Activation of Rac1 by phosphatidylinositol 3-kinase in vivo: role in activation of mitogen-activated protein kinase (MAPK) pathways and retinoic acid-induced neuronal differentiation of SH-SY5Y cells

Rho GTPases such as RhoA, Rac1 and Cdc42 are crucial players in the regulation of signal transduction pathways required for neuronal differentiation. Using an in vitro cell culture model of neuroblastoma SH-SY5Y cells, we demonstrated previously that RhoA is an in vivo substrate of tissue transglutaminase (TGase) and retinoic acid (RA) promoted activation of RhoA by transamidation. Although activation of RhoA promoted cytoskeletal rearrangement in SH-SY5Y cells, it was not involved in induction of neurite outgrowth. Here, we demonstrate that RA promotes activation of Rac1 in SH-SY5Y cells in a transamidation-independent manner. RA-induced activation of Rac1 is mediated by phosphatidylinositol 3-kinase (PI3K), probably because of phosphorylation of the p85 regulatory subunit by Src kinases. Over-expression of constitutively active PI3K or Rac1-V12 induces neurite outgrowth, activation of mitogen activated protein kinases (MAPKs), and expression of neuronal markers. The PI3K inhibitor LY294002, or over-expression of dominant negative Rac1-N17, blocks RA-induced neurite outgrowth, activation of MAPKs, and expression of neuronal markers, suggesting that activation of PI3K/Rac1 signaling represents a potential mechanism for regulation of neuronal differentiation in SH-SY5Y cells.     

A PKCbeta isoform mediates phorbol ester-induced activation of Erk1/2 and expression of neuronal differentiation genes in neuroblastoma cells.

Protein kinase C (PKC) activation induces neuronal differentiation of SH-SY5Y neuroblastoma cells. This study examines the role of PKCbeta isoforms in this process. The PKCbeta-specific inhibitor LY379196 had no effect on 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced neurite outgrowth from SH-SY5Y neuroblastoma cells. On the other hand, PKCbeta inhibition suppressed the TPA-stimulated increase in neuropeptide Y mRNA, activation of neuropeptide Y gene promoter elements, and phosphorylation of Erk1/2. The TPA-induced increase in neuropeptide Y expression was also inhibited by the MEK inhibitor PD98059. These data indicate that activation of a PKCbeta isoform, through a pathway involving Erk1/2, leads to increased expression of neuronal differentiation genes in neuroblastoma cells.     

Differences in gene expression profile among SH-SY5Y neuroblastoma subclones with different neurite outgrowth responses to nerve growth factor

Nerve growth factor (NGF) plays a key role in the differentiation of neurons. In this study, we established three NGF-induced neurite-positive (NIN+) subclones that showed high responsiveness to NGF-induced neurite outgrowth and three NGF-induced neurite-negative (NIN-) subclones that abolished NGF-induced neurite outgrowth from parental SH-SY5Y cells, and analyzed differences in the NGF signaling cascade. The NIN+ subclones showed enhanced responsiveness to FK506-mediated neurite outgrowth as well. To clarify the mechanism behind the high frequency of NGF-induced neurite outgrowth, we investigated differences in NGF signaling cascade among subclones. Expression levels of the NGF receptor TrkA, and NGF-induced increases in mRNAs for the immediate-early genes (IEGs) c-fos and NGF inducible (NGFI) genes NGFI-A, NGFI-B and NGFI-C, were identical among subclones. Microarray analysis revealed that the NIN+ cell line showed a very different gene expression profile to the NIN- cell line, particularly in terms of axonal vesicle-related genes and growth cone guidance-related genes. Thus, the difference in NGF signaling cascade between the NIN+ and NIN- cell lines was demonstrated by the difference in gene expression profile. These differentially expressed genes might play a key role in neurite outgrowth of SH-SY5Y cells in a region downstream from the site of induction of IEGs, or in a novel NGF signaling cascade.     

The atRA-responsive gene neuron navigator 2 functions in neurite outgrowth and axonal elongation

Neuron navigator 2 (Nav2) was first identified as an all-trans retinoic acid (atRA)-responsive gene in human neuroblastoma cells (retinoic acid-induced in neuroblastoma 1, RAINB1) that extend neurites after exposure to atRA. It is structurally related to the Caenorhabditis elegans unc-53 gene that is required for cell migration and axonal outgrowth. To gain insight into NAV2 function, the full-length human protein was expressed in C. elegans unc-53 mutants under the control of a mechanosensory neuron promoter. Transgene expression of NAV2 rescued the defects in unc-53 mutant mechanosensory neuron elongation, indicating that Nav2 is an ortholog of unc-53. Using a loss-of-function approach, we also show that Nav2 induction is essential for atRA to induce neurite outgrowth in SH-SY5Y cells. The NAV2 protein is located both in the cell body and along the length of the growing neurites of SH-SY5Y cells in a pattern that closely mimics that of neurofilament and microtubule proteins. Transfection of Nav2 deletion constructs in Cos-1 cells reveals a region of the protein (aa 837-1065) that directs localization with the microtubule cytoskeleton. Collectively, this work supports a role for NAV2 in neurite outgrowth and axonal elongation and suggests this protein may act by facilitating interactions between microtubules and other proteins such as neurofilaments that are key players in the formation and stability of growing neurites.     

Heterogeneity in retinoic acid signaling in neuroblastomas: Role of matrix metalloproteinases in retinoic acid-induced differentiation

Causes of retinoid resistance often observed in neuroblastomas are unknown. We studied all trans-retinoic acid (RA) signaling in neuroblastoma cells differing in N-myc levels in terms of neurite formation, expression of tissue transglutaminase, neuronal marker proteins, matrix metalloproteinases (MMPs), and activation of Rac1 and Cdc42. Poor invasiveness observed in SH-SY5Y, LA-N-5, and SMS-KCNR cells was associated with RA-induced neurite formation, Cdc42 activation and N-myc down regulation; expression of constitutively active Cdc42 down regulated N-myc expression and reduced invasion in RA-resistant SK-N-BE(2) and IMR32 cells. RA treatment for 24 h transiently increased invasion and expression of MMP9 in SH-SY5Y, LA-N-5 and MMP2 in SMS-KCNR cells. MMP inhibition prevented RA-induced neurite formation indicating a role in differentiation. Variation in RA signaling thus follows a defined pattern and relates to invasive potential. A defective RA signaling might result in retinoid resistance and unpredictable clinical outcome observed in some neuroblastomas.     

WW domain-containing oxidoreductase promotes neuronal differentiation via negative regulation of glycogen synthase kinase 3β

WW domain-containing oxidoreductase (WWOX), a putative tumour suppressor, is suggested to be involved in the hyperphosphorylation of Alzheimer's Tau. Tau is a microtubule-associated protein that has an important role in microtubule assembly and stability. Glycogen synthase kinase 3β (GSK3β) has a vital role in Tau hyperphosphorylation at its microtubule-binding domains. Hyperphosphorylated Tau has a low affinity for microtubules, thus disrupting microtubule stability. Bioinformatics analysis indicated that WWOX contains two potential GSK3β-binding FXXXLI/VXRLE motifs. Immunofluorescence, immunoprecipitation and molecular modelling showed that WWOX interacts physically with GSK3β. We demonstrated biochemically that WWOX can bind directly to GSK3β through its short-chain alcohol dehydrogenase/reductase domain. Moreover, the overexpression of WWOX inhibited GSK3β-stimulated S396 and S404 phosphorylation within the microtubule domains of Tau, indicating that WWOX is involved in regulating GSK3β activity in cells. WWOX repressed GSK3β activity, restored the microtubule assembly activity of Tau and promoted neurite outgrowth in SH-SY5Y cells. Conversely, RNAi-mediated knockdown of WWOX in retinoic acid (RA)-differentiated SH-SY5Y cells inhibited neurite outgrowth. These results suggest that WWOX is likely to be involved in regulating GSK3β activity, reducing the level of phosphorylated Tau, and subsequently promoting neurite outgrowth during neuron differentiation. In summary, our data reveal a novel mechanism by which WWOX promotes neuronal differentiation in response to RA.     

SH-SY5Y细胞α-突触核蛋白的过表达可部分抵抗鱼藤酮诱导的氧化应激

帕金森病（Parkinson’s disease,PD）的发病机制涉及到遗传和环境因素。环境因素通过线粒休导致氧化应激和α-突触核蛋白（α—synuclein）聚集,但其确切的作用机制尚不明确。本文利用过表达α-突触核蛋白-增强型绿色荧光蛋白（enhanced green fluorescent protein．EGFP）的人多巴胺能神经母细胞瘤细胞株SH—SY5Y为模型,研究α-突触核蛋白对鱼藤酮诱导氧化应激的影响,从而进一步了解α-突触核蛋白和细胞存活之间的关系。（1）用荧光显微镜观察融合绿色荧光蛋白的α-突触核蛋白的表达情况;（2）用实时定量PCR检测α-突触核蛋白基因的表达;（3）用免疫细胞化学测定α-突触核蛋白的分布;（4）用不同浓度的鱼藤酮作用细胞后,以MTT法测细胞的活力、DCF法检测细胞的氧化应激状态、黄嘌呤氧化酶法检测超氧化物歧化酶的活力,并用流式细胞仪分析细胞的凋亡。实时定量PCR结果显示,α-突触核蛋白基因表达量在α-突触核蛋白过表达的细胞要高于SH—SY5Y细胞,在荧光显微镜下可见绿色荧光蛋白和α-突触核蛋白的表达。鱼藤酮可使细胞活力下降、线粒体complex Ⅰ的活性降低,诱导细胞内氧化应激,而过表达α-突触核蛋白的细胞可以部分抵抗鱼藤酮的毒性作用,表现为细胞抗氧化能力迅速增高（P〈0．05）和鱼藤酮诱导的细胞凋亡数目明显降低。本研究证明α-突触核蛋白对鱼藤酮产生的氧化应激有部分抵抗作用,而使过表达α-突触核蛋白的SH—SY5Y细胞对鱼藤酮的毒性作用表现出一定的耐受性。这种耐受性也可能是细胞对外界损害的一种代偿反应,从而促进细胞的存活。     

Reprint of "on the size of the active site in proteases. I. Papain"

α-Synuclein is the major component of Lewy bodies and Lewy neurites, the pathological hallmarks of surviving neuronal cells in Parkinson's disease patients. However, the physiological role played by α-synuclein remains unclear. In this study, spectrin beta non-erythrocyte 1 (SPTBN1) interacted with α-synuclein in phage display assays using a normalized human brain cDNA library. A direct interaction between α-synuclein and SPTBN1 was confirmed by GST pull-down and co-immunoprecipitation assays. SPTBN1 and α-synuclein proteins colocalized in N2a neuronal cells. Transfection of SPTBN1 caused human SH-SY5Y dopaminergic neuron cells to inappropriately induce neurites, which extended from cell bodies. Cotransfection with α-synuclein reversed SPTBN1-induced excessive neurite branching in SH-SY5Y cells, and only a single neurite extended from each neuron. These results suggest that α-synuclein modulates neurite outgrowth by interacting with cytoskeletal proteins such as SPTBN1.     

The Neurotrophic Effects and Mechanism of Action for FK1706 in Neurorrhaphy Rat Models and SH-SY5Y Cells

FK1706 is a novel non-immunosuppressive immunophilin ligand with neurotrophic activity and exerts its neurotrophic effect through NGF. The present study aimed to elaborate on the neurotrophic activity and the mechanism of action of FK1706 in end-to-side neurorrhaphy rats and SH-SY5Y cells. In the regenerating nerves of neurorrhaphy rats, FK1706 increased the thickness of myelin sheath and the level of nerve regeneration-related proteins. The mechanism of action of FK1706 on neurite regrowth was elucidated in vitro by incubating SH-SY5Y cells in different conditions (Control, NGF, FK1706, NGF?+?FK1706, NGF?+?FK1706?+?geldanamycin). Under the conditions where NGF was used, the phosphorylation level of major proteins (Raf-1 and ERK) in the Ras/Raf/MAPK/ERK signaling pathway related to SH-SY5Y cell proliferation was significantly enhanced following the application of FK1706. The number of viable cells, cell viability and neurite length of SH-SY5Y cells was maximal when NGF and FK1706 were used simultaneously. The binding level of HSP90 and Raf-1 in FK1706 group was the highest. These results indicated that FK1706 could significantly promote nerve regeneration after neurorrhaphy. The putative mechanism of action stated that FK1706 could promote the binding of HSP90 and Raf-1, make Raf-1 continue to be activated, thereby affecting key proteins in the Ras/Raf/MAPK/ERK signaling pathway related to the neurotrophic effects of NGF to promote the proliferation and neurite regrowth of nerve cells.

     

TIMP-1基因缄默削弱SH-SY5Y细胞的增殖潜能并诱导其凋亡增强

前期研究发现,人基质金属蛋白酶组织抑制剂-1(tissue inhibitors of metalloproteinases-1,TIMP-1)在唐氏综合征（Down’s syndrome ,DS)胎儿脑组织内表达下调．为了探讨TIMP-1表达下调参与DS脑病变发生的可能机制,本研究以人神经母细胞瘤细胞（SH-SY5Y）为模型,观察TIMP-1基因沉默后对其增殖和凋亡的影响．应用LipofectaminTM2000将TIMP-1特异性短发卡 RNA( short hairpin RNA,shRNA)导入SH-SY5Y细胞,经嘌呤霉素筛选获得稳定表达TIMP-1-shRNA细胞株;应用RT-PCR、real-time PCR和Western 印迹对干扰效率进行鉴定：与SH-SY5Y细胞相比,无论在mRNA水平还是蛋白水平,SH-SY5Y-TIMP-1-shRNA细胞中TIMP-1的表达显著下调（下调率接近100%）．结果显示,已成功构建了TIMP-1基因沉默的SH-SY5Y细胞模型．在此基础上,通过MTT检测发现,TIMP-1基因沉默后SH-SY5Y细胞增殖减慢;流式细胞仪和荧光显微镜凋亡检测显示,TIMP-1基因沉默后SH-SY5Y细胞凋亡明显增加．这些研究结果表明,TIMP-1基因沉默能削弱SH-SY5Y细胞的增殖能力并增强SH-SY5Y的凋亡效应,提示TIMP-1可能是通过影响神经细胞的增殖和凋亡参与DS智力低下的发病过程．     

Low-dose/dose-rate γ radiation depresses neural differentiation and alters protein expression profiles in neuroblastoma SH-SY5Y cells and C17.2 neural stem cells

The effects of low doses of ionizing radiation on cellular development in the nervous system are presently unclear. The focus of the present study was to examine low-dose γ-radiation-induced effects on the differentiation of neuronal cells and on the development of neural stem cells to glial cells. Human neuroblastoma SH-SY5Y cells were exposed to (137)Cs γ rays at different stages of retinoic acid-induced neuronal differentiation, and neurite formation was determined 6 days after exposure. When SH-SY5Y cells were exposed to low-dose-rate γ rays at the onset of differentiation, the number of neurites formed per cell was significantly less after exposure to either 10, 30 or 100 mGy compared to control cells. Exposure to 10 and 30 mGy attenuated differentiation of immature C17.2 mouse-derived neural stem cells to glial cells, as verified by the diminished expression of glial fibrillary acidic protein. Proteomic analysis of the neuroblastoma cells by 2D-PAGE after 30 mGy irradiation showed that proteins involved in neuronal development were downregulated. Proteins involved in cell cycle and proliferation were altered in both cell lines after exposure to 30 mGy; however, the rate of cell proliferation was not affected in the low-dose range. The radiation-induced attenuation of differentiation and the persistent changes in protein expression is indicative of an epigenetic rather than a cytotoxic mechanism.     

The prevention of the staurosporine-induced apoptosis by Bcl-XL, but not by Bcl-2 or caspase inhibitors, allows the extensive differentiation of human neuroblastoma cells

Staurosporine is one of the best apoptotic inducers in different cell types including neuroblastomas. In this study we have compared the efficiency and final outcome of three different anti-apoptotic strategies in staurosporine-treated SH-SY5Y human neuroblastoma cells. At staurosporine concentrations up to 500 nm, z-VAD.fmk a broad-spectrum, noncompetitive inhibitor of caspases, reduced apoptosis in SH-SY5Y cells. At higher concentrations, z-VAD.fmk continued to inhibit caspases and the apoptotic phenotype but not cell death which seems to result from oxidative damage. Stable over-expression of Bcl-2 in SH-SY5Y protected cells from death at doses of staurosporine up to 1 microm. At higher doses, cytochrome c release from mitochondria occurred, caspases were activated and cells died by apoptosis. Therefore, we conclude that Bcl-2 increased the threshold for apoptotic cell death commitment. Over-expression of Bcl-X(L) was far more effective than Bcl-2. Bcl-X(L) transfected cells showed a remarkable resistance staurosporine-induced cytochrome c release and associated apoptotic changes and survived for up to 15 days in 1 microm staurosporine. In these conditions, SH-SY5Y displayed a remarkable phenotype of neuronal differentiation as assessed by neurite outgrowth and expression of neurofilament, Tau and MAP-2 neuronal specific proteins.     

Gliotoxin induces caspase-dependent neurite degeneration and calpain-mediated general cytotoxicity in differentiated human neuroblastoma SH-SY5Y cells

In this study, a significant increase by 50% in intracellular free calcium concentration ([Ca(2+)](i)) was observed in differentiated human neuroblastoma (SH-SY5Y) cells after exposure to 0.25microM of the fungal metabolite gliotoxin for 72h. Further, the involvement of caspases and calpains was demonstrated to underlie the gliotoxin-induced cytotoxic and neurite degenerative effects. The caspase inhibitor Z-VAD-fmk almost completely reduced the neurite degeneration from 40% degeneration of neurites to 5% as compared to control. Inhibition of calpains with calpeptin significantly attenuated gliotoxin-induced cytotoxicity, determined as reduction in total cellular protein content, from 43% to 14% as compared to control cells. Western blot analyses of alphaII-spectrin breakdown fragments confirmed activity of the proteases, and that alphaII-spectrin was cleaved by caspases in gliotoxin-exposed cells. These results show that calpains and caspases have a role in the toxicity of gliotoxin in differentiated SH-SY5Y cells and that the process may be Ca(2+)-mediated.     

The retinoic acid and brain-derived neurotrophic factor differentiated SH-SY5Y cell line as a model for Alzheimer's disease-like tau phosphorylation

The paired helical filaments of highly phosphorylated tau protein are the main components of neurofibrillary tangles (NFT) in Alzheimer's disease (AD). Protein kinases including glycogen synthase kinase 3 beta (GSK3beta), cyclin-dependent kinase 5 (Cdk5), and c-Jun N-terminal kinase (JNK) have been implicated in NFT formation making the use of selective kinase inhibitors an attractive treatment possibility in AD. When sequentially treated with retinoic acid (RA) and brain-derived neurotrophic factor (BDNF), the human neuroblastoma SH-SY5Y differentiates to neuron-like cells. We found that coincident with morphologically evident neurite outgrowth, both the content and phosphorylation state of tau increased in RA-BDNF differentiated SH-SY5Y cells. Tau phosphorylation increased at all the examined sites ser-199, ser-202, thr-205, ser-396, and ser-404, all of which are hyperphosphorylated in AD brain. We also investigated whether GSK3beta, Cdk5 or JNK was involved in tau phosphorylation in the differentiated SH-SY5Y cells. We found that GSK3beta contributed most and that Cdk5 made a minor contribution. JNK was not involved in tau phosphorylation in this system. The GSK3beta-inhibitor, lithium, inhibited tau phosphorylation in a concentration-dependent manner and with good reproducibility, which enables ranking of substances in this cell model. RA-BDNF differentiated SH-SY5Y cells could serve as a suitable model for studying the mechanisms of tau phosphorylation and for screening potential GSK3beta inhibitors.     

Role of autophagy in G2019S-LRRK2-associated neurite shortening in differentiated SH-SY5Y cells

Neuritic retraction represents a prominent feature of the degenerative phenotype associated with mutations in leucine rich repeat kinase 2 (LRRK2) that are implicated in autosomal dominant and some cases of sporadic Parkinson's disease. Alterations in macroautophagy, the vacuolar catabolism of cytoplasmic constituents, have been described in Parkinson's disease. In this study, we utilized retinoic-acid differentiated SH-SY5Y cells to determine whether autophagy contributes to mutant LRRK2-associated neurite degeneration. Transfection of pre-differentiated SH-SY5Y cells with LRRK2 cDNA containing the common G2019S mutation resulted in significant decreases in neurite length, which were not observed in cells transfected with wild type LRRK2 or its kinase-dead K1906M mutation. G2019S LRRK2 transfected cells also exhibited striking increases in autophagic vacuoles in both neuritic and somatic compartments, as demonstrated by fluorescence and western blot analysis of the autophagy marker green fluorescent protein-tagged microtubule-associated protein Light Chain 3 and by transmission electron microscopy. RNA interference knockdown of LC3 or Atg7 , two essential components of the conserved autophagy machinery, reversed the effects of G2019S LRRK2 expression on neuronal process length, whereas rapamycin potentiated these effects. The mitogen activated protein kinase/extracellular signal regulated protein kinase (MAPK/ERK) kinase (MEK) inhibitor 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126) reduced LRRK2-induced neuritic autophagy and neurite shortening, implicating MAPK/ERK-related signaling. These results indicate an active role for autophagy in neurite remodeling induced by pathogenic mutation of LRRK2.     

A cAMP-activated pathway, including PKA and PI3K, regulates neuronal differentiation

Neuronal differentiation is a complex process in which many different signalling pathways may be involved. An increase in the intracellular levels of cyclic AMP (cAMP) has been shown to induce neuronal differentiation and also to cooperate with NGF to induce PC12 neurite outgrowth in a Ras-dependent manner. However, the neuritogenic activities associated with cAMP are still not well understood. The purpose of this study was to investigate the potential neuritogenic activities mediated by cAMP. For this purpose, we used the human neuroblastoma cell line SH-SY5Y. These neuroblastoma cells respond to cAMP by forming neurite-like extensions. We tried to identify some essential pathways involved in the cAMP-induced neurite elongation of these cells. Our results indicated that PKA is transiently activated in this elongation model. When we blocked PKA activity, elongation did not take place. Similarly, PI3K also plays an essential role because when we blocked this kinase activity, there was no neurite elongation. Indeed, over-expression of the p110-catalytic subunit or an activating form of the p85-regulatory subunit (p65) is able to induce some degree of neurite extension. Moreover, our results showed that when elongation is initiated, PI3K is still essential for maintenance of the neuronal morphology, whereas PKA or MAPK (ERKs or p38) activation does not appear to be necessary during this process.     

Tissue transglutaminase mediates activation of RhoA and MAP kinase pathways during retinoic acid-induced neuronal differentiation of SH-SY5Y cells

All-trans-retinoic acid (RA) plays a crucial role in survival and differentiation of neurons. For elucidating signaling mechanisms involved in RA-induced neuronal differentiation, we have selected SH-SY5Y cells, which are an established in vitro cell model for studying RA signaling. Here we report that RA-induced neuronal differentiation of SH-SY5Y cells is coupled with increased expression/activation of TGase and in vivo transamidation and activation of RhoA. In addition, RA promotes formation of stress fibers and focal adhesion complexes, and activation of ERK1/2, JNK1, and p38alpha/beta/gamma MAP kinases. Using C-3 exoenzyme (RhoA inhibitor) or monodansylcadaverine (TGase inhibitor), we show that transamidated RhoA regulates cytoskeletal rearrangement and activation of ERK1/2 and p38gamma MAP kinases. Further, by using stable SH-SY5Y cell lines (overexpressing wild-type, C277S mutant, and antisense TGase), we demonstrate that transglutaminase activity is required for activation of RhoA, ERK1/2, JNK1, and p38gamma MAP kinases. Activated MAP kinases differentially regulate RA-induced neurite outgrowth and neuronal marker expression. The results of our studies suggest a novel mechanism of RA signaling, which involves activation of TGase and transamidation of RhoA. RA-induced activation of TGase is proposed to induce multiple signaling pathways that regulate neuronal differentiation.