Transforming Growth Factor β1-Regulated Cell Proliferation and Expression of Neural Cell Adhesion Molecule in B104 Neuroblastoma Cells

The expression and activity of factors influencing early neuronal development are altered by ethanol. Such factors include growth factors, for example, platelet-derived growth factor and basic fibroblast growth factor (for cell proliferation), and cell adhesion molecules (for neuronal migration). One agent, transforming growth factor beta1 (TGFbeta1), may affect both events. We tested the hypothesis that ethanol alters myriad TGFbeta1-mediated activities [i.e., cell proliferation and neural cell adhesion molecule (N-CAM) expression] using B104 neuroblastoma cells. TGFbeta1 inhibited the proliferation of B104 cells as evidenced by decreases in cell number and [3H]thymidine ([3H]dT) incorporation. TGFbeta1 induced sustained activation of extracellular signal-regulated kinases (ERKs), which are part of the family of mitogen-activated protein kinases (MAPKs). Treatment with PD98059 (a MAPK kinase blocker) abolished TGFbeta1-regulated inhibition of [3H]dT incorporation. TGFbeta1-mediated growth inhibition was potentiated by ethanol exposure. Ethanol also produced prolonged activation of ERK, an effect that was partially eliminated by treatment with PD98059. On the other hand, TGFbeta1 up-regulated N-CAM expression, and this up-regulation was not affected by treatment with PD98059. Ethanol inhibited the TGFbeta1-induced up-regulation of N-CAM expression in a concentration-dependent manner. Thus, TGFbeta1 affects ERK-dependent cell proliferation and ERK-independent N-CAM expression in B104 cells. Both activities are sensitive to ethanol and may underlie the ethanol-induced alterations in the proliferation and migration of CNS neurons.     

Autophagy is a protective response to ethanol neurotoxicity

Ethanol is a neuroteratogen and neurodegeneration is the most devastating consequence of developmental exposure to ethanol. The mechanisms underlying ethanol-induced neurodegeneration are complex. Ethanol exposure produces reactive oxygen species (ROS) which cause oxidative stress in the brain. We hypothesized that ethanol would activate autophagy to alleviate oxidative stress and neurotoxicity. Our results indicated that ethanol increased the level of the autophagic marker Map1lc3-II (LC3-II) and upregulated LC3 puncta in SH-SY5Y neuroblastoma cells. It also enhanced the levels of LC3-II and BECN1 in the developing brain; meanwhile, ethanol reduced SQSTM1 (p62) levels. Bafilomycin A₁, an inhibitor of autophagosome and lysosome fusion, increased p62 levels in the presence of ethanol. Bafilomycin A₁ and rapamycin potentiated ethanol-increased LC3 lipidation, whereas wortmannin and a BECN1-specific shRNA inhibited ethanol-promoted LC3 lipidation. Ethanol increased mitophagy, which was also modulated by BECN1 shRNA and rapamycin. The evidence suggested that ethanol promoted autophagic flux. Activation of autophagy by rapamycin reduced ethanol-induced ROS generation and ameliorated ethanol-induced neuronal death in vitro and in the developing brain, whereas inhibition of autophagy by wortmannin and BECN1-specific shRNA potentiated ethanol-induced ROS production and exacerbated ethanol neurotoxicity. Furthermore, ethanol inhibited the MTOR pathway and downregulation of MTOR offered neuroprotection. Taken together, the results suggest that autophagy activation is a neuroprotective response to alleviate ethanol toxicity. Ethanol modulation of autophagic activity may be mediated by the MTOR pathway.     

Autophagy is a protective response to ethanol neurotoxicity

Ethanol is a neuroteratogen and neurodegeneration is the most devastating consequence of developmental exposure to ethanol. The mechanisms underlying ethanol-induced neurodegeneration are complex. Ethanol exposure produces reactive oxygen species (ROS) which cause oxidative stress in the brain. We hypothesized that ethanol would activate autophagy to alleviate oxidative stress and neurotoxicity. Our results indicated that ethanol increased the level of the autophagic marker Map1lc3-II (LC3-II) and upregulated LC3 puncta in SH-SY5Y neuroblastoma cells. It also enhanced the levels of LC3-II and BECN1 in the developing brain; meanwhile, ethanol reduced SQSTM1 (p62) levels. Bafilomycin A₁, an inhibitor of autophagosome and lysosome fusion, increased p62 levels in the presence of ethanol. Bafilomycin A₁ and rapamycin potentiated ethanol-increased LC3 lipidation, whereas wortmannin and a BECN1-specific shRNA inhibited ethanol-promoted LC3 lipidation. Ethanol increased mitophagy, which was also modulated by BECN1 shRNA and rapamycin. The evidence suggested that ethanol promoted autophagic flux. Activation of autophagy by rapamycin reduced ethanol-induced ROS generation and ameliorated ethanol-induced neuronal death in vitro and in the developing brain, whereas inhibition of autophagy by wortmannin and BECN1-specific shRNA potentiated ethanol-induced ROS production and exacerbated ethanol neurotoxicity. Furthermore, ethanol inhibited the MTOR pathway and downregulation of MTOR offered neuroprotection. Taken together, the results suggest that autophagy activation is a neuroprotective response to alleviate ethanol toxicity. Ethanol modulation of autophagic activity may be mediated by the MTOR pathway.     

Cardiac overexpression of catalase antagonizes ADH-associated contractile depression and stress signaling after acute ethanol exposure in murine myocytes.

Alcohol dehydrogenase (ADH), which oxidizes ethanol into acetaldehyde, exacerbates ethanol-induced cardiac depression, although the mechanism of action remains unclear. This study was designed to examine the impact of antioxidant catalase (CAT) on cardiac contractile response to ethanol and activation of stress signaling. ADH-CAT double transgenic mice were generated by crossing CAT and ADH lines. Mechanical, intracellular Ca(2+) properties and reactive oxygen species generation were measured in ventricular myocytes. ADH-CAT, ADH, CAT and wild-type FVB myocytes exhibited similar mechanical and intracellular Ca(2+) properties. ADH or ADH-CAT myocytes had higher acetaldehyde-producing ability. Ethanol (80-640 mg/dl) suppressed FVB cell shortening and intracellular Ca(2+) transients with maximal inhibitions of 43.5 and 45.2%, respectively. Ethanol-induced depression on cell shortening and intracellular Ca(2+) was augmented in ADH group with maximal inhibitions of 66.8 and 69.6%, respectively. Interestingly, myocytes from CAT-ADH mice displayed normal ethanol response with maximal inhibitions of 46.0 and 47.2% for cell shortening and intracellular Ca(2+), respectively. CAT transgene lessened ethanol-induced inhibition on cell shortening (maximal inhibition of 30.3%) but not intracellular Ca(2+). ADH amplified ethanol-induced reactive oxygen species generation, which was nullified by the CAT transgene. Western blot analysis showed that ethanol reduced ERK phosphorylation and enhanced JNK phosphorylation without affecting p38 phosphorylation. The ethanol-induced changes in phosphorylation of ERK and JNK were amplified by ADH. CAT transgene itself did not affect ethanol-induced response in ERK and JNK phosphorylation, but it cancelled ADH-induced effects. These data suggest that antioxidant CAT may effectively antagonize ADH-induced enhanced cardiac depression in response to ethanol.     

Cdc42-dependent activation of NADPH oxidase is involved in ethanol-induced neuronal oxidative stress

It has been suggested that excessive reactive oxygen species (ROS) and oxidative stress play an important role in ethanol-induced damage to both the developing and mature central nervous system (CNS). The mechanisms underlying ethanol-induced neuronal ROS, however, remain unclear. In this study, we investigated the role of NADPH oxidase (NOX) in ethanol-induced ROS generation. We demonstrated that ethanol activated NOX and inhibition of NOX reduced ethanol-promoted ROS generation. Ethanol significantly increased the expression of p47(phox) and p67(phox), the essential subunits for NOX activation in cultured neuronal cells and the cerebral cortex of infant mice. Ethanol caused serine phosphorylation and membrane translocation of p47(phox) and p67(phox), which were prerequisites for NOX assembly and activation. Knocking down p47(phox) with the small interfering RNA was sufficient to attenuate ethanol-induced ROS production and ameliorate ethanol-mediated oxidative damage, which is indicated by a decrease in protein oxidation and lipid peroxidation. Ethanol activated cell division cycle 42 (Cdc42) and overexpression of a dominant negative (DN) Cdc42 abrogate ethanol-induced NOX activation and ROS generation. These results suggest that Cdc42-dependent NOX activation mediates ethanol-induced oxidative damages to neurons.     

Ethanol induces the production of cytokines via the Ca2+, MAP kinase, HIF-1alpha, and NF-kappaB pathway

In the present study, we sought to investigate the signal transduction pathways of expression of cytokines in the ethanol-stimulated human mast cell line, HMC-1. Ethanol significantly increased the intracellular calcium level in HMC-1. Ethanol also significantly enhanced IL-6, TNF-alpha, and TGF-beta1 production compared with media control, but did not significantly affect the IL-1beta production. After 8 h of stimulation, ethanol increased mRNA and protein expression levels of TNF-alpha and TGF-beta1 in HMC-1. The increased cytokine level was significantly inhibited by BAPTA-AM, PD98059, and SB203580. These inhibitors also inhibited ethanol-induced ERK and p38 MAPK phosphorylation. Ethanol resulted in a great increase in protein levels and promoter activity driving luciferase expression of HIF-1alpha and NF-kappaB in HMC-1 cells, but it did not affect on HIF-1alpha mRNA expression. Our observations show that calcium, MAPK activation, HIF-1alpha, and NF-kappaB are necessary for ethanol-induced TNF-alpha and TGF-beta1 expression. These results may have important implications for the study of alcohol-related diseases.     

Suppression of p75 neurotrophin receptor surface expression with intrabodies influences Bcl-xL mRNA expression and neurite outgrowth in PC12 cells

Background

Although p75 neurotrophin receptor (p75NTR) is the first neurotrophin receptor isolated, its diverse physiological functions and signaling have remained elusive for many years. Loss-of-function phenotypic analyses for p75NTR were mainly focused at the genetic level; however these approaches were impacted by off-target effect, insufficient stability, unspecific stress response or alternative active splicing products. In this study, p75NTR surface expression was suppressed for the first time at the protein level by endoplasmic reticulum (ER) retained intrabodies.

Results

Three monoclonal recombinant antibody fragments (scFv) with affinities in the low nanomolar range to murine p75NTR were isolated by antibody phage display. To suppress p75NTR cell surface expression, the encoding genes of these scFvs extended by the ER retention peptide KDEL were transiently transfected into the neuron-like rat pheochromocytoma cell line PC12 and the mouse neuroblastoma x mouse spinal cord hybrid cell line NSC19. The ER retained intrabody construct, SH325-G7-KDEL, mediated a downregulation of p75NTR cell surface expression as shown by flow cytometry. This effect was maintained over a period of at least eight days without activating an unfolded protein response (UPR). Moreover, the ER retention of p75NTR resulted in downregulation of mRNA levels of the anti-apoptotic protein Bcl-xL as well as in strong inhibition of NGF-induced neurite outgrowth in PC12 cells.

Conclusion

The ER retained intrabody SH325-G7-KDEL not only induces phenotypic knockdown of this p75NTR but also p75NTR-associated cellular responses in PC12 cells.     

The p35/CDK5 signaling is regulated by p75NTR in neuronal apoptosis after intracerebral hemorrhage

The p75 neurotrophin receptor (p75NTR), a member of tumor necrosis factor receptor superfamily, involves in neuronal apoptosis after intracerebral hemorrhage (ICH). It has been previously demonstrated that phosphorylation of p35 is a crucial factor for fighting against the proapoptotic p25/CDK5 signaling in neuronal apoptosis. Then, in ICH models of rats and primary cortical neurons, we found that the expressions of p75NTR, p-histone H1 (the kinase activity of CDK5), p25, Fas-associated phosphatase-1 (FAP-1), and phosphorylated myocyte enhancer factor 2D (p-MEF2D) were enhanced after ICH, whereas the expression of p35-Thr(138) was attenuated. Coimmunoprecipitation analysis indicated several interactions as follows: p35/p25 and CKD5, p75NTR and p35, as well as p75NTR and FAP-1. After p75NTR or FAP-1 depletion with double-stranded RNA interference in PC12 cells, the levels of p25 and p-histone H1 were attenuated, whereas p35-Thr(138) was elevated. Considering p75NTR has no effect of dephosphorylation, our results suggested that p75NTR might promote the dephosphorylation of p35-Thr(138) via interaction with FAP-1, and the p75NTR/p35 complex upregulated p25/CDK5 signaling to facilitate the neuronal apoptosis following ICH. So, in the study, we aimed to provide a theoretical and experimental basis that p75NTR could be regulated to reduce neuronal apoptosis following ICH for potential clinical treatment.     

TrkA mediates developmental sympathetic neuron survival in vivo by silencing an ongoing p75NTR-mediated death signal.

Developmental sympathetic neuron death is determined by functional interactions between the TrkA/NGF receptor and the p75 neurotrophin receptor (p75NTR). A key question is whether p75NTR promotes apoptosis by directly inhibiting or modulating TrkA activity, or by stimulating cell death independently of TrkA. Here we provide evidence for the latter model. Specifically, experiments presented here demonstrate that the presence or absence of p75NTR does not alter Trk activity or NGF- and NT-3-mediated downstream survival signaling in primary neurons. Crosses of p75NTR-/- and TrkA-/- mice indicate that the coincident absence of p75NTR substantially rescues TrkA-/- sympathetic neurons from developmental death in vivo. Thus, p75NTR induces death regardless of the presence or absence of TrkA expression. These data therefore support a model where developing sympathetic neurons are "destined to die" by an ongoing p75NTR-mediated apoptotic signal, and one of the major ways that TrkA promotes neuronal survival is by silencing this ongoing death signal.     

Functional interaction of Fas-associated phosphatase-1 (FAP-1) with p75(NTR) and their effect on NF-kappaB activation.

The common neurotrophin receptor p75(NTR), a member of the tumor necrosis factor (TNF) receptor superfamily, plays an important role in several cellular signaling cascades, including that leading to apoptosis. FAP-1 (Fas-associated phosphatase-1), which binds to the cytoplasmic tail of Fas, was originally identified as a negative regulator of Fas-mediated apoptosis. Here we have shown by co-immunoprecipitation that FAP-1 also binds to the p75(NTR) cytoplasmic domain in vivo through the interaction between the third PDZ domain of FAP-1 and C-terminal Ser-Pro-Val residues of p75(NTR). Furthermore, cells expressing a FAP-1/green fluorescent protein showed intracellular co-localization of FAP-1 and p75(NTR) at the plasma membrane. To elucidate the functional role of this physical interaction, we examined TRAF6 (TNF receptor-associated factor 6)-mediated NF-kappaB activation and tamoxifen-induced apoptosis in 293T cells expressing p75(NTR). The results revealed that TRAF6-mediated NF-kappaB activation was suppressed by p75(NTR) and that the p75(NTR)-mediated NF-kappaB suppression was reduced by FAP-1 expression. Interestingly, a mutant of the p75(NTR) intracellular domain with a single substitution of a Met for Val in its C-terminus, which cannot interact with FAP-1, displayed enhanced pro-apoptotic activity in 293T transfected cells. Thus, similar to Fas, FAP-1 may be involved in suppressing p75(NTR)-mediated pro-apoptotic signaling through its interaction with three C-terminal amino acids (tSPV). Thus, FAP-1 may regulate p75(NTR)-mediated signal transduction by physiological interaction through its third PDZ domain.     

Differential effects of ethanol on the expression of cyclo-oxygenase in cultured cortical astrocytes and neurons

The developing central nervous system is a primary target of ethanol toxicity. The teratogenic effect of ethanol may result from its action on prostaglandins. Prostaglandins are generated through the release of arachidonic acid (AA) by the action of cytosolic phospholipase A(2) (cPLA(2)) on membrane-bound phospholipids and the catalytic conversion of AA to prostaglandin E(2) (PGE(2)) by cyclo-oxygenase (COX). COX is expressed in two isoforms, constitutive COX1 and inducible COX2. Cultured astrocytes and neurons from immature cerebral cortex were used as in vitro models to investigate the effect of ethanol on PGE(2) synthesis. In both cell types, neither the activity nor the expression of cPLA(2) was affected by ethanol. PGE(2) was synthesized by astrocytes and neurons. Ethanol (200-400 mg/dL for 24 h) significantly increased PGE(2) production in both cell types and the ethanol-induced increase in PGE(2) accumulation in astrocytes was significantly greater than in neurons. These increases resulted from the effects of ethanol on COX. Overall COX activity was up-regulated by ethanol in astrocytes and neurons, and indomethacin, a nonselective blocker for COX, eliminated the ethanol-induced increases of COX activity in both cell types. Increased COX activity in astrocytes resulted from an increase in COX2 expression. NS-398, a selective COX2 blocker, completely inhibited ethanol-induced alterations in COX activity. In neurons, however, ethanol had a direct effect on COX activity in the absence of a change in COX expression. NS-398 only partially blocked ethanol-induced increases in neuronal COX activity. Thus, astrocytes are a primary target of ethanol and ethanol-induced increases in glial PGE(2) synthesis are mediated by COX, principally COX2. Ethanol toxicity may be mediated through PGE(2) in immature cortical cells.     

Tumor necrosis factor receptor-associated death domain protein is involved in the neurotrophin receptor-mediated antiapoptotic activity of nerve growth factor in breast cancer cells

The common neurotrophin receptor p75(NTR) has been shown to initiate intracellular signaling that leads either to cell survival or to apoptosis depending on the cell type examined; however, the mechanism by which p75(NTR) initiates its intracellular transduction remains unclear. We show here that the tumor necrosis factor receptor-associated death domain protein (TRADD) interacts with p75(NTR) upon nerve growth factor (NGF) stimulation. TRADD could be immunodetected after p75(NTR) immunoprecipitation from MCF-7 breast cancer cells stimulated by nerve growth factor. In addition, confocal microscopy indicated that NGF stimulation induced the plasma membrane localization of TRADD. Using a dominant negative form of TRADD, we also show that interactions between p75(NTR) and TRADD are dependent on the death domain of TRADD, thus demonstrating its requirement for binding. Furthermore, the p75(NTR)-mediated activation of NF-kappaB was inhibited by transfection with a dominant negative TRADD, resulting in an inhibition of NGF antiapoptotic activity. These results thus demonstrate that TRADD is involved in the p75(NTR)-mediated antiapoptotic activity of NGF in breast cancer cells.