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PubMed2002

Epidermal growth factor and transforming growth factor alpha mimic the effects of insulin in human fat cells and augment downstream signaling in insulin resistance.

The ability of the growth factors epidermal growth factor (EGF), transforming growth factor alpha, and platelet-derived growth factor to exert insulin-like effects on glucose transport and lipolysis were examined in human and rat fat cells. No effects were found in rat fat cells, whereas EGF (EC(50) for glucose transport approximately 0.02 nm) and transforming growth factor alpha (EC(50) approximately 0.2 nm), but not platelet-derived growth factor, mimicked the effects of insulin (EC(50) approximately 0.2 nm) on both pathways. EGF receptors, but not EGF, were abundantly expressed in human fat cells as well as in human skeletal muscle. EGF increased the tyrosine phosphorylation of several proteins (the EGF receptor, insulin receptor substrate (IRS)-1, IRS-2, and Grb2-associated binder 1), whereas Shc and Gab2 were only weakly and inconsistently phosphorylated. p85, the regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase), was also found to associate with all of these docking molecules, showing that EGF activated PI 3-kinase pools that were additional to those of insulin. EGF and/or insulin increased protein kinase B/Akt serine phosphorylation to a similar extent, whereas mitogen-activated protein kinase phosphorylation was more pronounced for EGF than for insulin. The impaired insulin-stimulated downstream signaling, measured as protein kinase B/Akt serine phosphorylation, in insulin-resistant cells (Type 2 diabetes) was improved by the addition of EGF. Thus, EGF receptors, but not EGF, are abundantly expressed in human fat cells and skeletal muscle. EGF mimics the effects of insulin on both the metabolic and mitogenic pathways but utilize in part different signaling pathways. Both insulin and EGF increase the tyrosine phosphorylation and activation of IRS-1 and IRS-2, whereas EGF is also capable of activating additional PI 3-kinase pools and, thus, can augment the downstream signaling of insulin in insulin-resistant states like Type 2 diabetes.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2002

Slug is a novel downstream target of MyoD. Temporal profiling in muscle regeneration.

Temporal expression profiling was utilized to define transcriptional regulatory pathways in vivo in a mouse muscle regeneration model. Potential downstream targets of MyoD were identified by temporal expression, promoter data base mining, and gel shift assays; Slug and calpain 6 were identified as novel MyoD targets. Slug, a member of the snail/slug family of zinc finger transcriptional repressors critical for mesoderm/ectoderm development, was further shown to be a downstream target by using promoter/reporter constructs and demonstration of defective muscle regeneration in Slug null mice.

Animals↗
PubMed2002

Downstream codons in the retinoic acid receptor beta -2 and beta -4 mRNAs initiate translation of a protein isoform that disrupts retinoid-activated transcription.

Retinoic acid receptors (RARs) are essential for the differentiation and maintenance of normal epithelium. In studies of RARs in breast cancer, there are striking differences in the expression of certain protein isoforms of the RARbeta gene between cells derived from normal human mammary glands and those derived from breast tumors. While the protein isoforms RARbeta2 and RARbeta4 consist of the longest open reading frames of the RARbeta2 and RARbeta4 mRNAs, respectively, we find that a fraction of scanning ribosomes bypass these upstream RARbeta2 and RARbeta4 protein start codons and initiate translation downstream. This downstream translation initiation site is identical in the RARbeta2 and RARbeta4 transcripts and generates a third RARbeta protein isoform, here termed RARbeta' (formerly human RARbeta4). RARbeta' lacks protein domains found in the N terminus of RARbeta2 and RARbeta4, including one of two zinc fingers required for DNA binding. However, RARbeta' retains the ability to heterodimerize with RXRalpha and interact with transcription cofactors. In reporter gene assays, RARbeta' repressed retinoic acid-activated transcription of co-transfected RARbeta2, RARbeta4, and RARalpha. This repression required the presence of acidic amino acids within the AF2 domain. These findings demonstrate an antagonistic role for RARbeta' in signaling by retinoic acid.

Base Sequence↗
PubMed2002

The poly(A) signal, without the assistance of any downstream element, directs RNA polymerase II to pause in vivo and then to release stochastically from the template.

Genes encoding polyadenylated mRNAs depend on their poly(A) signals for termination of transcription. Typically, transcription downstream of the poly(A) signal gradually declines to zero, but often there is a transient increase in polymerase density immediately preceding the decline. Special elements called pause sites are traditionally invoked to account for this increase. Using run-on transcription from the nuclei of transfected cells, we show that both the pause and the gradual decline that follow a poly(A) site are generated entirely by the poly(A) signal itself in a series of model constructs. We found no other elements to be involved and argue that the elements called pause sites do not function through pausing. Both the poly(A)-dependent pause and the subsequent decline occurred earlier for a stronger poly(A) signal than for a weaker one. Because the gradual decline resembles the abortive elongation that occurs downstream of many promoters, one model has proposed that the poly(A) signal flips the polymerase from the elongation mode to the abortive mode like a binary switch. We compared abortive elongators with poly(A) terminators and found a 4-fold difference in processivity. We conclude that poly(A) terminating polymerases do not merely revert to their prior state of low processivity but rather convert to a new termination-prone condition.

Animals↗
PubMed2002

A member of Forkhead transcription factor FKHRL1 is a downstream effector of STI571-induced cell cycle arrest in BCR-ABL-expressing cells.

A member of the Forkhead transcription factor family, FKHRL1, lies downstream of the phosphatidylinositol 3-kinase-Akt activation pathway in cytokine signaling. Because the phosphatidylinositol 3-kinase-Akt activation pathway is required for BCR-ABL-mediated transformation and survival signaling in chronic myelogenous leukemia (CML), in this study we examined the involvement of FKHRL1 in the BCR-ABL-mediated signaling pathway. FKHRL1 was constitutively phosphorylated in BCR-ABL-expressing cell lines KCL22 and KU812, and its phosphorylation was inhibited by treatment with STI571, a specific inhibitor of BCR-ABL tyrosine kinase. Concomitantly, STI571 induced cell cycle arrest at the G(0)/G(1) phase, accompanied by up-regulation of a cyclin-dependent kinase inhibitor p27/Kip1 in KCL22 cells. In addition, FKHRL1 was constitutively phosphorylated in the TF-1/bcr-abl cell line ectopically expressing BCR-ABL but not in the parent TF-1 cell line. Considering several lines of evidence that phosphorylated FKHRL1 has lost transcriptional activity and that p27/Kip1 expression is positively regulated by dephosphorylated "active" FKHRL1, BCR-ABL may down-regulate p27/Kip1 expression via the loss of FKHRL1 function as a transcription factor. To demonstrate this hypothesis, we generated a tamoxifen-inducible "active FKHRL1" FKHRL1-TM (a triple mutant of FKHRL1, in which all three Akt phosphorylation sites have been mutated), estrogen receptor system in the KCL22 cell line. The addition of tamoxifen inhibited the cell growth indicating that overexpression of FKHRL1 in the nucleus antagonized deregulated proliferation of CML cells. Collectively, FKHRL1 regulates the expression of p27/Kip1 as a downstream molecule of BCR-ABL signaling in CML cells. BCR-ABL-induced loss of FKHRL1 function may be involved in oncogenic transformation of CML partially via the down-regulation of p27/Kip1 proteins.

Animals↗
PubMed2003

Regulation of biglycan gene expression by transforming growth factor-beta requires MKK6-p38 mitogen-activated protein Kinase signaling downstream of Smad signaling.

Several signaling pathways have been implicated in mediating TGF-beta1-induced extracellular matrix production and fibrosis. We have shown recently that induction of biglycan (BGN) expression by TGF-beta1 depended on a functional Smad pathway (Chen, W.-B., Lenschow, W., Tiede, K., Fischer, J. W., Kalthoff, H., and Ungefroren, H. (2002) J. Biol. Chem. 277, 36118-36128). Here, we present evidence that the ability of TGF-beta 1 to induce BGN mRNA, in addition to Smads, requires p38 MAPK signaling, because 1) pharmacological inhibitors of p38 dose-dependently inhibited the TGF-beta effect without significantly affecting the transcriptional activity of a constitutively active mutant of the TGF-beta type I receptor or Smad2 phosphorylation at concentrations up to 10 microm, 2) the up-regulation of BGN mRNA was preceded by a delayed increase in the phosphorylation of p38 and its upstream activator MKK6 in TGF-beta 1-treated PANC-1 cells, 3) inhibition of the p38 pathway by stable retroviral transduction with a dominant negative mutant of either p38 or MKK6 reduced TGF-beta 1-induced BGN mRNA expression, and 4) overexpression of wild-type p38 or MKK6, but not MKK3, augmented the TGF-beta 1 effect on BGN mRNA. We further demonstrate that the (delayed) p38 activation by TGF-beta 1 is downstream of Smads and requires a functional Smad pathway, because blocking TGF-beta-induced p38 activity with SB202190 had no effect on Smad2 phosphorylation, but blocking Smad signaling by forced expression of Smad7 abolished TGF-beta1 induction of p38 activation and, as shown earlier, BGN mRNA expression; finally, re-expression of Smad4 in Smad4-null CFPAC-1 cells restored TGF-beta-induced p38 phosphorylation and, as demonstrated previously, BGN mRNA accumulation. These results clearly show that TGF-beta induction of BGN expression in pancreatic cells requires activation of MKK6-p38 MAPK signaling downstream of Smad signaling and provide a mechanistic clue to the up-regulation of BGN seen in inflammatory response-related fibrosis and desmoplasia.

Base Sequence↗
PubMed2003

Kruppel-like factor 4 (KLF4) represses histidine decarboxylase gene expression through an upstream Sp1 site and downstream gastrin responsive elements.

Histidine decarboxylase (HDC) is the enzyme that catalyzes the conversion of histidine to histamine, a bioamine that plays an important role in allergic responses, inflammation, neurotransmission, and gastric acid secretion. Previously, we demonstrated that gastrin activates HDC promoter activity in a gastric cancer (AGS-E) cell line through three overlapping downstream promoter elements. In the current study, we used the yeast one-hybrid strategy to identify nuclear factors that bind to these three elements. Among eight positives from the one-hybrid screen, we identified Kruppel-like factor 4 (KLF4) (previously known as gut-enriched Kruppel-like factor (GKLF)) as one factor that binds to the gastrin responsive elements in the HDC promoter. Electrophoretic mobility shift assays confirmed that KLF4 is able to bind all three gastrin responsive elements. In addition, transient cotransfection experiments showed that overexpression of KLF4 dose dependently and specifically inhibited HDC promoter activity. Regulation of HDC transcription by KLF4 was confirmed by changes in the endogenous HDC messenger RNA by KLF4 small interfering RNA and KLF4 overexpression. We further showed that KLF4 inhibits HDC promoter activity by competing with Sp1 at the upstream GC box and also independently by binding the three downstream gastrin responsive elements. Taken together, these results indicate that KLF4 can act to repress HDC gene expression by Sp1-dependent and -independent mechanisms.

DNA-Binding Proteins↗
PubMed2003

Vasodilator-stimulated phosphoprotein activation of serum-response element-dependent transcription occurs downstream of RhoA and is inhibited by cGMP-dependent protein kinase phosphorylation.

Vasodilator-stimulated phosphoprotein (VASP) associates with cytoskeletal structures and promotes F-actin formation. RhoA, a member of the Ras superfamily of proteins, activates serum response element (SRE)-dependent transcription through changes in actin dynamics. We now show that the F-actin binding region of VASP is required for VASP stimulation of SRE-dependent transcription, and that VASP is downstream of RhoA in stimulating SRE-dependent transcription. The isolated carboxyl-terminal coiled-coil region of VASP mediates protein tetramerization and has been used as a dominant negative form of VASP; we found that it forms complexes with endogenous VASP in vivo and inhibits in a dose-dependent fashion serum-, RhoA-, and VASP-stimulated SRE-dependent transcription. Cyclic GMP-dependent protein kinase (G-kinase) inhibits RhoA activation of SRE-dependent transcription (Gudi, T., Chen, J. C., Casteel, D. E., Seasholtz, T. M., Boss, G. R., and Pilz, R. B. (2002) J. Biol. Chem. 277, 37382-37393). We now show that the G-kinase inhibition that occurs downstream of RhoA can be explained, at least in part, by G-kinase phosphorylation of VASP on Ser(239) at the carboxyl-terminal end of the G-actin binding site, with some contribution by phosphorylation of Ser(157), which is proximal to the profilin binding site. A phosphorylation-deficient VASP mutant can partly prevent cGMP/G-kinase inhibition of serum- and RhoA-induced SRE-dependent transcription. These studies show that VASP, an important component of the cellular microfilament system, plays a major role in regulating SRE-dependent transcription, and that G-kinase regulates VASP activity.

Actins↗
PubMed2004

Identification of the Jak/Stat proteins as novel downstream targets of EphA4 signaling in muscle: implications in the regulation of acetylcholinesterase expression.

Eph receptors and their cognate ligands ephrins are important players in axon guidance and neural patterning during development of the nervous system. Much of our knowledge about the signal transduction pathways triggered by Eph receptors has been related to the modulation of actin cytoskeleton, which is fundamental in mediating the cellular responses in growth cone navigation, cell adhesion, and cell migration. In contrast, little was known about whether long term activation of Eph receptor would regulate gene expression. Here we report a novel signaling pathway of EphA4, which involves activation of the tyrosine kinase Jak2 and the transcriptional activator Stat3. Transfection of COS7 cells with EphA4, but not the kinase-dead mutant, induced tyrosine phosphorylation of Jak2, Stat1, and Stat3. Treatment of cultured C2C12 myotubes with ephrin-A1 also induced tyrosine phosphorylation of Stat3, which was abolished by the Jak2 inhibitor AG490. Moreover, Jak2 was co-immunoprecipitated with EphA4 in muscle, and both proteins were concentrated at the neuromuscular junction (NMJ) of adult muscle. By using microarray analysis, we have identified acetylcholinesterase, the critical enzyme that hydrolyzed the neurotransmitter acetylcholine at the NMJ, as a downstream target gene of the Jak/Stat pathway in muscle. More importantly, ephrin-A1 increased the expression of acetylcholinesterase protein in C2C12 myotubes, which was abolished by AG490. In contrast, ephrin-A1 reduced the expression of fibronectin mRNA in C2C12 myotubes independently of Jak2. Finally, the expression level of acetylcholinesterase in limb muscle of EphA4 null mice was significantly reduced compared with the wild-type control. Taken together, these results have identified Jak/Stat proteins as the novel downstream targets of EphA4 signaling. In addition, the present study provides the first demonstration of a potential function of Eph receptors and Jak/Stat proteins at the NMJ.

Acetylcholinesterase↗
PubMed2004

Essential role for phospholipase D2 activation downstream of ERK MAP kinase in nerve growth factor-stimulated neurite outgrowth from PC12 cells.

The signaling pathway that triggers morphological differentiation of PC12 cells is mediated by extracellular signal-regulated kinase (ERK), the classic mitogen-activated protein (MAP) kinase. However, mediators of the pathway downstream of ERK have not been identified. We show here that phospholipase D2 (PLD2), which generates the pleiotropic signaling lipid phosphatidic acid (PA), links ERK activation to neurite outgrowth in nerve growth factor (NGF)-stimulated PC12 cells. Increased expression of wild type PLD2 (WT-PLD2) dramatically elongated neurites induced by NGF stimulation or transient expression of the active form of MAP kinase-ERK kinase (MEK-CA). The response was activity-dependent, because it was inhibited by pharmacological suppression of the PLD-mediated PA production and by expression of a lipase-deficient PLD2 mutant. Furthermore, PLD2 was activated by MEK-CA, whereas NGF-stimulated PLD2 activation and hypertrophic neurite extension were blocked by an MEK-specific inhibitor. Taken together, these results provide evidence that PLD2 functions as a downstream signaling effector of ERK in the NGF signaling pathway, which leads to neurite outgrowth by PC12 cells.

Actins↗
PubMed2004

Mitochondrial function is required for hydrogen peroxide-induced growth factor receptor transactivation and downstream signaling.

The transactivation of growth factor receptors is an early event in H(2)O(2)-induced signaling, although proximal targets in this process remain unclear. We found that inhibition of flavin- or heme-containing proteins eliminated H(2)O(2)-induced transactivation of the epidermal growth factor receptor and stimulation of its downstream targets, JNK and Akt. Inhibition of mitochondrial function with rotenone, antimycin A, KCN, carbonylcyanide-m-chlorophenylhydrazone, or oligomycin reproduced this effect, as did generation of mitochondrial DNA-deficient (pseudo-rho(0)) cells. Mitochondrial function had no role in JNK activation in response to UV irradiation or tumor necrosis factor-alpha. The impact of mitochondrial function on H(2)O(2)-induced growth factor transactivation was ubiquitous and applied to both the vascular endothelial growth factor (VEGF)-2 receptor and the platelet-derived growth factor-beta receptor in endothelium and fibroblasts, respectively. In contrast, ligand-induced growth factor activation was unrelated to mitochondrial function. Growth factor receptor transactivation and its downstream signaling in response to H(2)O(2) appeared to involve redox-sensitive mitochondrial events as they were abrogated by a mitochondrial-targeted antioxidants but not their nontargeted counterparts. Functionally, we found that mitochondrial-targeted antioxidants inhibited H(2)O(2)-induced apoptosis and cell death but had no effect with UV irradiation. These data establish a novel role for the mitochondrion as a proximal target specific to H(2)O(2)-induced signaling and growth factor transactivation.

Animals↗
PubMed2004

Oxidants inhibit ERK/MAPK and prevent its ability to delay neutrophil apoptosis downstream of mitochondrial changes and at the level of XIAP.

Normal spontaneous apoptosis in neutrophils is enhanced by "stress" stimuli such as tumor necrosis factor-alpha, Fas ligand, and oxidants, and this effect is inhibited by anti-apoptotic stimuli including granulocyte-macrophage colony-stimulating factor, lipopolysaccharide, and formylmethionine-leucine-phenylalanine. In this report we demonstrate that anti-apoptotic stimuli protect neutrophils from stress-induced apoptosis via activation of the ERK/MAPK pathway. The protection occurs downstream of mitochondrial alterations assessed as a decrease in membrane potential concomitant with enhanced cytochrome c release. ERK activation was shown to inhibit apoptosis by maintaining levels of XIAP, which is normally decreased in the presence of the pro-apoptotic/stress stimuli. This report also demonstrates that potent intra- and extracellular oxidants inhibit the protective effect of ERK. Oxidant-dependent inhibition of ERK was because of activation of p38 MAPK and activation of the protein phosphatases PP1 and PP2A. Our data suggest that ERK suppresses stress-induced apoptosis downstream of mitochondrial alterations by maintaining XIAP levels and that oxidants block this effect through activation of p38 and protein phosphatases.

Antioxidants↗
PubMed2004

Bisphosphonates and estrogens inhibit osteocyte apoptosis via distinct molecular mechanisms downstream of extracellular signal-regulated kinase activation.

Both estrogens and bisphosphonates attenuate osteocyte apoptosis by activating the extracellular signal-regulated kinases (ERKs). However, whereas estrogens activate ERKs via an extranuclear function of the estrogen receptor, bisphosphonates do so by opening connexin 43 hemichannels. Here, we demonstrated that the signaling events downstream of ERKs induced by these two stimuli are also distinct. Inhibition of osteocyte apoptosis by estrogens requires nuclear accumulation of ERKs and activation of downstream transcription factors. On the other hand, anti-apoptosis induced by bisphosphonates requires neither transcription nor ERK-dependent transcription factors. Instead, the effect of bisphosphonates is abolished when ERKs are restricted to the nucleus by blocking CRM1/exportin1-mediated nuclear protein export or by expressing nuclear-anchored ERKs, but it is unaffected in cells expressing cytoplasmic-anchored ERKs. Connexin 43/ERK-mediated anti-apoptosis induced by bisphosphonates requires the kinase activity of the cytoplasmic target of ERKs, p90(RSK), which in turn phosphorylates the pro-apoptotic protein BAD and C/EBPbeta. Phosphorylation of BAD renders it inactive, whereas phosphorylation of C/EBPbeta leads to binding of pro-caspases, thus inhibiting apoptosis independently of the transcriptional activity of this transcription factor. Consistent with the evidence that estrogens and bisphosphonates phosphorylate diverse targets of ERKs, probably resulting from activation of spatially distinct pools of these kinases, the two agents had additive effects on osteocyte survival.

Animals↗
PubMed2005

Protein kinase A regulates caspase-9 activation by Apaf-1 downstream of cytochrome c.

The cyclic AMP signal transduction pathway modulates apoptosis in diverse cell types, although the mechanism is poorly understood. A critical component of the intrinsic apoptotic pathway is caspase-9, which is activated by Apaf-1 in the apoptosome, a large complex assembled in response to release of cytochrome c from mitochondria. Caspase-9 cleaves and activates effector caspases, predominantly caspase-3, resulting in the demise of the cell. Here we identified a distinct mechanism by which cyclic AMP regulates this apoptotic pathway through activation of protein kinase A. We show that protein kinase A inhibits activation of caspase-9 and caspase-3 downstream of cytochrome c in Xenopus egg extracts and in a human cell-free system. Protein kinase A directly phosphorylates human caspase-9 at serines 99, 183, and 195. However, mutational analysis demonstrated that phosphorylation at these sites is not required for the inhibitory effect of protein kinase A on caspase-9 activation. Importantly, protein kinase A inhibits cytochrome c-dependent recruitment of procaspase-9 to Apaf-1 but not activation of caspase-9 by a constitutively activated form of Apaf-1. These data indicate that extracellular signals that elevate cyclic AMP and activate protein kinase A may suppress apoptosis by inhibiting apoptosome formation downstream of cytochrome c release from mitochondria.

Amino Acid Sequence↗
PubMed2006

Control of fibroblast growth factor (FGF) 7- and FGF1-induced mitogenesis and downstream signaling by distinct heparin octasaccharide motifs.

Variation in length, disaccharide composition, and sulfation of heparan sulfate (HS) affects fibroblast growth factor (FGF) signaling. However, it is unclear whether the specific distribution of groups within oligosaccharides or random variations in charge density underlies the effects. Recently we showed that a mixture of undersulfated octasaccharides exhibiting 7 and 8 sulfates (7,8-S-OctaF7) generated from heparin had the highest affinity for FGF7 monitored by salt resistance (>0.60 M salt) of octasaccharide-FGF7 complexes. 7,8-S-OctaF7 also had the highest specific activity for formation of a complex with dimeric FGFR2IIIb competent to bind FGF7. Here we show that when endogenous HS was inhibited by chlorate treatment, 7,8-S-OctaF7 specifically supported FGF7-stimulated DNA synthesis and downstream signaling in FGFR2IIIb-expressing mouse keratinocytes. It failed to support FGF1 signaling in both HS-deficient mouse keratinocytes and 3T3 fibroblasts. In contrast, abundant, more highly sulfated and heterogenous mixtures of octasaccharides with lower affinity (0.30-0.60 M salt) for FGF7 supported FGF1-induced signaling in both cell types. In contrast to the two-component 7,8-S-OctaF7 mixture from FGF7, the high affinity octasaccharide fraction from FGF1 was a heterogeneous mixture with components ranging from 8 to 12 sulfates with 11-S-octasaccharides the most abundant. The high affinity fraction exhibited similar properties to the lower affinity fractions from both FGF1 and FGF7. Octasaccharide mixtures eluting from FGF1 between 0.30 and 0.60 M and above 0.60 M salt were nearly equal in support of FGF1 signaling in fibroblasts and keratinocytes. Both were deficient in support of FGF7-induced signaling in keratinocytes. The results show that both variations in overall charge density and specific distribution of charged groups within HS motifs exhibit FGF-specific control over formation of FGF-HS-FGFR complexes and downstream signaling.

3T3 Cells↗
PubMed2006

STAT3 as a downstream mediator of Trk signaling and functions.

Signal transducer and activator of transcription 3 (STAT3) has long been shown to regulate gene transcription in response to cytokines and growth factors. Recent evidence suggests that STAT3 activation may also occur downstream of receptor-tyrosine kinase activation. In the current study we have identified STAT3 as a novel signal transducer for TrkA, the receptor-tyrosine kinase that mediates the functions of nerve growth factor (NGF). Activation of TrkA by NGF triggered STAT3 phosphorylation at Ser-727, and enhanced the DNA binding and transcriptional activities of STAT3. More importantly, neurotrophin-induced increase in STAT3 activation was observed to underlie several downstream functions of neurotrophin signaling. First of all, knockdown of STAT3 expression using the RNA interference approach attenuated NGF-induced transcription of immediate early genes in PC12 cells. Furthermore, reduced STAT3 expression in PC12 cells suppressed NGF-induced cyclin D1 expression, thereby inhibiting growth arrest normally triggered by NGF treatment. Finally, inhibition of STAT3 expression decreased brain-derived neurotrophic factor-promoted neurite outgrowth in primary hippocampal neurons. Together, our findings have identified STAT3 as an essential component of neurotrophin signaling and functions.

Animals↗
PubMed2000

Heat shock protein 70 inhibits apoptosis downstream of cytochrome c release and upstream of caspase-3 activation.

Heat shock protein 70 (HSP70) has been shown to act as an inhibitor of apoptosis. We have also observed an inhibitory effect of HSP70 on apoptotic cell death both in preheated U937 and stably transfected HSP70-overexpressing U937 (U937/HSP70) cells. However, the molecular mechanism whereby HSP70 prevents apoptosis still remains to be solved. To address this issue, we investigated the effect of HSP70 on apoptotic processes in an in vitro system. Caspase-3 cleavage and DNA fragmentation were detected in cytosolic fractions from normal cells upon addition of dATP, but not from preheated U937 or U937/hsp70 cells. Moreover, the addition of purified recombinant HSP70 to normal cytosolic fractions prevented caspase-3 cleavage and DNA fragmentation, suggesting that HSP70 prevents apoptosis upstream of caspase-3 processing. Because cytochrome c was still released from mitochondria into the cytosol by lethal heat shock despite prevention of caspase-3 activation and cell death in both preheated U937 and U937/hsp70 cells, it was evident that HSP70 acts downstream of cytochrome c release. Results obtained in vitro with purified deletion mutants of HSP70 showed that the carboxyl one-third region (from amino acids 438 to 641) including the peptide-binding domain and the carboxyl-terminal EEVD sequence was essential to prevent caspase-3 processing. From these results, we conclude that HSP70 acts as a strong suppressor of apoptosis acting downstream of cytochrome c release and upstream of caspase-3 activation.

Animals↗
PubMed1999

Radionuclides and trace elements in fish collected upstream and downstream of Los Alamos National Laboratory and the doses to humans from the consumption of muscle and bone.

The purpose of this study was to determine radionuclide and trace element concentrations in bottom-feeding fish (catfish, carp, and suckers) collected from the confluences of some of the major canyons that cross Los Alamos National Laboratory (LANL) lands with the Rio Grande (RG) and the potential radiological doses from the ingestion of these fish. Samples of muscle and bone (and viscera in some cases) were analyzed for 3H, 90Sr, 137Cs, totU, 238Pu, 239,240Pu, and 241Am and Ag, As, Ba, Be, Cr, Cd, Cu, Hg, Ni, Pb, Sb, Se, and Tl. Most radionuclides, with the exception of 90Sr, in the muscle plus bone portions of fish collected from LANL canyons/RG were not significantly (p < 0.05) higher from fish collected upstream (San Ildefonso/background) of LANL. Strontium-90 in fish muscle plus bone tissue significantly (p < 0.05) increases in concentration starting from Los Alamos Canyon, the most upstream confluence (fish contained 3.4E-02 pCi g-1 [126E-02 Bq kg-1]), to Frijoles Canyon, the most downstream confluence (fish contained 14E-02 pCi g-1 [518E-02 Bq kg-1]). The differences in 90Sr concentrations in fish collected downstream and upstream (background) of LANL, however, were very small. Based on the average concentrations (+/- 2SD) of radionuclides in fish tissue from the four LANL confluences, the committed effective dose equivalent from the ingestion of 46 lb (21 kg) (maximum ingestion rate per person per year) of fish muscle plus bone, after the subtraction of background, was 0.1 +/- 0.1 mrem y-1 (1.0 +/- 1.0 microSv y-1), and was far below the International Commission on Radiological Protection (all pathway) permissible dose limit of 100 mrem y-1 (1000 microSv y-1). Of the trace elements that were found above the limits of detection (Ba, Cu, and Hg) in fish muscle collected from the confluences of canyons that cross LANL and the RG, none were in significantly higher (p < 0.05) concentrations than in muscle of fish collected from background locations.

Animals↗