matrix metalloproteinase-9 (MMP-9), which degrades the matrix and leads to blood–brain barrier dysfunction. Hcy competitively binds to ?-aminbutyric acid (GABA) receptors, which are excitatory neurotransmitter receptors. However, the role of GABA-A receptor in Hcy-induced cerebrovascular remodeling is not clear. We hypothesized that Hcy causes cerebrovascular remodeling by increasing redox stress and MMP-9 activity via the extracellular signal-regulated kinase (ERK) signaling pathway and by inhibition of GABA-A receptors, thus behaving as an inhibitory neurotransmitter. Hcy-induced reactive oxygen species production was detected using the fluorescent probe, 2?–7?-dichlorodihydrofluorescein diacetate. Hcy increased nicotinamide adenine dinucleotide phosphate-oxidase-4 concomitantly suppressing thioredoxin. Hcy caused activation of MMP-9, measured by gelatin zymography. The GABA-A receptor agonist, muscimol ameliorated the Hcy-mediated MMP-9 activation. In parallel, Hcy caused phosphorylation of ERK and selectively decreased levels of tissue inhibitors of metalloproteinase-4 (TIMP-4). Treatment of the endothelial cell with muscimol restored the levels of TIMP-4 to the levels in control group. Hcy induced expression of iNOS and decreased eNOS expression, which lead to a decreased NO bioavailability. Furthermore muscimol attenuated Hcy-induced MMP-9 via ERK signaling pathway. These results suggest that Hcy competes with GABA-A receptors, inducing the oxidative stress transduction pathway and leading to ERK activation.




Soluble Forms of the Notch Ligands Delta1 and Jagged1 Promote in Vivo Tumorigenicity in NIH3T3 Fibroblasts with Distinct Phenotypes  

PubMed Central

We previously found that soluble forms of the Notch ligands Jagged1 and Delta1 induced fibroblast growth factor receptor-dependent cell transformation in NIH3T3 fibroblasts. However, the phenotypes of these lines differed, indicating distinct functional differences among these Notch ligands. In the present study, we used allografts to test the hypothesis that NIH3T3 fibroblasts that express soluble forms of Delta1 and Jagged1 accelerate tumorigenicity in vivo. With the exception of the full-length Jagged1 transfectant, all other cell lines, including the control, generated tumors when injected subcutaneously in athymic mice. Suppression of Notch signaling by the soluble ligands significantly increased tumor onset and growth, whereas full-length Jagged1 completely suppressed tumor development. In addition, there were striking differences in tumor pathology with respect to growth kinetics, vascularization, collagen content, size and number of necrotic foci, and invasiveness into the underlying tissue. Further, the production of angiogenic factors, including vascular endothelial growth factor, also differed among the tumor types. Lastly, both Jagged1- and Delta1-derived tumors contained phenotypically distinct populations of lipid-filled cells that corresponded with increased expression of adipocyte markers. The divergence of tumor phenotype may be attributed to ligand-specific alterations in Notch receptor responses in exogenous and endogenous cell populations within the allographs. Our findings demonstrate distinct functional properties for these Notch ligands in the promotion of tumorigenicity in vivo.

Urs, Sumithra; Roudabush, Alice; O'Neill, Christine F.; Pinz, Ilka; Prudovsky, Igor; Kacer, Doreen; Tang, Yuefang; Liaw, Lucy; Small, Deena



Characterization of thyrotropin receptor antibody-induced signaling cascades.  


The TSH receptor (TSHR) is constitutively active and is further enhanced by TSH ligand binding or by stimulating TSHR antibodies (TSHR-Abs) as seen in Graves' disease. TSH is known to activate the thyroid epithelial cell via both Galphas-cAMP/protein kinase A/ERK and Galphaq-Akt/protein kinase C coupled signaling networks. The recent development of monoclonal antibodies to the TSHR has enabled us to investigate the hypothesis that different TSHR-Abs may have unique signaling imprints that differ from TSH ligand itself. We have, therefore, performed sequential studies, using rat thyrocytes (FRTL-5, passages 5-20) as targets, to examine the signaling pathways activated by a series of monoclonal TSHR-Abs in comparison with TSH itself. Activation of key signaling molecules was estimated by specific immunoblots and/or enzyme immunoassays. Continuing constitutive TSHR activity in thyroid cells, deprived of TSH and serum for 48 h, was demonstrated by pathway-specific chemical inhibition. Under our experimental conditions, TSH ligand and TSHR-stimulating antibodies activated both Galphas and Galphaq effectors. Importantly, some TSHR-blocking and TSHR-neutral antibodies were also able to generate signals, influencing primarily the Galphaq effectors and induced cell proliferation. Most strikingly, antibodies that used the Galphaq cascades used c-Raf-ERK-p90RSK as a unique signaling cascade not activated by TSH. Our study demonstrated that individual TSHR-Abs had unique molecular signatures which resulted in sequential preferences. Because downstream thyroid cell signaling by the TSHR is both ligand dependent and independent, this may explain why TSHR-Abs are able to have variable influences on thyroid cell biology. PMID:18719020

Morshed, Syed A; Latif, Rauf; Davies, Terry F



Contribution of PGE2 EP1 receptor in hemin-induced neurotoxicity.  


Although hemin-mediated neurotoxicity has been linked to the production of free radicals and glutamate excitotoxicity, the role of the prostaglandin E2 (PGE2)-EP1 receptor remains unclear. Activation of the EP1 receptor in neurons results in increased intracellular calcium levels; therefore, we hypothesize that the blockade of the EP1 receptor reduces hemin neurotoxicity. Using postnatal primary cortical neurons cultured from wild-type (WT) and EP1(-/-) mice, we investigated the EP1 receptor role in hemin neurotoxicity measured by lactate dehydrogenase (LDH) cell survival assay. Hemin (75 ?M) induced greater release of LDH in WT (34.7 ± 4.5%) than in EP1(-/-) (27.6 ± 3.3%) neurons. In the presence of the EP1 receptor antagonist SC-51089, the hemin-induced release of LDH decreased. To further investigate potential mechanisms of action, we measured changes in the intracellular calcium level [Ca(2+)]i following treatment with 17-phenyl trinor PGE2 (17-pt-PGE2) a selective EP1 agonist. In the WT neurons, 17-pt-PGE2 dose-dependently increased [Ca(2+)]i. However, in EP1(-/-) neurons, [Ca(2+)]i was significantly attenuated. We also revealed that hemin dose-dependently increased [Ca(2+)]i in WT neurons, with a significant decrease in EP1(-/-) neurons. Both 17-pt-PGE2 and hemin-induced [Ca(2+)]i were abolished by N-methyl-D-aspartic (NMDA) acid receptor and ryanodine receptor blockers. These results suggest that blockade of the EP1 receptor may be protective against hemin neurotoxicity in vitro. We speculate that the mechanism of hemin neuronal death involves [Ca(2+)]i mediated by NMDA acid receptor-mediated extracellular Ca(2+) influx and EP1 receptor-mediated intracellular release from ryanodine receptor-operated Ca(2+) stores. Therefore, blockade of the EP1 receptor could be used to minimize neuronal damage following exposure to supraphysiological levels of hemin. PMID:24109429

Mohan, Shekher; Glushakov, Alexander V; Decurnou, Alexander; Narumiya, Shuh; Doré, Sylvain



Contribution of PGE2 EP1 receptor in hemin-induced neurotoxicity  

PubMed Central

Although hemin-mediated neurotoxicity has been linked to the production of free radicals and glutamate excitotoxicity, the role of the prostaglandin E2 (PGE2)-EP1 receptor remains unclear. Activation of the EP1 receptor in neurons results in increased intracellular calcium levels; therefore, we hypothesize that the blockade of the EP1 receptor reduces hemin neurotoxicity. Using postnatal primary cortical neurons cultured from wild-type (WT) and EP1?/? mice, we investigated the EP1 receptor role in hemin neurotoxicity measured by lactate dehydrogenase (LDH) cell survival assay. Hemin (75 ?M) induced greater release of LDH in WT (34.7 ± 4.5%) than in EP1?/? (27.6 ± 3.3%) neurons. In the presence of the EP1 receptor antagonist SC-51089, the hemin-induced release of LDH decreased. To further investigate potential mechanisms of action, we measured changes in the intracellular calcium level [Ca2+]i following treatment with 17-phenyl trinor PGE2 (17-pt-PGE2) a selective EP1 agonist. In the WT neurons, 17-pt-PGE2 dose-dependently increased [Ca2+]i. However, in EP1?/? neurons, [Ca2+]i was significantly attenuated. We also revealed that hemin dose-dependently increased [Ca2+]i in WT neurons, with a significant decrease in EP1?/? neurons. Both 17-pt-PGE2 and hemin-induced [Ca2+]i were abolished by N-methyl-D-aspartic (NMDA) acid receptor and ryanodine receptor blockers. These results suggest that blockade of the EP1 receptor may be protective against hemin neurotoxicity in vitro. We speculate that the mechanism of hemin neuronal death involves [Ca2+]i mediated by NMDA acid receptor-mediated extracellular Ca2+ influx and EP1 receptor-mediated intracellular release from ryanodine receptor-operated Ca2+ stores. Therefore, blockade of the EP1 receptor could be used to minimize neuronal damage following exposure to supraphysiological levels of hemin.

Mohan, Shekher; Glushakov, Alexander V.; deCurnou, Alexander; Narumiya, Shuh; Dore, Sylvain



Effects of the local administration of selective ?-, ?-and ?-opioid receptor agonists on osteosarcoma-induced hyperalgesia  

Microsoft Academic Search

The stimulation of peripheral opioid receptors yields analgesic responses in a model of bone cancer-induced pain in mice.\\u000a In order to know the type(s) of peripheral opiate receptors involved, the paw thermal withdrawal latencies were measured in\\u000a C3H\\/HeJ mice bearing a tibial osteosarcoma, after administering selective agonists of ?-,?-and ?-opiate receptors. The peritumoral\\u000a administration of DAGO (0.6–6 ?g) inhibited the osteosarcoma-induced

Ana Baamonde; Ana Lastra; Lucía Juárez; Verónica García; Agustín Hidalgo; Luis Menéndez



Neurokinin 1 receptors regulate morphine-induced endocytosis and desensitization of mu-opioid receptors in CNS neurons.  


mu-Opioid receptors (MORs) are G-protein-coupled receptors (GPCRs) that mediate the physiological effects of endogenous opioid neuropeptides and opiate drugs such as morphine. MORs are coexpressed with neurokinin 1 receptors (NK1Rs) in several regions of the CNS that control opioid dependence and reward. NK1R activation affects opioid reward specifically, however, and the cellular basis for this specificity is unknown. We found that ligand-induced activation of NK1Rs produces a cell-autonomous and nonreciprocal inhibition of MOR endocytosis induced by diverse opioids. Studies using epitope-tagged receptors expressed in cultured striatal neurons and a neuroblastoma cell model indicated that this heterologous regulation is mediated by NK1R-dependent sequestration of arrestins on endosome membranes. First, endocytic inhibition mediated by wild-type NK1Rs was overcome in cells overexpressing beta-arrestin2, a major arrestin isoform expressed in striatum. Second, NK1R activation promoted sequestration of beta-arrestin2 on endosomes, whereas MOR activation did not. Third, heterologous inhibition of MOR endocytosis was prevented by mutational disruption of beta-arrestin2 sequestration by NK1Rs. NK1R-mediated regulation of MOR trafficking was associated with reduced opioid-induced desensitization of adenylyl cyclase signaling in striatal neurons. Furthermore, heterologous regulation of MOR trafficking was observed in both amygdala and locus ceruleus neurons that naturally coexpress these receptors. These results identify a cell-autonomous mechanism that may underlie the highly specific effects of NK1R on opioid signaling and suggest, more generally, that receptor-specific trafficking of arrestins may represent a fundamental mechanism for coordinating distinct GPCR-mediated signals at the level of individual CNS neurons. PMID:19129399

Yu, Y Joy; Arttamangkul, Seksiri; Evans, Christopher J; Williams, John T; von Zastrow, Mark



Ligand binding to somatostatin receptors induces receptor-specific oligomer formation in live cells  

Microsoft Academic Search

Heptahelical receptors (HHRs) are generally thought to function as monomeric entities. Several HHRs such as somatostatin receptors (SSTRs), however, form homo- and heterooligomers when activated by ligand binding. By using dual fluorescent ligands simultaneously applied to live cells monotransfected with SSTR5 (R5) or SSTR1 (R1), or cotransfected with R5 and R1, we have analyzed the ligand receptor stoichiometry and aggregation

Ramesh C. Patel; Ujendra Kumar; Don C. Lamb; John S. Eid; Magalie Rocheville; Michael Grant; Aruna Rani; Theodore Hazlett; Shutish C. Patel; Enrico Gratton; Yogesh C. Patel



Aldosterone deficiency and mineralocorticoid receptor antagonism prevent angiotensin II-induced cardiac, renal, and vascular injury  

PubMed Central

Angiotensin II causes cardiovascular injury in part by aldosterone-induced mineralocorticoid receptor activation, and it can also activate the mineralocorticoid receptor in the absence of aldosterone in vitro. Here we tested whether endogenous aldosterone contributes to angiotensin II/salt-induced cardiac, vascular, and renal injury by the mineralocorticoid receptor. Aldosterone synthase knockout mice and wild type littermates were treated with angiotensin II or vehicle plus the mineralocorticoid receptor antagonist spironolactone or regular diet while drinking 0.9- saline. Angiotensin II/salt caused hypertension in both the knockout and wild type mice; an effect significantly blunted in the knockout mice. Either genetic aldosterone deficiency or mineralocorticoid receptor antagonism reduced cardiac hypertrophy, aortic remodeling, and albuminuria, as well as cardiac, aortic, and renal plasminogen activator inhibitor-1 mRNA expression during angiotensin II treatment. Mineralocorticoid receptor antagonism reduced angiotensin II/salt-induced glomerular hypertrophy, but aldosterone deficiency did not. Combined mineralocorticoid receptor antagonism and aldosterone deficiency reduced blood urea nitrogen and restored nephrin immunoreactivity. Angiotensin II/salt also promoted glomerular injury through the mineralocorticoid receptor in the absence of aldosterone. Thus, mineralocorticoid antagonism may have protective effects in the kidney beyond aldosterone synthase inhibition.

Luther, James M.; Luo, Pengcheng; Wang, Zuofei; Cohen, Samuel E.; Kim, Hyung-Suk; Fogo, Agnes B.; Brown, Nancy J.



Involvement of non-muscarinic receptors in phosphoinositide signalling during soman-induced seizures.  


Previous investigations have indicated that soman-induced convulsions involve the inositol lipid signalling system. We previously reported that 10 min after the onset of seizures, inositol 1,4,5-triphosphate (IP3) build-up was coupled to activation of non-muscarinic receptor subtypes. In the present study, we demonstrate that (1) in addition to muscarinic receptors, histamine H1 subtypes and glutamate metabotropic receptors contribute to the first IP3 increase (first 10 min of seizures) and (2) the histamine H1 subtype and glutamate metabotropic receptors are also involved in the second step of inositol phosphate response (after 10 min of seizures). alpha 1-adrenoceptor and 5-HT2 receptors, known to be coupled to phosphoinositide turnover, did not participate in soman-induced IP3 response. Neurochemical interactions between cholinergic, histamine H1 and glutamate metabotropic systems, responsible of the phosphoinositide hydrolysis under soman are envisaged. PMID:7621903

Bodjarian, N; Carpentier, P; Baubichon, D; Blanchet, G; Lallement, G



Role of dopamine D2 receptors in plasticity of stress-induced addictive behaviours.  


Dopaminergic systems are implicated in stress-related behaviour. Here we investigate behavioural responses to chronic stress in dopamine D2 receptor knockout mice and find that anxiety-like behaviours are increased compared with wild-type mice. Repeated stress exposure suppresses cocaine-induced behavioural sensitization, cocaine-seeking and relapse behaviours in dopamine D2 receptor knockout mice. Cocaine challenge after drug withdrawal in cocaine-experienced wild-type or dopamine D2 receptor knockout mice is associated with inhibition of long-term depression in the nucleus accumbens, and chronic stress during withdrawal prevents inhibition after cocaine challenge in cocaine-experienced dopamine D2 receptor knockout mice, but not in wild-type mice. Lentiviral-induced knockdown of dopamine D2 receptors in the nucleus accumbens of wild-type mice does not affect basal locomotor activity, but confers stress-induced inhibition of the expression of cocaine-induced behavioural sensitization. Stressed mice depleted of dopamine D2 receptors do not manifest long-term depression inhibition. Our results suggest that dopamine D2 receptors have roles in regulating synaptic modification triggered by stress and drug addiction. PMID:23481387

Sim, Hye-Ri; Choi, Tae-Yong; Lee, Hyo Jin; Kang, Eun Young; Yoon, Sehyoun; Han, Pyung-Lim; Choi, Se-Young; Baik, Ja-Hyun



Type I IL-4 Receptors Selectively Activate IRS-2 to Induce Target Gene Expression in Macrophages  

PubMed Central

Although interleukin (IL)-4 and IL-13 participate in allergic inflammation and share a receptor subunit (IL-4R?), differential functions for these cytokines have been reported. Therefore, we compared cells expressing type I and II IL-4 receptors with cells expressing only type II receptors for their responsiveness to these cytokines. IL-4 induced highly efficient, ?C-dependent tyrosine phosphorylation of insulin receptor substrate 2 (IRS-2), whereas IL-13 was less effective, even when phosphorylation of signal transducer and activator of transcription 6 (STAT6) was maximal. Only type I receptor-?C+ signaling induced efficient association of IRS-2 with p85 or GRB2. IL-4 signaling through type I receptor complexes induced more robust expression of a subset of genes associated with alternatively activated macrophages than did IL-13, despite equivalent activation of STAT6. Thus, IL-4 activates signaling pathways through the type I receptor complex, qualitatively differently from IL-13, which cooperate to induce optimal gene expression.

Heller, Nicola M.; Qi, Xiulan; Junttila, Ilkka S.; Shirey, Kari Ann; Vogel, Stefanie N.; Paul, William E.; Keegan, Achsah D.



Deoxynivalenol induces ectodomain shedding of TNF receptor 1 and thereby inhibits the TNF-?-induced NF-?B signaling pathway.  


Trichothecene mycotoxins are known to inhibit eukaryotic translation and to trigger the ribotoxic stress response, which regulates gene expression via the activation of the mitogen-activated protein (MAP) kinase superfamily. In this study, we found that deoxynivalenol induced the ectodomain shedding of tumor necrosis factor (TNF) receptor 1 (TNFRSF1A) and thereby inhibited the TNF-?-induced signaling pathway. In human lung carcinoma A549 cells, deoxynivalenol and 3-acetyldeoxynivalenol inhibited the expression of intercellular adhesion molecule-1 (ICAM-1) induced by TNF-? more strongly than that induced by interleukin 1? (IL-1?), whereas T-2 toxin and verrucarin A exerted nonselective inhibitory effects. Deoxynivalenol and 3-acetyldeoxynivalenol also inhibited the nuclear factor ?B (NF-?B) signaling pathway induced by TNF-?, but not that induced by IL-1?. Consistent with these findings, deoxynivalenol and 3-acetyldeoxynivalenol induced the ectodomain shedding of TNF receptor 1 by TNF-?-converting enzyme (TACE), also known as a disintegrin and metalloproteinase 17 (ADAM17). In addition to the TACE inhibitor TAPI-2, the MAP kinase or extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor U0126 and the p38 MAP kinase inhibitor SB203580, but not the c-Jun N-terminal kinase (JNK) inhibitor SP600125, suppressed the ectodomain shedding of TNF receptor 1 induced by deoxynivalenol and reversed its selective inhibition of TNF-?-induced ICAM-1 expression. Our results demonstrate that deoxynivalenol induces the TACE-dependent ectodomain shedding of TNF receptor 1 via the activation of ERK and p38 MAP kinase, and thereby inhibits the TNF-?-induced NF-?B signaling pathway. PMID:23357557

Hirano, Seiya; Kataoka, Takao



Metabotropic glutamate receptors are involved in calcium-induced LTP of AMPA and NMDA receptor-mediated responses in the rat hippocampus  

Microsoft Academic Search

Effects of metabotropic glutamate (mGlu) receptors on calcium-induced long-term potentiation (LTP) of ?-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA) and N-methyl-d-aspartate (NMDA) receptor-mediated components were investigated in rat hippocampal slices using whole-cell patch-clamp recordings of excitatory postsynaptic currents (EPSCs). Calcium-induced LTP comprises a parallel, long-lasting increase of AMPA and NMDA receptor-mediated components. The calcium-induced LTP of the AMPA receptor-mediated component can be significantly attenuated by

San-Nan Yang; Jian-Nan Wu; Demeral Liu; Che-Se Tung



Adhesion of Metastatic, ras-transformed NIH 3T3 Cells to Osteopontin, Fibronectin, and Laminin1  

Microsoft Academic Search

We previously reported that H-ras-induced metastatic ability in murine NIH 3T3 cells is accompanied by increased expression of osteopontin (OPN). OPN is a secreted phosphoprotein that contains a GRGDS amino acid sequence, suggesting adhesive function, but the function of OPN in tumor cells remains poorly understood. Here we report that PAP2 cells (ras-transformed, metastatic NIH 3T3 cells) adhere and spread

Ann F. Chambers; Charulata Hota; Charles W. Prince



Temporal and spatial assembly of lipid droplet-associated proteins in 3T3-L1 preadipocytes  

Microsoft Academic Search

This study aimed to investigate the relationship between newly formed lipid droplets and lipid droplet surface proteins, including perilipin, adipose differentiation related protein (ADRP), and p200 kDa protein (p200) in 3T3-L1 preadipocytes, during lipogenesis. Sterol ester was used to induce nascent lipid droplets in 3T3-L1 preadipocytes and the sequence of lipids and lipid droplet surface proteins was studied using a combination

Seu-Mei Wang; Ran-Der Hwang; Andrew S. Greenberg; Hui-Ling Yeo



Protein kinase Ce promotes adipogenic commitment and isessential for terminal differentiation of 3T3-F442A preadipocytes  

Microsoft Academic Search

The role of protein kinase C (PKC) isoforms in the commitment of multipotent fibroblasts to the adipocyte lineage and in their terminal differentiation into mature adipocytes was investigated. Ectopic overexpression of PKC-e, but not other PKC isoforms, committed multipotent NIH-3T3 cells to adipogenic differentiation in the presence of hormonal inducers. In committed 3T3-F442A preadipocytes, PKC-e protein expression increased during the

P. R. Webb; C. Doyle; N. G. Anderson



Endothelin-A Receptor Antagonism Attenuates Carcinoma-induced Pain through Opioids in Mice  

PubMed Central

We previously reported that endothelin A (ET-A) receptor antagonism attenuates carcinoma-induced pain in a cancer pain mouse model. In this study, we investigated the mechanism of ET-A receptor-mediated antinociception and evaluated the role of endogenous opioid analgesia. Squamous cell carcinoma (SCC) cell culture treated with the ET-A receptor antagonist (BQ-123) at 10?6 M and 10?5 M significantly increased production and secretion of ?-endorphin and leu-enkephalin, respectively. Behavioral studies were performed by inducing tumors in the hind paw of female nude mice with local injection of cells derived from a human oral SCC. Significant pain, as indicated by reduction in withdrawal thresholds in response to mechanical stimulation, began at four days after SCC inoculation and lasted to 18 days, the last day of measurement. Local administration of either naloxone methiodide (500 µg/kg), selective antagonists for µ-opioid receptor (CTOP, 500 µg/kg) or ?-opioid receptor (naltrindole, 11 mg/kg), but not ?-opioid receptor (nor-BNI, 2.5 mg/kg), significantly reversed antinociception observed from ET-A receptor antagonism (BQ-123, 92 mg/kg) in cancer animals. These results demonstrate that antagonism of peripheral endothelin-A receptor attenuates carcinoma pain by modulating release of endogenous opioids to act on opioid receptors in the cancer microenvironment.

Quang, Phuong N.; Schmidt, Brian L.



Inducible viral receptor, A possible concept to induce viral protection in primitive immune animals  

PubMed Central

A pseudolysogen (PL) is derived from the lysogenic Vibrio harveyi (VH) which is infected with the VHS1 (Vibrio harveyi Siphoviridae-like 1) bacteriophage. The lysogenic Vibrio harveyi undergoes an unequivalent division of the extra-chromosomal VHS1 phage genome and its VH host chromosome and produces a true lysogen (TL) and pseudolysogen (PL). The PL is tolerant to super-infection of VHS1, as is of the true lysogen (TL), but the PL does not contain the VHS1 phage genome while the TL does. However, the PL can become susceptible to VHS1 phage infection if the physiological state of the PL is changed. It is postulated that this is due to a phage receptor molecule which can be inducible to an on-and-off regulation influence by an alternating condition of the bacterial host cell. This characteristic of the PL leads to speculate that this phenomenon can also occur in high organisms with low immunity such as shrimp. This article proposes a hypothesis that the viral receptor molecule on the target cell can play a crucial role in which the invertebrate aquaculture animals can become tolerant to viral infection. A possible mechanism may be that the target cell disrupts the viral receptor molecule to prevent super infection. This concept can explain a mechanism for the prevention of viral infection in invertebrate animals which do not have acquired immunity in response to pathogens. It can guide us to develop a mechanism of immunity to viral infection in low-evolved-immune animals. Also, it can be an additional mechanism that exists in high immune organism, as in human for the prevention of viral infection



RasGRP1 sensitizes an immature B cell line to antigen receptor-induced apoptosis.  


RasGRP1 is a guanine nucleotide exchange factor that activates Ras GTPases and is activated downstream of antigen receptors on both T and B lymphocytes. Ras-GRP1 provides signals to immature T cells that confer survival and proliferation, but RasGRP1 also promotes T cell receptor-mediated deletion of mature T cells. We used the WEHI-231 cell line as an experimental system to determine whether RasGRP1 can serve as a quantitative modifier of B cell receptor-induced deletion of immature B cells. A 2-fold elevation in RasGRP1 expression markedly increased apoptosis of WEHI-231 cells following B cell receptor ligation, whereas a dominant negative mutant of RasGRP1 suppressed B cell receptor-induced apoptosis. Activation of ERK1 or ERK2 kinases was not required for RasGRP1-mediated apoptosis. Instead, elevated RasGRP1 expression caused down-regulation of NF-kappaB and Bcl-x(L), which provide survival signals counter-acting apoptosis induction by B cell receptor. Inhibition of NF-kappaB was sufficient to enhance B cell receptor-induced apoptosis of WEHI-231 cells, and ligation of co-stimulatory receptors that activate NF-kappaB suppressed the ability of RasGRP1 to promote B cell receptor-induced apoptosis. These experiments define a novel apoptosis-promoting pathway leading from B cell receptor to the inhibition of NF-kappaB and demonstrate that differential expression of RasGRP1 has the potential to modulate the sensitivities of B cells to negative selection following antigen encounter. PMID:14970203

Guilbault, Benoit; Kay, Robert J



T=3/2 states in {sup 13}C  

SciTech Connect

Background: Experimental information on the structure of low-lying levels with isospin 3/2 in systems with 13 nucleons can be used to evaluate the quality of modern theoretical predictions for the light exotic systems. This level structure is poorly known at present. Purpose: Search for T=3/2 states in {sup 13}C was performed in this work. Method: These states were observed in the resonance elastic scattering of radioactive beam {sup 12}B on protons using the thick target inverse kinematics technique. Results: Six new states in {sup 13}C were identified as T=3/2 states. Tentative spin-parity assignments are suggested. Comparison to the previous knowledge of the level structure of T=3/2 A=13 system and to shell-model predictions is given. Conclusion: The elastic scattering of neutron-rich beams on proton target is a convenient tool for isobaric analog states search. The observed {sup 12}B+p excitation function is determined by the T=3/2 states and no features associated with T=1/2 states were found. Although the level scheme of T=3/2 states in A=13 systems is still far from being complete the emerging picture shows significant disagreements with predictions of contemporary shell models.

Skorodumov, B. B.; Aprahamian, A.; Almaraz, S.; Lamm, L. O.; Quinn, M.; Teymurazyan, A.; Woehr, A. [Institute for Nuclear Structure and Astrophysics, University of Notre Dame, Notre Dame, Indiana 46556 (United States); Rogachev, G. V.; Johnson, E. D. [Department of Physics, Florida State University, Tallahassee, Florida 32306 (United States); Goldberg, V. Z. [Texas A and M University, College Station, Texas 77843 (United States); Kolata, J. J. [Physics Department, University of Notre Dame, Notre Dame, Indiana 46556 (United States); Amro, H. [Physics Department, University of Michigan, Ann Arbor, Michigan 48109 (United States)



Ejaculatory response induced by a 5HT 2 receptor agonist mCPP in rats: Differential roles of 5HT 2 receptor subtypes  

Microsoft Academic Search

It has been reported that systemic administration of m-CPP (1-[3-chlorophenyl] piperazine hydrochloride), a 5-HT2 receptor agonist, produces a 5-HT2C receptor-mediated penile erections and self-grooming in rats. In the present study, we examined the ability of m-CPP to induce ejaculation in rats and determined which 5-HT2 receptor subtypes may be involved in the m-CPP-induced ejaculation. The ejaculatory response was assessed by

Akihiko Yonezawa; Masaru Yoshizumi; Manabu Ebiko; Shin-nosuke Ise; Chizuko Watanabe; Hirokazu Mizoguchi; Yukio Kimura; Shinobu Sakurada



Increased depressive behaviour in mice harboring the mutant thyroid hormone receptor alpha 1.  


Clinical evidence indicates that hypothyroidism contributes to mood disorders. The present study tested if the mutant thyroid hormone receptor alpha 1 (TRalpha1) that causes a receptor-mediated hypothyroidism in the brain affects depressive and anxious behaviour in mice. Mice heterozygous for the TRalpha1 allele (TRalpha1+/m), yielding a receptor protein with a 10-fold reduced affinity to triiodothyronine (T3), and wildtype (wt) mice were subjected to several paradigms specifically testing depressive and anxious behaviour. Mutant and wt mice were either treated with T3 or vehicle. Untreated TRalpha1+/m animals displayed reduced locomotion, higher rates of helplessness in the shuttle box-, greater levels of anxiety in the startle response- and dark light box behavioural paradigms when compared to wt mice. Continuous T3-substitution therapy was effective in alleviating anxious and depressive behaviour without affecting locomotion in mutant mice. Notably, continuous T3-substitution reduced overall locomotion and increased helpless behaviour in wt mice when compared to untreated wt mice. The data suggest that receptor-mediated hypothyroidism caused by an unliganded thyroid hormone receptor alpha 1 leads to a depressive and anxious phenotype in mice, which is responsive to continuous T3-substitution and that an iatrogeneously induced hyperthyreoidism by continuous T3-administration leads to a hypolocomotive and depressive phenotype. PMID:20580649

Pilhatsch, Maximilian; Winter, Christine; Nordström, Kristina; Vennström, Björn; Bauer, Michael; Juckel, Georg



Allopregnanolone prevents dieldrin-induced NMDA receptor internalization and neurotoxicity by preserving GABA(A) receptor function.  


Dieldrin is an endocrine disruptor that accumulates in mammalian adipose tissue and brain. It induces convulsions due to its antagonism of the ?-aminobutyric acid A receptor (GABA(A)R). We have previously reported that long-term exposure to dieldrin causes the internalization of the N-methyl-D-aspartate receptor (NMDAR) as a result of persistent GABA(A)R inhibition. Because the neurosteroids 17?-estradiol (E2) and allopregnanolone are known to modulate the function and trafficking of GABA(A)R and NMDAR, we examined the effects of E2 and allopregnanolone on dieldrin-induced GABA(A)R inhibition, NMDAR internalization, and neuronal death in cortical neurons. We found that 1 nM E2 increased the membrane expression of NR1/NR2B receptors and postsynaptic density 95 but did not induce their physical association. In contrast, 10 nM E2 had no effect on these proteins but reduced NR2A membrane expression. We also found that exposure to 60 nM dieldrin for 6 d in vitro caused the internalization of NR1 and NR2B but not NR2A. Treatment with either 1 nM E2 or 10 ?M allopregnanolone prevented the dieldrin-induced reduction in membrane levels of the NR1/NR2B receptors. Furthermore, prolonged exposure to 200 nM dieldrin down-regulated the expression of NR2A; this was inhibited only by allopregnanolone. Although both hormones restored NMDAR function, as measured by the NMDA-induced rise in intracellular calcium, allopregnanolone (but not E2) reversed the inhibition of GABA(A)R and neuronal death caused by prolonged exposure to dieldrin. Our results indicate that allopregnanolone protects cortical neurons against the neurotoxicity caused by long-term exposure to dieldrin by maintaining GABA(A)R and NMDAR functionality. PMID:22166974

Briz, Víctor; Parkash, Jyoti; Sánchez-Redondo, Sara; Prevot, Vincent; Suńol, Cristina



Gastrin and d1 dopamine receptor interact to induce natriuresis and diuresis.  


Oral NaCl produces a greater natriuresis and diuresis than the intravenous infusion of the same amount of NaCl. Gastrin is the major gastrointestinal hormone taken up by renal proximal tubule (RPT) cells. We hypothesized that renal gastrin and dopamine receptors interact to synergistically increase sodium excretion, an impaired interaction of which may be involved in the pathogenesis of hypertension. In Wistar-Kyoto rats, infusion of gastrin induced natriuresis and diuresis, which was abrogated in the presence of a gastrin (cholecystokinin B receptor [CCKBR]; CI-988) or a D1-like receptor antagonist (SCH23390). Similarly, the natriuretic and diuretic effects of fenoldopam, a D1-like receptor agonist, were blocked by SCH23390, as well as by CI-988. However, the natriuretic effects of gastrin and fenoldopam were not observed in spontaneously hypertensive rats. The gastrin/D1-like receptor interaction was also confirmed in RPT cells. In RPT cells from Wistar-Kyoto but not spontaneously hypertensive rats, stimulation of either D1-like receptor or gastrin receptor inhibited Na(+)-K(+)-ATPase activity, an effect that was blocked in the presence of SCH23390 or CI-988. In RPT cells from Wistar-Kyoto and spontaneously hypertensive rats, CCKBR and D1 receptor coimmunoprecipitated, which was increased after stimulation of either D1 receptor or CCKBR in RPT cells from Wistar-Kyoto rats; stimulation of one receptor increased the RPT cell membrane expression of the other receptor, effects that were not observed in spontaneously hypertensive rats. These data suggest that there is a synergism between CCKBR and D1-like receptors to increase sodium excretion. An aberrant interaction between the renal CCK?BR and D1-like receptors (eg, D1 receptor) may play a role in the pathogenesis of hypertension. PMID:24019399

Chen, Yue; Asico, Laureano D; Zheng, Shuo; Villar, Van Anthony M; He, Duofen; Zhou, Lin; Zeng, Chunyu; Jose, Pedro A



ET A receptor mediates the signaling of endothelin-1 in osteoblast-like cells  

Microsoft Academic Search

We previously reported that endothelin-1 (ET-1) stimulates phosphatidylcholine-hydrolyzing phospholipase D independently of phosphoinositide hydrolysis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the characteristics of the receptors mediating ET-1-induced intracellular signaling pathway in MC3T3-E1 cells. Cyclo-d-Trp-d-Asp-Pro-d-Val-Leu (BQ123), a selective ETA receptor antagonist, significantly inhibited the ET-1-induced formation of inositol phosphates in a dose-dependent manner in the range between

A. Suzuki; J. Shinoda; Y. Watanabe-Tomita; N. Ozaki; Y. Oiso; O. Kozawa



Expression of human epidermal growth factor pressures cDNA in transfected mouse NIH 3T3 cells  

SciTech Connect

Stable cell lines expressing the human epidermal growth factor (EGF) precursor have been prepared by transfection of mouse NIH 3T3 cells with a bovine papillomavirus-based vector in which the human kidney EGF precursor cDNA has been placed under the control of the inducible mouse metallothionein I promoter. Synthesis of the EGF precursor can be induced by culturing the cells in 5 mM butyric acid or 100 ZnCl/sub 2/. The EGF precursor synthesized by these cells appears to be membrane associated; none is detectable in the cytoplasm. The size of the EGF precursor expressed by these cells is approx. = 150-180 kDa, which is larger than expected from its amino acid sequence, suggesting that it is posttranslationally modified, presumably by glycosylation. The EGF precursor was also detected in the conditioned medium from these cells, indicating that some fraction of the EGF precursor synthesized by these transfected cells may be secreted. Preliminary data suggest that this soluble form of the EGF precursor may compete with /sup 125/I-labeled EGF for binding to the EGF receptor. These cell lines should be useful for studying the processing of the EGF precursor to EGF as well as determining the properties and possible functions of the EGF precursor itself.

Mroczkowski, B.; Reich, M.; Whittaker, J.; Bell, G.I.; Cohen, S.



Hepatitis C Virus Induces the Cannabinoid Receptor 1  

Microsoft Academic Search

BackgroundActivation of hepatic CB1 receptors (CB1) is associated with steatosis and fibrosis in experimental forms of liver disease. However, CB1 expression has not been assessed in patients with chronic hepatitis C (CHC), a disease associated with insulin resistance, steatosis and metabolic disturbance. We aimed to determine the importance and explore the associations of CB1 expression in CHC.MethodsCB1 receptor mRNA was

David van der Poorten; Mahsa Shahidi; Enoch Tay; Jayshree Sesha; Kayla Tran; Duncan McLeod; Jane S. Milliken; Vikki Ho; Lionel W. Hebbard; Mark W. Douglas; Jacob George



Localization of type I interferon receptor limits interferon-induced TLR-3 in epithelial cells  

EPA Science Inventory

This study aimed to expand on the role of type I IFNs in the influenza-induced upregulation of TLR3 and determine whether and how the localization of the IFN-alpha/beta receptor (IFNAR) in respiratory epithelial cells could modify IFN-induced responses. Using differentiated prima...


Opposing roles of glucocorticoid receptor and mineralocorticoid receptor in trimethyltin-induced cytotoxicity in the mouse hippocampus.  


The organotin trimethyltin (TMT) is known to cause neuronal degeneration in the murine brain. Earlier studies indicate that TMT-induced neuronal degeneration is enhanced by adrenalectomy and prevented by exogenous glucocorticoid. The aim of this study was to investigate the regulation of TMT neuroxicity by corticosterone receptors including type I (mineralocorticoid receptor, MR) and type II (glucocorticoid receptor, GR) in adult mice. The systemic injection of TMT at the dose of 2.0 or 2.8 mg/kg produced a marked elevation in the level of plasma corticosterone that was both dose and time dependent. The MR agonist aldosterone had the ability to exacerbate TMT cytotoxicity in the dentate granule cell layer, whereas its antagonist spironolactone protected neurons from TMT cytotoxicity there. In contrast, the GR antagonist mifepristone exacerbated the TMT cytotoxicity. Taken together, our data suggest TMT cytotoxicity is oppositely regulated by GR and MR signals, being exacerbated by MR activation in adult mice. PMID:22309794

Ogita, Kiyokazu; Sugiyama, Chie; Acosta, Gabriela Beatriz; Kuramoto, Nobuyuki; Shuto, Makoto; Yoneyama, Masanori; Nakamura, Yukary; Shiba, Tatsuo; Yamaguchi, Taro



Direct involvement of the receptor-mediated apoptotic pathways in cisplatin-induced renal tubular cell death  

Microsoft Academic Search

Direct involvement of the receptor-mediated apoptotic pathways in cisplatin-induced renal tubular cell death.BackgroundTumor necrosis factor (TNF) receptor family members, such as Fas and TNF receptor 1 (TNFR1), are thought to induce apoptosis in a variety of cells and organs. Although a number of potential scenarios have been postulated for the involvement of these receptors in the pathogenesis of acute renal

Kazuhiko Tsuruya; Toshiharu Ninomiya; Masanori Tokumoto; Makoto Hirakawa; Kohsuke Masutani; Masatomo Taniguchi; Kyoichi Fukuda; Hidetoshi Kanai; Kenji Kishihara; Hideki Hirakata; Mitsuo Iida



Coenzyme Q10 increases the fatty acid oxidation through AMPK-mediated PPAR? induction in 3T3-L1 preadipocytes.  


Coenzyme Q10(CoQ10) is a known anti-adipogenic factor. However, the mechanism by which CoQ10 acts is unclear. In this study, we found that CoQ10 increased the phosphorylation of AMP-activated protein kinase (AMPK) in 3T3-L1preadipocytes. CoQ10 induced an increase in cytoplasmic calcium concentrations, which is reflected by increased Fluo-3 intensity under confocal microscopy recording. Either inhibition of Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) or knock-down CaMKK blocked CoQ10-induced AMPK phosphorylation, suggesting the involvement of calcium in CoQ10-mediated AMPK signaling. CoQ10 also increased the expression of peroxisome proliferator-activated receptor alpha (PPAR?) at both the mRNA and protein levels. Knock down of AMPK with siRNA or inhibition of AMPK using an AMPK inhibitor compound C blocked CoQ10-induced expression of PPAR?, indicating that AMPK plays a critical role in PPAR? induction. In addition, CoQ10 increased fatty acid oxidation in 3T3-L1preadipocytes. The promoter activity of PPAR? was increased by CoQ10 in an AMPK-dependent fashion. Moreover, the induction of acyl-CoA oxidase (ACO), a target gene of PPAR?, was blocked under the PPAR? knock down condition. Furthermore, treatment with CoQ10 blocked differentiation-induced adipogenesis. This blockade was not observed under the PPAR? knock-down condition. Collectively, these results demonstrate that CoQ10 induces PPAR? expression via the calcium-mediated AMPK signal pathway and suppresses differentiation-induced adipogenesis. PMID:22885103

Lee, Soo Kyung; Lee, Jung Ok; Kim, Ji Hae; Kim, Nami; You, Ga Young; Moon, Ji Wook; Sha, Jie; Kim, Su Jin; Lee, Yong Woo; Kang, Ho Jin; Park, Sun Hwa; Kim, Hyeon Soo



Agonist-induced receptor internalization in Chinese hamster ovary cells stably co-expressing ?(1)- and ?(2)-adrenergic receptors.  


?(1)- and ?(2)-Adrenergic receptors (?(1)-AR and ?(2)-AR) are co-expressed in numerous tissues, for example, heart and bladder. They play a very important role in the responses of a variety of organs to sympathetic nerve stimulation. Recent studies suggest that many G protein-coupled receptors, such as ?(1)-AR, ?(2)-AR, ? opioid receptor and ? opioid receptor, can form homo- and heterooligomers. Previous studies demonstrated that the ?(1)-AR and ?(2)-AR formed dimers in living HEK 293 cells. The aim of the present study is to investigate whether such heterooligomerization affect the agonist-induced receptor internalization in the CHO-K1 cells stably co-expressing ?(1)-AR and ?(2)-AR. Using co-immunoprecipitation, we confirmed that ?(1)-AR and ?(2)-AR formed heterooligomers in the CHO-K1 cells. In cells co-expressing ?(1)-AR and ?(2)-AR, 30% of ?(1)-AR was internalized by isoproterenol, whereas only 20% of ?(1)-AR was internalized in cells expressing the ?(1)-AR alone. Heterooligomerization did not affect the ratio of internalized ?(2)-AR. Salmeterol, a specific ?(2)-AR agonist, broke ?(1)-AR/?(2)-AR heterooligomers, and induced ?(2)-AR-specific internalization in cells co-expressing ?(1)-AR and ?(2)-AR. The present study demonstrated that heterooligomerization between ?(1)-AR and ?(2)-AR accelerates the isoproterenol-promoted internalization of the ?(1)-AR, and that salmeterol induces ?(2)-AR-specific internalization in Chinese hamster ovary (CHO) cells stably co-expressing ?(1)-AR and ?(2)-AR. PMID:23302644

Yoshihara, Takako; Yonoki, Yuzuru; Saito, Maki; Nakahara, Tsutomu; Sakamoto, Kenji; Ishii, Kunio



Magnetic moments of T=3/2 mirror pairs  

SciTech Connect

We predict values of the magnetic moments of T=3/2 proton-rich fp-shell nuclei in the mass range A=43-53, by using known values for their neutron-rich mirrors together with shell-model estimates for small quantities. We extend the analysis to those T=3/2 sd-shell mirror pairs for which both the T{sub z}=-3/2 and T{sub z}=+3/2 magnetic moments have been measured. We find that these obey the same linear relation as previously deduced for T=1/2 mirror pairs.

Perez, S. M. [Department of Physics, University of Cape Town, Private Bag, Rondebosch 7700 (South Africa); iThemba LABS, P. O. Box 722, Somerset West 7129 (South Africa); Richter, W. A. [Department of Physics, University of the Western Cape, Private Bag X17, Bellville 7535 (South Africa); Brown, B. A. [Department of Physics and Astronomy, and National Superconducting Cyclotron Laboratory, Michigan State University, East Lansing, Michigan 48824-1321 (United States); Horoi, M. [Department of Physics, Central Michigan University, Mount Pleasant, Michigan 48859 (United States)



Disabling TNF receptor signaling by induced conformational perturbation of tryptophan-107  

PubMed Central

We have disabled TNF receptor (TNFR) function by inducing allosteric modulation of tryptophan-107 (W107) in the receptor. The allosteric effect operates by means of an allosteric cavity found a short distance from a previously identified loop involved in ligand binding. Occupying this cavity by small molecules leads to perturbation of distal W107 and disables functions of the TNFR, a molecule not known to undergo conformational change upon binding TNF-?. TNF-?-induced NF-?B and p38 kinase activities and clinical symptoms of collagen-induced arthritis in mice were all diminished. Thus, disabling receptor function by induced conformational changes of active binding surfaces represents an innovative paradigm in structure-based drug design.

Murali, Ramachandran; Cheng, Xin; Berezov, Alan; Du, Xiulian; Schon, Arnie; Freire, Ernesto; Xu, Xiaowei; Chen, Youhai H.; Greene, Mark I.



Transformation of NIH 3T3 cells by microinjection of Haras p21 protein  

Microsoft Academic Search

Alteration in gene structure has been shown to occur in some human tumours1-5. These altered genes, termed oncogenes, were originally identified by their ability to induce foci of transformed cells on transfected mouse 3T3 cultures. The oncogene identified in the EJ\\/T24 human bladder carcinoma is similar to the transforming gene of BALB and Harvey murine sarcoma virus (MSV)6,7 and differs

Dennis W. Stacey; Hsiang-Fu Kung



Corticosteroids as long-term regulators of the insulin effectiveness in mouse 3T3 adipocytes  

Microsoft Academic Search

Summary  Since corticosteroid treatment is often accompanied by insulin resistance, we explored the role of corticosteroids in the regulation of the insulin effectiveness in cultured 3T3 (mouse) adipocytes. Exposure of the fat cells to dexamethasone or corticosterone (0–5 days) induced a time-, concentration-, and protein synthesis-dependent and reversible decrease in insulin binding and in basal and insulin-stimulated 2-deoxyglucose uptake. The decrease

J. P. M. van Putten; Tj. Wieringa; H. M. J. Krans



Spinal muscarinic receptors are activated during low or high frequency TENS-induced antihyperalgesia in rats  

PubMed Central

Transcutaneous electrical nerve stimulation (TENS) is a non-pharmacological modality used clinically to relieve pain. Central involvement of serotonin and endogenous opioids are implicated in TENS-induced analgesia. Activation of spinal cholinergic receptors is antinociceptive and these receptors interact with opioid and serotonin receptors. In the current study, the possible involvement of spinal cholinergic receptors in TENS analgesia was investigated in rats. Hyperalgesia was induced by inflaming one knee joint with 3% kaolin—carrageenan and assessed by measuring paw withdrawal latency (PWL) to heat before and 4 h after injection. The non-selective nicotinic antagonist mecamylamine (50 ?g), non-selective muscarinic antagonist atropine (30 ?g) or one of the muscarinic subtype antagonists: pirenzepine (M1, 10 ?g), methoctramine (M2, 10 ?g), 4-DAMP (M3, 10 ?g), or saline was administered intrathecally just prior to TENS treatment. Low or high frequency TENS was then applied to the inflamed knee and PWL was determined again. Atropine, pirenzepine and 4-DAMP significantly attenuated the antihyperalgesic effects of low and high frequency TENS while mecamylamine and methoctramine had no effects, compared to saline control. The results show that TENS-induced antihyperalgesia is mediated partially by activation of spinal muscarinic receptors but not spinal nicotinic receptors. Further, the results also indicate that spinal M1 and M3 muscarinic receptor subtypes mediate the muscarinic component of TENS antihyperalgesia.

Radhakrishnan, R.; Sluka, K.A.



Haloperidol induces neurotoxicity by the NMDA receptor downstream signaling pathway, alternative from glutamate excitotoxicity.  


The NMDA receptor is believed to be important in a wide range of nervous system functions including neuronal migration, synapse formation, learning and memory. In addition, it is involved in excitotoxic neuronal cell death that occurs in a variety of acute and chronic neurological disorders. Besides of agonist/coagonist sites, other modulator sites, including butyrophenone site may regulate the N-methyl-D-aspartate receptor. It has been shown that haloperidol, an antipsychotic neuroleptic drug, interacts with the NR2B subunit of NMDA receptor and inhibits NMDA response in neuronal cells. We found that NMDA receptor was co-immunoprecipitated by anti-Ras antibody and this complex, beside NR2 subunit of NMDA receptor contained haloperidol-binding proteins, nNOS and Ras-GRF. Furthermore, we have shown that haloperidol induces neurotoxicity of neuronal cells via NMDA receptor complex, accompanied by dissociation of Ras-GRF from membranes and activation of c-Jun-kinase. Inclusion of insulin prevented relocalization of Ras-GRF and subsequent neuronal death. Haloperidol-induced dissociation of Ras-GRF leads to inhibition of membrane-bound form of Ras protein and changes downstream regulators activity that results in the initiation of the apoptotic processes via the mitochondrial way. Our results suggest that haloperidol induces neuronal cell death by the interaction with NMDA receptor, but through the alternative from glutamate excitotoxicity signaling pathway. PMID:17092607

Zhuravliova, E; Barbakadze, T; Natsvlishvili, N; Mikeladze, D G



Cholinergic Interneurons Mediate Fast VGluT3-Dependent Glutamatergic Transmission in the Striatum  

PubMed Central

The neurotransmitter glutamate is released by excitatory projection neurons throughout the brain. However, non-glutamatergic cells, including cholinergic and monoaminergic neurons, express markers that suggest that they are also capable of vesicular glutamate release. Striatal cholinergic interneurons (CINs) express the Type-3 vesicular glutamate transporter (VGluT3), although whether they form functional glutamatergic synapses is unclear. To examine this possibility, we utilized mice expressing Cre-recombinase under control of the endogenous choline acetyltransferase locus and conditionally expressed light-activated Channelrhodopsin2 in CINs. Optical stimulation evoked action potentials in CINs and produced postsynaptic responses in medium spiny neurons that were blocked by glutamate receptor antagonists. CIN-mediated glutamatergic responses exhibited a large contribution of NMDA-type glutamate receptors, distinguishing them from corticostriatal inputs. CIN-mediated glutamatergic responses were insensitive to antagonists of acetylcholine receptors and were not seen in mice lacking VGluT3. Our results indicate that CINs are capable of mediating fast glutamatergic transmission, suggesting a new role for these cells in regulating striatal activity.

Higley, Michael J.; Balthasar, Nina; Seal, Rebecca P.; Edwards, Robert H.; Lowell, Bradford B.; Kreitzer, Anatol C.; Sabatini, Bernardo L.



Effect of Gambisan on the Inhibition of Adipogenesis in 3T3-L1 Adipocytes  

PubMed Central

This study was conducted to explore the antiadipogenic effect and possible mechanism of Gambisan on 3T3-L1 cells. For quality control, Gambisan was standardized by HPLC and the standard compounds ephedrine, epigallocatechin-3-gallate, and caffeine were screened. Cultured 3T3-L1 cells that had been induced to differentiate were treated with various concentrations of Gambisan or its major component extracts (Ephedra intermedia Schrenk, Atractylodes lancea DC., and Thea sinensis L.) for 72 hours for MTT assay to determine cell viability or 10 days for LDH assay, triglyceride assay, DNA content measurement, Oil red O staining, RT-PCR, and western blot. Gambisan significantly inhibited adipogenesis in 3T3-L1 cells by reducing triglyceride contents and lipid accumulation in a dose-dependent manner without obvious cytotoxicity. Viability and DNA content in 3T3-L1 cells treated with Gambisan were significantly higher than cells treated with the major component extracts at every concentration. The anti-adipogenic effects of Gambisan appeared to be mediated by a significant downregulation of the expression of lipoprotein lipase mRNA and PPAR?, C/EBP?, and SREBP-1 protein apart from the expression of hormone-sensitive lipase. Gambisan could act as a possible therapeutic agent for obesity. However, further studies including in vivo assays and clinical trials are needed to confirm the efficacy, safety and mechanisms of the antiobesity effects of Gambisan.

Kang, Jung Won; Nam, Dongwoo; Kim, Kun Hyung; Huh, Jeong-Eun; Lee, Jae-Dong



Effect of Gambisan on the Inhibition of Adipogenesis in 3T3-L1 Adipocytes.  


This study was conducted to explore the antiadipogenic effect and possible mechanism of Gambisan on 3T3-L1 cells. For quality control, Gambisan was standardized by HPLC and the standard compounds ephedrine, epigallocatechin-3-gallate, and caffeine were screened. Cultured 3T3-L1 cells that had been induced to differentiate were treated with various concentrations of Gambisan or its major component extracts (Ephedra intermedia Schrenk, Atractylodes lancea DC., and Thea sinensis L.) for 72 hours for MTT assay to determine cell viability or 10 days for LDH assay, triglyceride assay, DNA content measurement, Oil red O staining, RT-PCR, and western blot. Gambisan significantly inhibited adipogenesis in 3T3-L1 cells by reducing triglyceride contents and lipid accumulation in a dose-dependent manner without obvious cytotoxicity. Viability and DNA content in 3T3-L1 cells treated with Gambisan were significantly higher than cells treated with the major component extracts at every concentration. The anti-adipogenic effects of Gambisan appeared to be mediated by a significant downregulation of the expression of lipoprotein lipase mRNA and PPAR ? , C/EBP ? , and SREBP-1 protein apart from the expression of hormone-sensitive lipase. Gambisan could act as a possible therapeutic agent for obesity. However, further studies including in vivo assays and clinical trials are needed to confirm the efficacy, safety and mechanisms of the antiobesity effects of Gambisan. PMID:24069055

Kang, Jung Won; Nam, Dongwoo; Kim, Kun Hyung; Huh, Jeong-Eun; Lee, Jae-Dong



Intracellular NAADP increase induced by extracellular NAADP via the P2Y11-like receptor.  


The aim of the study was to identify a signalling pathway allowing NAADP-induced intracellular NAADP increase and involving the P2Y11-like receptor. P2Y11-like and ?-adrenergic receptors may play important regulatory roles within the cardiovascular system. Both receptors have been shown to be involved in triggering myocardial preconditioning. Using a Langendorff model we report a positive inotropic response induced by extracellular NAADP via P2Y11-like receptor stimulation. In cardiomyocyte cultures, P2Y11-like receptor stimulation by extracellular NAADP ([NAADP]e) increased intracellular cADP-ribose and NAADP concentration as evidenced by direct measurements. NF546, a new selective P2Y11 receptor agonist, increased intracellular cAMP, cADP-ribose and NAADP concentration confirming the involvement of the P2Y11-like receptor in this signalling pathway. NF157, a P2Y11 receptor antagonist, suppressed the increase in intracellular cADPr, NAADP and NAAD induced by either [NAADP]e or NF546. The response profile for intracellular cADP-ribose and NAADP concentration following P2Y11-like stimulation with NF546 was similar to reported data relating ?-adrenergic stimulation with isoprenaline. This response represents the signature of the Gs/ADP-ribosyl cyclase activity. Moreover, this study provides a signalling pathway: intracellular NAADP increase induced by extracellular NAADP via metabotropic activity of P2Y11-like receptor. This pathway implying P2Y11-like could take part in the intracellular calcium rise reported for extracellular NAADP. PMID:23726915

Djerada, Zoubir; Millart, Hervé



Castor oil induces laxation and uterus contraction via ricinoleic acid activating prostaglandin EP3 receptors  

PubMed Central

Castor oil is one of the oldest drugs. When given orally, it has a laxative effect and induces labor in pregnant females. The effects of castor oil are mediated by ricinoleic acid, a hydroxylated fatty acid released from castor oil by intestinal lipases. Despite the wide-spread use of castor oil in conventional and folk medicine, the molecular mechanism by which ricinoleic acid acts remains unknown. Here we show that the EP3 prostanoid receptor is specifically activated by ricinoleic acid and that it mediates the pharmacological effects of castor oil. In mice lacking EP3 receptors, the laxative effect and the uterus contraction induced via ricinoleic acid are absent. Although a conditional deletion of the EP3 receptor gene in intestinal epithelial cells did not affect castor oil-induced diarrhea, mice lacking EP3 receptors only in smooth-muscle cells were unresponsive to this drug. Thus, the castor oil metabolite ricinoleic acid activates intestinal and uterine smooth-muscle cells via EP3 prostanoid receptors. These findings identify the cellular and molecular mechanism underlying the pharmacological effects of castor oil and indicate a role of the EP3 receptor as a target to induce laxative effects.

Tunaru, Sorin; Althoff, Till F.; Nusing, Rolf M.; Diener, Martin; Offermanns, Stefan



NMDA Receptor Blockade Alters Stress-Induced Dendritic Remodeling in Medial Prefrontal Cortex  

PubMed Central

The development and relapse of many psychopathologies can be linked to both stress and prefrontal cortex dysfunction. Glucocorticoid stress hormones target medial prefrontal cortex (mPFC) and either chronic stress or chronic administration of glucocorticoids produces dendritic remodeling in prefrontal pyramidal neurons. Exposure to stress also causes an increase in the release of the excitatory amino acid glutamate, which binds to N-methyl-D-aspartate (NMDA) receptors, which are plentiful in mPFC. NMDA receptor activation is crucial for producing hippocampal dendritic remodeling due to stress and for dendritic reorganization in frontal cortex after cholinergic deafferentation. Thus, NMDA receptors could mediate stress-induced dendritic retraction in mPFC. To test this hypothesis, dendritic morphology of pyramidal cells in mPFC was assessed after blocking NMDA receptors with the competitive NMDA antagonist ±3-(2-carboxypiperazin-4yl)propyl-1-phosphonic acid (CPP) during restraint stress. Administration of CPP prevented stress-induced dendritic atrophy. Instead, CPP-injected stressed rats showed hypertrophy of apical dendrites compared with controls. These results suggest that NMDA activation is crucial for stress-induced dendritic atrophy in mPFC. Furthermore, NMDA receptor blockade uncovers a new pattern of stress-induced dendritic changes, suggesting that other neurohormonal changes in concert with NMDA receptor activation underlie the net dendritic retraction seen after chronic stress.

Martin, Kathryn P.



Nucleotide receptors involved in UTP-induced rat arterial smooth muscle cell migration.  


Many factors have been shown to be involved in the development of hyperplasic lesions of vessels, but the role of extracellular nucleotides remains largely unknown. The presence of P2Y and P2X nucleotide receptors on arterial endothelial and smooth muscle cells suggests a potential role for nucleotides in the vessel pathophysiology. Although the role of P2X in physiology of vessels is well documented, that of P2Y is not completely understood. We recently demonstrated that extracellular nucleotides, and particularly UTP, induced migration of cultured arterial smooth muscle cells (ASMCs). This migration is dependent on osteopontin expression and involves the Rho and mitogen-activated protein (MAP) kinase pathways. An important question is to determine the specific role of the different P2Y receptors of rat ASMCs in the UTP-induced migration process. Therefore, we first quantified mRNA levels of P2Y(2), P2Y(4), and P2Y(6) nucleotide receptors in cultured rat ASMCs by a competitive RT-PCR approach and demonstrated that P2Y(2) is the most highly expressed among these receptors potentially involved in the UTP-mediated response. In addition to UTP, UDP also induced ASMC migration even when UTP regeneration was inhibited, suggesting the involvement of UDP receptor P2Y(6). Moreover, suramin, a specific antagonist of rat P2Y(2) receptor, acted as an inhibitor of UTP-induced migration. Taken together, these results suggest a prominent role for the UTP receptor, P2Y(2), and for the UDP receptor, P2Y(6), in UTP-induced rat ASMC migration. PMID:11934835

Pillois, Xavier; Chaulet, Hervé; Belloc, Isabelle; Dupuch, Françoise; Desgranges, Claude; Gadeau, Alain-Pierre



Direct evidence for ligand-induced internalization of the yeast alpha-factor pheromone receptor.  

PubMed Central

When Saccharomyces cerevisiae a cells bind alpha-factor pheromone, the ligand is internalized and its binding sites are lost from the cell surface in a time-, energy-, and temperature-dependent manner. This report presents direct evidence for alpha-factor-induced internalization of cell surface receptors. First, membrane fractionation on Renografin density gradients indicated that the alpha-factor receptors were predominantly found in the plasma membrane peak before alpha-factor treatment and then appeared in membranes of lesser buoyant density after alpha-factor exposure. Second, receptors were susceptible to cleavage by extracellular proteases before alpha-factor treatment and then became resistant to proteolysis after exposure to pheromone, consistent with the transit of receptors from the cell surface to an internal compartment. The median transit time in both assays was approximately 8 min. The ultimate target of the internalized receptors was identified as the vacuole, since the membranes containing internalized receptors cofractionated with vacuolar membranes, since the turnover of receptors was stimulated by alpha-factor exposure, and since receptor degradation was blocked in a pep4 mutant that is deficient for vacuolar proteases. The carboxy-terminal domain of the receptor that is required for ligand internalization was also found to be essential for endocytosis of the receptor. A receptor mutant, ste2-L236H, which is defective for pheromone response but capable of ligand internalization, was found to be proficient for receptor endocytosis. Hence, separate structural features of the receptor appear to specify its signal transduction and internalization activities. Images

Schandel, K A; Jenness, D D



Sciadopitysin protects osteoblast function via its antioxidant activity in MC3T3-E1 cells.  


Age-related osteoblast dysfunction is the main cause of age-related bone loss in both men and women. In the present study, the effect of sciadopitysin, a type of biflavonoids, on osteoblast function was investigated in osteoblastic MC3T3-E1 cells. Sciadopitysin caused a significant elevation of alkaline phosphatase activity, collagen synthesis, osteocalcin production, mineralization, and glutathione content in the cells (P<0.05). Sciadopitysin also decreased the production of tumor necrosis factor-a (TNF-?) induced by antimycin A, a mitochondrial electron transport inhibitor. We investigated the protective effects of sciadopitysin on antimycin A-induced toxicity in osteoblastic MC3T3-E1 cells. Exposure of MC3T3-E1 cells to antimycin A caused a significant reduction in osteoblast dysfunction. However, pretreatment with sciadopitysin prior to antimycin A exposure significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, adenosine triphosphate (ATP) loss, reactive oxygen species (ROS) release, and nitrotyrosine increase, suggesting that sciadopitysin may be useful for protecting mitochondria against a burst of oxidative stress. Moreover, sciadopitysin increased phosphorylation of cAMP-response element-binding protein (CREB) inhibited by antimycin A. Our results demonstrate that sciadopitysin may reduce or prevent osteoblasts degeneration. PMID:23624148

Suh, Kwang Sik; Lee, Young Soon; Kim, Young Seol; Choi, Eun Mi



Drug-induced mild therapeutic hypothermia obtained by administration of a transient receptor potential vanilloid type 1 agonist  

PubMed Central

Background The use of mechanical/physical devices for applying mild therapeutic hypothermia is the only proven neuroprotective treatment for survivors of out of hospital cardiac arrest. However, this type of therapy is cumbersome and associated with several side-effects. We investigated the feasibility of using a transient receptor potential vanilloid type 1 (TRPV1) agonist for obtaining drug-induced sustainable mild hypothermia. Methods First, we screened a heterogeneous group of TRPV1 agonists and secondly we tested the hypothermic properties of a selected candidate by dose-response studies. Finally we tested the hypothermic properties in a large animal. The screening was in conscious rats, the dose-response experiments in conscious rats and in cynomologus monkeys, and the finally we tested the hypothermic properties in conscious young cattle (calves with a body weight as an adult human). The investigated TRPV1 agonists were administered by continuous intravenous infusion. Results Screening: Dihydrocapsaicin (DHC), a component of chili pepper, displayed a desirable hypothermic profile with regards to the duration, depth and control in conscious rats. Dose-response experiments: In both rats and cynomologus monkeys DHC caused a dose-dependent and immediate decrease in body temperature. Thus in rats, infusion of DHC at doses of 0.125, 0.25, 0.50, and 0.75 mg/kg/h caused a maximal ?T (°C) as compared to vehicle control of -0.9, -1.5, -2.0, and -4.2 within approximately 1 hour until the 6 hour infusion was stopped. Finally, in calves the intravenous infusion of DHC was able to maintain mild hypothermia with ?T > -3°C for more than 12 hours. Conclusions Our data support the hypothesis that infusion of dihydrocapsaicin is a candidate for testing as a primary or adjunct method of inducing and maintaining therapeutic hypothermia.



Opposite modulation of apomorphine- or amphetamine-induced stereotypy by antagonists of CCK receptors.  


Stereotyped behavior is elicited by activation of dopaminergic systems with drugs such as apomorphine and amphetamine. In previous studies, we have reported that the sulfated cholecystokinin octapeptide (CCK-8) decreased apomorphine-induced stereotypy in animals with normal and supersensitive dopamine receptors. The aim of the present study was to evaluate the effects of CCK(1) and CCK(2) receptor antagonists on stereotyped behavior induced by apomorphine or amphetamine. Rats were pretreated with the CCK(1) (SR 27897B; 1-[[2-(4-(2-chlorophenyl) thiazol-2-yl) aminocarbonyl]indolyl]acetic acid; 500 microg/kg; i.p.) or CCK(2) (L-365,260; 3R-(+)-N-(2,3-dihydro-1-methyl-2-oxo-5 phenyl-1H-1, 4-benzodiazepine-3-yl)-N'-(3-methyl phenyl)-urea; 500 microg/kg; i.p. ) receptor antagonists or saline 15 min before apomorphine (0.6 mg/kg; s.c.) or amphetamine (9.0 mg/kg; i.p.) injection. Both CCK(1) and CCK(2) receptor antagonists significantly increased apomorphine-induced stereotypy. In contrast, only the blockade of CCK(2) receptors significantly decreased amphetamine-induced stereotypy. The results suggest a dual opposite mechanism for CCK-dopamine interactions. These data also suggest that both apomorphine- and amphetamine-induced stereotypy should be used whenever effects of drugs acting on dopaminergic systems are being assessed. PMID:10650159

Tieppo, C A; Ferreira, F S; Sassatani, A S; Felicio, L F; Nasello, A G



Multiple CNS nicotinic receptors mediate l-dopa-induced dyskinesias: Studies with parkinsonian nicotinic receptor knockout mice.  


Accumulating evidence supports the idea that drugs acting at nicotinic acetylcholine receptors (nAChRs) may be beneficial for Parkinson's disease, a neurodegenerative movement disorder characterized by a loss of nigrostriatal dopaminergic neurons. Nicotine administration to parkinsonian animals protects against nigrostriatal damage. In addition, nicotine and nAChR drugs improve l-dopa-induced dyskinesias, a debilitating side effect of l-dopa therapy which remains the gold-standard treatment for Parkinson's disease. Nicotine exerts its antidyskinetic effect by interacting with multiple nAChRs. One approach to identify the subtypes specifically involved in l-dopa-induced dyskinesias is through the use of nAChR subunit null mutant mice. Previous work with ?2 and ?6 nAChR knockout mice has shown that ?6?2* nAChRs were necessary for the development/maintenance of l-dopa-induced abnormal involuntary movements (AIMs). The present results in parkinsonian ?4 nAChR knockout mice indicate that ?4?2* nAChRs also play an essential role since nicotine did not reduce l-dopa-induced AIMs in such mice. Combined analyses of the data from ?4 and ?6 knockout mice suggest that the ?6?4?2?3 subtype may be critical. In contrast to the studies with ?4 and ?6 knockout mice, nicotine treatment did reduce l-dopa-induced AIMs in parkinsonian ?7 nAChR knockout mice. However, ?7 nAChR subunit deletion alone increased baseline AIMs, suggesting that ?7 receptors exert an inhibitory influence on l-dopa-induced AIMs. In conclusion, ?6?2*, ?4?2* and ?7 nAChRs all modulate l-dopa-induced AIMs, although their mode of regulation varies. Thus drugs targeting one or multiple nAChRs may be optimal for reducing l-dopa-induced dyskinesias in Parkinson's disease. PMID:23831952

Quik, Maryka; Campos, Carla; Grady, Sharon R



(+)-Episesamin inhibits adipogenesis and exerts anti-inflammatory effects in 3T3-L1 (pre)adipocytes by sustained Wnt signaling, down-regulation of PPAR? and induction of iNOS.  


Obesity and its associated health risks still demand for effective therapeutic strategies. Drugs and compositions derived from Oriental medicine such as green tea polyphenols attract growing attention. Previously, an extract from the Japanese spice bush Lindera obtusiloba (L. obtusiloba) traditionally used for treatment of inflammation and prevention of liver damage was shown to inhibit adipogenesis. Aiming for the active principle of this extract (+)-episesamin was identified, isolated and applied in adipogenic research using 3T3-L1 (pre)adipocytes, an established cell line for studying adipogenesis. With an IC50 of 10?M (+)-episesamin effectively reduced the growth of 3T3-L1 preadipocytes and decreased hormone-induced 3T3-L1 differentiation as shown by reduced accumulation of intracellular lipid droplets and diminished protein expression of GLUT-4 and vascular endothelial growth factor. Mechanistically, the presence of (+)-episesamin during hormone-induced differentiation provoked a reduced phosphorylation of ERK1/2 and ?-catenin along with a reduced protein expression of peroxisome proliferator-activated receptor ? and a strongly increased protein expression of iNOS. Treatment of mature adipocytes with (+)-episesamin resulted in a reduction of intracellular stored lipid droplets and induced the proapoptotic enzymes caspases-3/-7. Besides interfering with adipogenesis, (+)-episesamin showed anti-inflammatory activity by counteracting the lipopolysaccharide- and tumor necrosis factor ?-induced secretion of interleukin 6 by 3T3-L1 preadipocytes. In conclusion, (+)-episesamin seems to be the active drug in the L. obtusiloba extract being responsible for the inhibition of adipogenesis and, thus, should be evaluated as a novel potential complementary treatment for obesity. PMID:22818712

Freise, Christian; Trowitzsch-Kienast, Wolfram; Erben, Ulrike; Seehofer, Daniel; Kim, Ki Young; Zeitz, Martin; Ruehl, Martin; Somasundaram, Rajan



Teachers Teaching Teachers (T3). Volume 6, Number 1  

ERIC Educational Resources Information Center

|"Teachers Teaching Teachers" ("T3") focuses on coaches' roles in the professional development of teachers. Each issue also explores the challenges and rewards that teacher leaders encounter. This issue includes: (1) Collective Responsibility Makes All Teachers the Best (Stephanie Hirsh); (2) Tools: How Our School Measures up/Exploring Our…

Armstrong, Anthony, Ed.



Teachers Teaching Teachers (T3)[TM]. Volume 4, Number 6  

ERIC Educational Resources Information Center

|"Teachers Teaching Teachers" ("T3") focuses on coaches' roles in the professional development of teachers. Each issue also explores the challenges and rewards that teacher leaders encounter. This issue includes: (1) Values and Clarity Build Classroom Language (Valerie von Frank); (2) Tools: Identifying and Clarifying Beliefs about Learning; (3)…

von Frank, Valerie, Ed.



Teachers Teaching Teachers (T3) [TM]. Vol. 2 No. 7  

ERIC Educational Resources Information Center

This issue of "Teachers Teaching Teachers" ("T3") focuses on coaches' role in the professional development of teachers. It contains the following articles: (1) An Excerpt from "Taking the Lead" (Joellen Killion and Cindy Harrison); (2) Be Like a Virus and Connect (Bill Ferriter); (3) No. 1 Resource Has a Human Face (Joellen Killion); (4) With This…

Richardson, Joan, Ed.



T3: On Mapping Text To Time Series  

Microsoft Academic Search

We investigate if the mapping between text and time series data is feasible such that relevant data mining problems in text can find their counterparts in time series (and vice versa). As a preliminary work, we present the T3 (Text To Time series) framework that utilizes dierent

Tao Yang; Dongwon Lee



Teachers Teaching Teachers (T3)[TM]. Volume 4, Number 7  

ERIC Educational Resources Information Center

"Teachers Teaching Teachers" ("T3") focuses on coaches' roles in the professional development of teachers. Each issue also explores the challenges and rewards that teacher leaders encounter. This issue includes: (1) Learning Cycle Spins Individuals into a Team (Valerie von Frank); (2) NSDC Tool: The Professional Teaching and Learning Cycle; (3)…

von Frank, Valerie, Ed.



Numerical Tokamak Turbulence Calculations on the CRAY T3E  

SciTech Connect

Full cross section calculations of ion-temperature-gradient-driven turbulence with Landau closure are being carried out as part of the Numerical Tokamak Turbulence Project, one of the U.S. Department of Energy`s Phase II Grand Challenges. To include the full cross section of a magnetic fusion device like the tokamak requires more memory and CPU time than is available on the National Energy Research Scientific Computing Center`s (NERSC`s) shared-memory vector machines such as the CRAY C90 and J90. Calculations of cylindrical multi-helicity ion-temperature-gradient-driven turbulence were completed on NERSC`s 160-processor distributed-memory CRAY T3E parallel computer with 256 Mbytes of memory per processor. This augurs well for yet more memory and CPU intensive calculations on the next-generation T3E at NERSC. This paper presents results on benchmarks with the current T3E at NERSC. Physics results pertaining to plasma confinement at the core of tokamaks subject to ion-temperature-gradient-driven-turbulence are also highlighted. Results at this resolution covering this extent of physical time were previously unattainable. Work is in progress to increase the resolution, improve the performance of the parallel code, and include toroidal geometry in these calculations in anticipation of the imminent arrival of a fully configured,512-processor, T3E-900 model.

Lynch, V.E., Leboeuf, J.N., Carreras, B.A. [Oak Ridge National Lab., TN (United States)], Alvarez, J.D., Garcia, L. [Universidad `Carlos III` de Madrid (Spain)



T 4 , T 3 and rT 3 levels in serum and cerebrospinal fluid of patients with amyotrophic lateral sclerosis  

Microsoft Academic Search

Thyronine (T4), triiodothyronine (T3), and reversetriiodothyronine (rT3) levels were evaluated in cerebrospinal fluid (CSF) and in serum of 12 patients with definite amyotrophic lateral sclerosis (ALS) by specific radioimmunoassays. Circulating microsomal and thyroglobulin antibodies were also evaluated. In all patients serum levels of T4, T3 and rT3 were within normal limits. In CSF, the rT3 levels were significantly elevated to

J.-P. Malin; R. Ködding; H. Fuhrmann; A. Mühlen



NMDA receptor activation induces translocation and activation of Rac in mouse hippocampal area CA1  

PubMed Central

Neuronal development requires several discrete morphological steps that are believed to involve the small GTPase Rac. For example, neural activity, through NMDA receptors and/or AMPA receptors, activates Rac leading to elaboration of dendritic arbors. In the current study, we have conducted studies which indicate that Rac might be an important molecule involved in morphological plasticity in the adult mouse. We demonstrate that Rac is expressed at synapses in the adult mouse hippocampus. We also demonstrate that treatment of hippocampal slices with NMDA induces membrane translocation and activation of Rac in area CA1. Interestingly, we also find that there is an increase in Rac that is associated with NMDA receptor complexes following NMDA receptor activation. Taken together, our data are consistent with the idea that Rac could be participating in NMDA receptor-dependent changes in morphology that occur during synaptic plasticity and memory formation in the adult mouse hippocampus.

Tejada-Simon, Maria V.; Villasana, Laura E.; Serrano, Faridis; Klann, Eric



Dual Roles for Endothelin-B Receptors in Modulating Adjuvant-Induced Inflammatory Hyperalgesia in Rats.  


Injection of endothelin-1 (ET-1) into the plantar rat hindpaw causes acute pain at high concentrations and tactile sensitization at low concentrations. The pro-nociceptive actions are driven through ET(A) receptors for both levels of [ET-1], but the ET(B) receptors are only pro-nociceptive for allodynia from low [ET-1] and anti-nociceptive for pain from high [ET-1]. The goal of the present work was to discriminate the roles of the ET receptors in the acute hyperalgesia from inflammation by complete Freund's adjuvant (CFA, 20 mg/paw) into the rat hindpaw. Selective antagonists were injected l0 min before and then together with CFA. An ET(A) receptor antagonist, BQ-123, reduced CFA-induced thermal hyperalgesia (by up to 50%), as did an ET(B) receptor antagonist, BQ-788 (by up to 66%). BQ-123 and BQ-788 also delayed the onset (by 1.5 - 2 h) but insignificantly reduced the maximum degree of CFA-induced allodynia (~10%). Surprisingly, an ET(B) receptor agonist, IRL-1620, also reduced maximum thermal hyperalgesia induced by CFA, suppressed peak allodynia and delayed its occurrence by ~ 3 h. The latter actions of IRL-1620 were reversed by co-administration of BQ-788, naloxone hydrochloride and the peripherally restricted opiate receptor antagonist naloxone methiodide, and by antiserum against ?-endorphin. These findings demonstrate an important role for endogenous ET-1 in acute inflammatory pain and a dual action of ET(B) receptors, including a pro-algesic action along with the important activation of a local analgesic pathway, implying that at least two different ET(B) receptors contribute to modulation of inflammatory pain. PMID:20559459

Khodorova, Alla; Zou, Shiping; Ren, Ke; Dubner, Ronald; Davar, Gudarz; Strichartz, Gary



Dual Roles for Endothelin-B Receptors in Modulating Adjuvant-Induced Inflammatory Hyperalgesia in Rats  

PubMed Central

Injection of endothelin-1 (ET-1) into the plantar rat hindpaw causes acute pain at high concentrations and tactile sensitization at low concentrations. The pro-nociceptive actions are driven through ETA receptors for both levels of [ET-1], but the ETB receptors are only pro-nociceptive for allodynia from low [ET-1] and anti-nociceptive for pain from high [ET-1]. The goal of the present work was to discriminate the roles of the ET receptors in the acute hyperalgesia from inflammation by complete Freund's adjuvant (CFA, 20 mg/paw) into the rat hindpaw. Selective antagonists were injected l0 min before and then together with CFA. An ETA receptor antagonist, BQ-123, reduced CFA-induced thermal hyperalgesia (by up to 50%), as did an ETB receptor antagonist, BQ-788 (by up to 66%). BQ-123 and BQ-788 also delayed the onset (by 1.5 – 2 h) but insignificantly reduced the maximum degree of CFA-induced allodynia (~10%). Surprisingly, an ETB receptor agonist, IRL-1620, also reduced maximum thermal hyperalgesia induced by CFA, suppressed peak allodynia and delayed its occurrence by ~ 3 h. The latter actions of IRL-1620 were reversed by co-administration of BQ-788, naloxone hydrochloride and the peripherally restricted opiate receptor antagonist naloxone methiodide, and by antiserum against ?-endorphin. These findings demonstrate an important role for endogenous ET-1 in acute inflammatory pain and a dual action of ETB receptors, including a pro-algesic action along with the important activation of a local analgesic pathway, implying that at least two different ETB receptors contribute to modulation of inflammatory pain.

Khodorova, Alla; Zou, Shiping; Ren, Ke; Dubner, Ronald; Davar, Gudarz; Strichartz, Gary



Toll-Like Receptor 2 Modulates the Proinflammatory Milieu in Staphylococcus aureus-Induced Brain Abscess  

Microsoft Academic Search

Toll-like receptor 2 (TLR2) is a pattern recognition receptor (PRR) that plays an important role in innate immune recognition of conserved structural motifs on a wide array of pathogens, including Staphylococcus aureus. To ascertain the functional significance of TLR2 in the context of central nervous system (CNS) parenchymal infection, we evaluated the pathogenesis of S. aureus-induced experimental brain abscess in

Tammy Kielian; Anessa Haney; Patrick M. Mayes; Sarita Garg; Nilufer Esen



Intra-accumbens infusion of D 3 receptor agonists reduces spontaneous and dopamine-induced locomotion  

Microsoft Academic Search

The present study investigated whether PD 128907 and 7-OH-DPAT, described as preferential dopamine (DA) D3 receptor agonists, produce hypolocomotion by acting at postsynaptic dopaminergic receptors within the nucleus accumbens. Bilateral infusion of PD 128907 (1.5 and 3 ?g\\/0.5 ?l) induced a dose-dependent hypolocomotion, whereas its enantiomer, PD 128908, was inactive. Local infusion of 7-OH-DPAT and the preferential DA autoreceptor agonist,

Abdel-Mouttalib Ouagazzal; Ian Creese



Stimulation of 131 Integrins on Fibroblasts Induces PDGF Independent Tyrosine Phosphorylation of PDGF (3Receptors  

Microsoft Academic Search

We report that integrin-mediated signaling induces a rapid and transient tyrosine phosphorylation of platelet-derived growth factor (PDGF) 13-receptors in human diploid foreskin AG 1518 fibroblasts. A tran- sient tyrosine phosphorylation of PDGF i3-receptors was evident one and two hours after cells had been plated on collagen type I and fibronectin, as well as on immobilized anti-integrin subunit IgG, but not

Christian Sundberg; Kristofer Rubin



Ontogeny of dopamine agonist-induced sensitization: role of NMDA receptors  

Microsoft Academic Search

In contrast to adults, preweanling rats exhibit behavioral sensitization for only a few days after cessation of dopamine\\u000a (DA) agonist treatment. The reasons for this ontogenetic difference are uncertain, but maturational changes in the N-methyl-d-aspartate (NMDA) receptor may be responsible, since stimulation of these receptors is necessary for the development of DA\\u000a agonist-induced sensitization in adult rats. The purpose of

M. A. Duke; Jason O’Neal; S. A. McDougall



Swim training improves leptin receptor deficiency-induced obesity and lipid disorder by activating uncoupling proteins  

Microsoft Academic Search

Leptin receptor deficiency causes morbid obesity and hyperlipidemia in mice. Since physical exercise enhances energy expenditure, it is an important part of successful weight-control regimens. We investi- gated the mechanism by which swim training regulates leptin receptor deficiency-induced obesity and lipid disorder in a mouse model of obesity (obese db\\/db mouse). Swim training for 6 weeks significantly decreased body weight

Ki Sook Oh; Eun Young Kim; Michung Yoon; Chung Moo Lee


Methamphetamine-induced behavioral sensitization in mice: alterations in  ? -opioid receptor  

Microsoft Academic Search

Summary  We had previously demonstrated that opioid receptors contribute to the induction and expression of behavioral sensitization induced by repeated daily injection with 2.5 mg\\/kg of methamphetamine for 7 days. Using the same regimen, the present study investigated the alterations in ?-opioid receptor during the induction (on days 2, 5, and 8) and expression (on days 11 and 21) periods of behavioral sensitization. Radioligand

Chi-Tso Chiu; Tangeng Ma; Ing K. Ho



Tumor Necrosis Factor Receptor Family Member RANK Mediates Osteoclast Differentiation and Activation Induced by Osteoprotegerin Ligand  

Microsoft Academic Search

A receptor that mediates osteoprotegerin ligand (OPGL)-induced osteoclast differentiation and activation has been identified via genomic analysis of a primary osteoclast precursor cell cDNA library and is identical to the tumor necrosis factor receptor (TNFR) family member RANK. The RANK mRNA was highly expressed by isolated bone marrow-derived osteoclast progenitors and by mature osteoclasts in vivo. Recombinant OPGL binds specifically

Hailing Hsu; David L. Lacey; Colin R. Dunstan; Irina Solovyev; Anne Colombero; Emma Timms; Hong-Lin Tan; Gary Elliott; Michael J. Kelley; Ildiko Sarosi; Ling Wang; Xing-Zhong Xia; Robin Elliott; Laura Chiu; Tabitha Black; Sheila Scully; Casey Capparelli; Sean Morony; Grant Shimamoto; Michael B. Bass; William J. Boyle



Endogenous b-Adrenergic Receptors Inhibit Lipopolysaccharide-Induced Pulmonary Cytokine Release and Coagulation  

Microsoft Academic Search

b2-adrenergic receptors are expressed on different cell types in the lung, including respiratory epithelial cells, smooth muscle cells, and macrophages.Theaimofthecurrentstudywastodeterminetherole of b-adrenergic receptors in the regulation of lung inflammation induced by instillation via the airways of lipopolysaccharide (LPS) (a constituent of the gram-negative bacterial cell wall) or lipoteichoic acid (LTA) (a component of the gram-positive bacterial cell wall). Mice inhaled

Ida A. J. Giebelen; Masja Leendertse; Mark C. Dessing; Joost C. M. Meijers; Marcel Levi; Christian Draing; Sonja von Aulock; Tom van der Poll


Can a serotonin type 3 (5HT3) receptor antagonist reduce experimentally-induced itch?  

Microsoft Academic Search

Background: Serotonin type 3 (5-HT3) receptor antagonists have been reported to be a novel therapeutic principle for the treatment of cholestatic and uremic pruritus.¶Objective: To determine the antipruritic effect of a 5-HT3 receptor antagonist (tropisetron) on histamine and serotonin-induced itch under experimental conditions in comparison to native skin and after pretreatment with an orally applied antihistamine (cetirizine).¶Methods: Histamine and serotonin

E. Weisshaar; B. Ziethen; H. Gollnick



Metabotropic Glutamate Receptors Modulate Ischemia-Induced GABA Release in Mouse Hippocampal Slices  

Microsoft Academic Search

The involvement of glutamate receptors in GABA release in ischemia was investigated in hippocampal slices from adult (3-month-old) and developing (7-day-old) mice. For in vitro ischemia, the slices were superfused in glucose-free media under nitrogen. Ionotropic glutamate receptor agonists failed to affect the ischemia-induced basal GABA release at either age. The K+-stimulated release in the immature hippocampus was potentiated by

Pirjo Saransaari; Simo S. Oja



The platelet fibrinogen receptor: an immunogold-surface replica study of agonist-induced ligand binding and receptor clustering  

PubMed Central

Platelet aggregation requires the binding of fibrinogen to its receptor, a heterodimer consisting of the plasma-membrane glycoproteins (GP) IIb and IIIa. Although the GPIIb-IIIa complex is present on the surface of unstimulated platelets, it binds fibrinogen only after platelet activation. We have used an immunogold-surface replica technique to study the distribution of GPIIb-IIIa and bound fibrinogen over broad areas of surface membranes in unstimulated, as well as thrombin-activated and ADP-activated human platelets. We found that the immunogold-labeled GPIIb-IIIa was monodispersed over the surface of unstimulated platelets, although the cell surface lacked immunoreactive fibrinogen. On thrombin-stimulated platelets, approximately 65% of the GPIIb-IIIa molecules were in clusters within the plane of the membrane. Fibrinogen, which had been released from the alpha-granules of these cells, bound to GPIIb-IIIa on the cell surface and was similarly clustered. To determine whether the receptors clustered before ligand binding, or as a consequence thereof, we studied the surface distribution of GPIIb-IIIa after stimulation with ADP, which causes activation of the fibrinogen receptor function of GPIIb-IIIa without inducing the release of fibrinogen. In the absence of added fibrinogen, the unoccupied, yet binding-competent receptors on ADP-stimulated platelets were monodispersed. The addition of fibrinogen caused the GPIIb-IIIa molecules to cluster on the cell surface. Clustering was also induced by the addition of the GPIIb-IIIa-binding domains of fibrinogen, namely the tetrapeptide Arg-Gly-Asp-Ser on the alpha-chain or the gamma-chain decapeptide gamma 402-411. These results show that receptor occupancy causes clustering of GPIIb-IIIa in activated platelets.



Characterization of Ligand-Induced Endocytosis of EGF - Receptors.  

National Technical Information Service (NTIS)

We have made progress this year in engineering and using a cell line that is conditionally defective in receptor-mediated endocytosis to directly assess the role of endocytosis in controlling cellular responses to activated EGF-R. We find, not surprisingl...

S. L. Schmid C. LAmaze



Galantamine improves apomorphine-induced deficits in prepulse inhibition via muscarinic ACh receptors in mice  

PubMed Central

Background and purpose Galantamine, a weak acetylcholine esterase (AChE) inhibitor and allosteric potentiator of nicotinic ACh receptors (nAChRs), improves apomorphine-induced deficits in prepulse inhibition (PPI), sensory information-processing deficits, via a nAChR-independent mechanism. The present study examined the role of muscarinic ACh receptors (mAChRs) in the effect of galantamine, and studied the mechanism of galantamine-induced increases in prefrontal ACh levels in mice. Experimental approach Apomorphine (1 mg kg?1) was administered to male ddY mice (9–10 weeks old) to create a PPI deficit model. Extracellular ACh concentrations in the prefrontal cortex were measured by in vivo microdialysis. Key results Galantamine- and donepezil-mediated improvements in apomorphine-induced PPI deficits were blocked by the preferential M1 mAChR antagonist telenzepine. The mAChR agonist oxotremorine also improved apomorphine-induced PPI deficits. Galantamine, like donepezil, increased extracellular ACh concentrations in the prefrontal cortex. Galantamine-induced increases in prefrontal ACh levels were partially blocked by the dopamine D1 receptor antagonist SCH23390, but not by antagonists of mAChRs (telenzepine) and nAChRs (mecamylamine). Galantamine increased dopamine, but not 5-HT, release in the prefrontal cortex. Conclusions and implications Galantamine improves apomorphine-induced PPI deficits by stimulating mAChRs through increasing brain ACh levels via a dopamine D1 receptor-dependent mechanism and AChE inhibition.

Yano, K; Koda, K; Ago, Y; Kobayashi, H; Kawasaki, T; Takuma, K; Matsuda, T



Alpha7 nicotinic receptor mediated protection against ethanol-induced cytotoxicity in PC12 cells.  


Ethanol caused a concentration-dependent loss of PC12 cells over a 24 h interval, accompanied by an increase in intracellular calcium. The specific alpha7 nicotinic receptor partial agonist DMXB attenuated both of these ethanol-induced actions at a concentration (3 microM) found previously to protect against apoptotic and necrotic cell loss. The alpha7 nicotinic receptor antagonist methylylaconitine blocked the neuroprotective action of DMXB when applied with but not 30 min after the agonist. These results indicate that activation of alpha7 nicotinic receptors may be therapeutically useful in preventing ethanol-neurotoxicity. PMID:9878750

Li, Y; King, M A; Grimes, J; Smith, N; de Fiebre, C M; Meyer, E M



17?-Estradiol induces vasorelaxation in a G-protein-coupled receptor 30-independent manner.  


17?-Estradiol (E2) exerts rapid non-genomic vascular effects through activation of its plasma membrane receptors. We tested the hypothesis that E2 induces vasorelaxation through activation of the G-protein-coupled receptor 30 (GPR30) in rat aorta. Rat aortic rings were mounted in organ baths and subjected to contraction followed by relaxation. Whether endothelium was intact or denuded, both E2 and G1, a GPR30 agonist, induced vasorelaxation in concentration-dependent manners. Although G15, a specific GPR30 antagonist, blocked G1-induced vasorelaxation, it did not block E2-induced vasorelaxation. In conclusion, 17?-estradiol induces vasorelaxation in a GPR30-independent manner in rat aorta. PMID:22688596

Seok, Young Mi; Jang, Eun Jin; Reiser, Oliver; Hager, Markus; Kim, In Kyeom



Bacterial Chaperone Protein, Skp, Induces Leukocyte Chemotaxis via C5a Receptor  

PubMed Central

C5a receptor has been identified as a leukocyte chemotactic receptor to two intrinsic chemical mediators, C5a and the S19 ribosomal protein dimer, so far. We found an Escherichia coli protein that also induced the chemotactic responses of monocytes and polymorphonuclear leukocytes via the C5a receptor. We identified the E. coli-derived chemoattractant to be Skp by the molecular size and the N-terminal amino acid sequence. Skp is a periplasmic chaperone protein widely present in gram-negative bacterial species. Immunoabsorption experiments indicated that Skp was the major leukocyte chemotactic factor in the E. coli extract. Receptor-antagonizing experiments with analogue peptides of S19 ribosomal protein and of C5a suggested that Skp induced the receptor activation by the two-step binding mechanism as in the cases of the intrinsic mediators, sharing the ligand-binding site of the receptor among them at each binding step. The C5a receptor would play a role in the host defense directly recognizing the bacteria-derived protein, besides identifying the signals of the intrinsic chemical mediators.

Shrestha, Arjun; Shi, Lei; Tanase, Sumio; Tsukamoto, Makiko; Nishino, Norikazu; Tokita, Kazutaka; Yamamoto, Tetsuro



Gs-coupled adenosine receptors differentially limit antigen-induced mast cell activation.  


Mast cell activation results in the immediate release of proinflammatory mediators prestored in cytoplasmic granules, as well as initiation of lipid mediator production and cytokine synthesis by these resident tissue leukocytes. Allergen-induced mast cell activation is central to the pathogenesis of asthma and other allergic diseases. Presently, most pharmacological agents for the treatment of allergic disease target receptors for inflammatory mediators. Many of these mediators, such as histamine, are released by mast cells. Targeting pathways that limit antigen-induced mast cell activation may have greater therapeutic efficacy by inhibiting the synthesis and release of many proinflammatory mediators produced in the mast cell. In vitro studies using cultured human and mouse mast cells, and studies of mice lacking A(2B) receptors, suggest that adenosine receptors, specifically the G(s)-coupled A(2A) and A(2B) receptors, might provide such a target. Here, using a panel of mice lacking various combinations of adenosine receptors, and mast cells derived from these animals, we show that adenosine receptor agonists provide an effective means of inhibition of mast cell degranulation and induction of cytokine production both in vitro and in vivo. We identify A(2B) as the primary receptor limiting mast cell degranulation, whereas the combined activity of A(2A) and A(2B) is required for the inhibition of cytokine synthesis. PMID:23149337

Hua, Xiaoyang; Chason, Kelly D; Jania, Corey; Acosta, Tatiana; Ledent, Catherine; Tilley, Stephen L



The Role of Purinergic Receptors in Cancer-Induced Bone Pain  

PubMed Central

Cancer-induced bone pain severely compromises the quality of life of many patients suffering from bone metastasis, as current therapies leave some patients with inadequate pain relief. The recent development of specific animal models has increased the understanding of the molecular and cellular mechanisms underlying cancer-induced bone pain including the involvement of ATP and the purinergic receptors in the progression of the pain state. In nociception, ATP acts as an extracellular messenger to transmit sensory information both at the peripheral site of tissue damage and in the spinal cord. Several of the purinergic receptors have been shown to be important for the development and maintenance of neuropathic and inflammatory pain, and studies have demonstrated the importance of both peripheral and central mechanisms. We here provide an overview of the current literature on the role of purinergic receptors in cancer-induced bone pain with emphasis on some of the difficulties related to studying this complex pain state.

Falk, Sarah; Uldall, Maria; Heegaard, Anne-Marie



Substance P, a potent bombesin antagonist in murine Swiss 3T3 cells, inhibits the growth of human small cell lung cancer cells in vitro  

SciTech Connect

In the search for a more potent bombesin antagonist, the authors found (D-Arg{sup 1},D-Phe{sup 5},D-Trp{sup 7,9},Leu{sup 11})substance P to be effective in mouse fibroblasts and to inhibit the growth of small cell lung cancer, a tumor that secretes bombesin-like peptides that may act as autocrine growth factors. In murine Swiss 3T3 cells, substance P proved to be a bombesin antagonist as judged by the following criteria: (i) inhibition of DNA synthesis induced by gastrin-releasing peptide and other bombesin-like peptides; (ii) inhibition of {sup 125}I-labeled gastrin-releasing peptide binding to the bombesin/gastrin-releasing peptide receptor; (iii) reduction in cross-linking of the M{sub r} 75,000-85,000 protein putatively a component of the bombesin/gastrin-releasing peptide receptor; (iv) blocking of early cellular events that precede mitogenesis-calcium mobilization and inhibition of epidermal growth factor binding. Substance P also inhibits mitogenesis induced by vasopressin but not that induced by a variety of other mitogens. Both antagonists reversibly inhibited the growth of small cell lung cancer in vitro in a concentration-dependent manner. Peptide antagonists could, therefore, have far-reaching therapeutic implications.

Woll, P.J.; Rozengurt, E. (Imperial Cancer Research Fund, London (England))



Lysophosphatidic acid receptor 1 modulates lipopolysaccharide-induced inflammation in alveolar epithelial cells and murine lungs  

PubMed Central

Lysophosphatidic acid (LPA), a bioactive phospholipid, plays an important role in lung inflammation by inducing the release of chemokines and lipid mediators. Our previous studies have shown that LPA induces the secretion of interleukin-8 and prostaglandin E2 in lung epithelial cells. Here, we demonstrate that LPA receptors contribute to lipopolysaccharide (LPS)-induced inflammation. Pretreatment with LPA receptor antagonist Ki16425 or downregulation of LPA receptor 1 (LPA1) by small-interfering RNA (siRNA) attenuated LPS-induced phosphorylation of p38 MAPK, I-?B kinase, and I-?B in MLE12 epithelial cells. In addition, the blocking of LPA1 also suppressed LPS-induced IL-6 production. Furthermore, LPS treatment promoted interaction between LPA1 and CD14, a LPS coreceptor, in a time- and dose-dependent manner. Disruption of lipid rafts attenuated the interaction between LPA1 and CD14. Mice challenged with LPS increased plasma LPA levels and enhanced expression of LPA receptors in lung tissues. To further investigate the role of LPA receptors in LPS-induced inflammation, wild-type, or LPA1-deficient mice, or wild-type mice pretreated with Ki16425 were intratracheally challenged with LPS for 24 h. Knock down or inhibition of LPA1 decreased LPS-induced IL-6 release in bronchoalveolar lavage (BAL) fluids and infiltration of cells into alveolar space compared with wild-type mice. However, no significant differences in total protein concentration in BAL fluids were observed. These results showed that knock down or inhibition of LPA1 offered significant protection against LPS-induced lung inflammation but not against pulmonary leak as observed in the murine model for lung injury.

Zhao, Jing; He, Donghong; Su, Yanlin; Berdyshev, Evgeny; Chun, Jerold; Natarajan, Viswanathan



Mg2+-induced adenosine-receptor mediated relaxations in mesenteric vascular beds of diabetic rats.  


Our previous studies showed that the magnesium Mg2+-induced relaxations were completely dependent on concentration of nitric oxide (NO) in non-diabetic rat mesenteric vascular beds, in diabetic rats other mechanisms may be involved. The present study was designed to determine the role of adenosine receptor in Mg2+-induced relaxation in streptozotocin (STZ)-induced diabetic rats vessels. Diabetes was induced by the intravenous injection of 60 mg/kg STZ. Eight weeks after diabetes induction, superior mesenteric arteries were isolated and perfused according to the McGregor method. Prepared vascular beds were constricted with phenylephrine to induce 70-75% of maximal constriction (0.001 M). Mg2+ at concentrations of 10(-4) to 10(-1) M were added into the medium and perfusion pressure was recorded. Theophylline (1 mM), and 3,7- dimethyl-1- propargylxanthine (0.01 ?M) were added into medium 20 min before phenylephrine administration. In the presence of theophylline, vasorelaxation induced by high dose of Mg2+ (from 0.03 to 0.1 M) was totally suppressed. In presence of N(?)-nitro-L-arginine methyl ester (L-NAME), the response of Mg2+ was completely inhibited at low dose of Mg2+. But, Mg2+-induced relaxation in the presence of adenosine A2a receptor blocker was significantly suppressed in high dose of Mg2+. Mg2+-induced relaxation in the presence of an A2a receptor blocker was not suppressed either by denudation of endothelium or presence of L-NAME. From the results of this study it may be concluded that Mg2+-induced relaxation at high concentrations is mediated by adenosine A2a receptors, but at low concentrations Mg2+-induced relaxation is dependent on NO. PMID:23255667

Tavasoli, Roya A; Soltani, Nepton; Keshavarz, Mansoor; Shorabipour, Shahla



Haemolysis induced by ?-toxin from Staphylococcus aureus requires P2X receptor activation.  


Recently, it was documented that ?-haemolysin (HlyA) from Escherichia coli uses erythrocyte P2 receptors cause lysis. This finding was surprising as it appeared firmly established that HlyA-dependent pore formation per se is sufficient for full cell lysis. We discovered that HlyA induced a sequential process of shrinkage and swelling and that the final haemolysis is completely prevented by blockers of P2X receptors and pannexin channels. This finding has potential clinical relevance as it may offer specific pharmacological interference to ameliorate haemolysis inflicted by pore-forming bacterial toxins. In this context, it is essential to know whether this is specific to HlyA-induced cell damage or if other bacterial pore-forming toxins involve purinergic signals to orchestrate haemolysis. Here, we investigate if the haemolysis produced by ?-toxin from Staphylococcus aureus involves P2 receptor activation. We observed that ?-toxin-induced haemolysis is completely blocked by the unselective P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid. Moreover, several selective blockers of P2X(1) and P2X(7) ionotropic receptors abolished haemolysis in murine and equine erythrocytes. Inhibitors of pannexin channels partially reduced the ?-toxin induced lysis. Thus, we conclude that ?-toxin, similar to HlyA from E. coli produces cell damage by specific activation of a purinergic signalling cascade. These data indicate that pore-forming toxins in general require purinergic signalling to elicit their toxicity. PMID:21847558

Skals, Marianne; Leipziger, Jens; Praetorius, Helle A



The insulin-like growth factor I receptor-induced interaction of insulin receptor substrate-4 and Crk-II.  


Stimulation of the insulin or insulin-like growth factor (IGF)-I receptor results in activation of several signaling pathways. Proteins of the insulin receptor substrate (IRS) family play important roles in mediating these signaling cascades. To date, four members of the IRS family of docking proteins have been characterized. Recently, we have reported that stimulation of the IGF-I receptor in 293 HEK cells regulates interaction of the newly discovered IRS-4 molecule with the Crk family of proteins. In the present study, we characterize the molecular basis of these interactions. C- and N termini truncation analysis of IRS-4 demonstrated that the region between amino acids 678 and 800 of the IRS-4 molecule is involved in this interaction. This region contains a cluster of four tyrosines (Y(700), Y(717), Y(743), and Y(779)). We hypothesize that one or more of these tyrosines are involved in the interaction between the SH2 domain of the Crk-II molecule when IRS-4 is phosphorylated upon IGF-I receptor activation. Additional mutational analyses confirmed this hypothesis. Interestingly, none of these four tyrosines was individually critical for the interaction between Crk-II and IRS-4, but when all four tyrosines were simultaneously mutated to phenylalanine, the IGF-I induced interaction between these molecules was abolished. Taken together, these results suggest a novel mechanism of Crk-II binding to tyrosine phosphorylated proteins. PMID:11316748

Karas, M; Koval, A P; Zick, Y; LeRoith, D