GSKIP,an inhibitor of GSK3β, mediates the N‐cadherin/β‐catenin pool in the differentiation of SH‐SY5Y cells

Emerging evidence has shown that GSK3β plays a pivotal role in regulating the specification of axons and dendrites. Our previous study has shown a novel GSK3β interaction protein (GSKIP) able to negatively regulate GSK3β in Wnt signaling pathway. To further characterize how GSKIP functions in neurons, human neuroblastoma SH‐SY5Y cells treated with retinoic acid (RA) to differentiate to neuron‐like cells was used as a model. Overexpression of GSKIP prevents neurite outgrowth in SH‐SY5Y cells. GSKIP may affect GSK3β activity on neurite outgrowth by inhibiting the specific phosphorylation of tau (ser396). GSKIP also increases β‐catenin in the nucleus and raises the level of cyclin D1 to promote cell‐cycle progression in SH‐SY5Y cells. Additionally, overexpression of GSKIP downregulates N‐cadherin expression, resulting in decreased recruitment of β‐catenin. Moreover, depletion of β‐catenin by small interfering RNA, neurite outgrowth is blocked in SH‐SY5Y cells. Altogether, we propose a model to show that GSKIP regulates the functional interplay of the GSK3β/β‐catenin, β‐catenin/cyclin D1, and β‐catenin/N‐cadherin pool during RA signaling in SH‐SY5Y cells. J. Cell. Biochem. 108: 1325–1336, 2009. © 2009 Wiley‐Liss, Inc.     

Synuclein-1 is selectively up-regulated in response to nerve growth factor treatment in PC12 cells

Mutations in the alpha-synuclein gene have recently been identified in families with inherited Parkinson's disease and the protein product of this gene is a component of Lewy bodies, indicating that alpha-synuclein is involved in Parkinson's disease pathogenesis. A role for normal alpha-synuclein in synaptic function, apoptosis or plasticity responses has been suggested. We show here that in rat pheochromocytoma PC12 cells synuclein-1, the rat homolog of human alpha-synuclein, is highly and selectively up-regulated at the mRNA and protein levels after 7 days of nerve growth factor treatment. Synuclein-1 expression appears neither sufficient nor necessary for the neuritic sprouting that occurs within 1-2 days of nerve growth factor treatment. Rather, it likely represents a component of a late neuronal maturational response. Synuclein-1 redistributes diffusely within the cell soma and the neuritic processes in nerve growth factor-treated PC12 cells. Cultured neonatal rat sympathetic neurones express high levels of synuclein-1, with a diffuse intracellular distribution, similar to neuronal PC12 cells. These results suggest that levels of synuclein-1 may be regulated by neurotrophic factors in the nervous system and reinforce a role for alpha-synuclein in plasticity-maturational responses. In contrast, there is no correlation between synuclein expression and apoptotic death following trophic deprivation.     

Synthesis and secretion of β-nerve growth factor by mouse teratocarcinoma cell lines

Using a cDNA probe and a two-site enzyme immunoassay, β-nerve growth factor (βNGF) synthesis was monitored in several mouse teratocarcinoma cell lines. Trace amounts of NGF mRNA were detected in the embryonal carcinoma (EC) PCC4, F9 and 1003 clones, whereas the myocardial (PCD1), myogenic (1168) and adipogenic (1246) clones contained significantly higher levels of NGF mRNA and secreted mature βNGF peptide in the culture medium. The 1003, 1168 and 1246 strains were derived from the same teratocarcinoma cell line and their ability or inability to synthesize the neurotrophic factor may reflect a developmental decision for divergent differentiation programs. Induction of NGF mRNA and protein synthesis was observed in a differentiated derivative of an SV40-transformed F9 clone which expresses the viral T antigen. Southern blot analysis of the genomic DNAs revealed no structural alterations of the NGF locus between teratocarcinoma cells that express the NGF gene and those that do not. Similar analysis of the DNA methylation pattern in C-C-G-G sequences using the Hpa II and Msp I isoschizomers indicated no methylation changes of the NGF gene in the teratocarcinoma DNAs. At least two, and probably all four, of the already mapped Msp I sites within the NGF gene are methylated in all teratocarcinoma DNAs examined, as well as in the male mouse submaxillary gland DNA, the organ richest in this factor.     

Synthesis of the biologically active β-subunit of human nerve growth factor in Escherichia coli

The gene(NGFB) encoding the β subunit of mature human nerve growth factor (hNGFB) was subcloned into the pJLA503 expression vector under the control of bacteriophage promoters p_R and p_L, and expressed in Escherichia coli. The recombinant protein represented approximately 3% of the total cellular protein. Biologically active hNGFB was solubilized (0.2% total NGFB) and purified by cation-exchange chromatography and it yielded two bands on polyacrylamide-gel electrophoresis under nonreducing conditions, corresponding to the monomeric (14 kDa) and homodimeric (26.5 kDa) forms of the molecule. Both hNGFB forms were immunopositive on Western blots with rabbit anti-NGFB antibodies; however, following additional purification, only the species corresponding to the hNGFB homodimer was biologically active on cultured chicken dorsal root ganglion neurons. These results demonstrate the feasibility of synthesizing the biologically active form of hNGFB in E. coli.     

Sequence and chromosomal localisation of the gene encoding ovine latent transforming growth factor-β1

Transforming growth factor-β1 (TGF-β1) plays important roles in pathologic processes. To further investigate the actions of this cytokine in sheep, the entire 1170-bp ovine TGF-βl pro-protein-encoding sequence has been determined by the cloning and sequencing of specific polymerase-chain-reaction amplification products of TGF-β1 cDN A sequences. In addition, these sequences have been used to estimate the length of the TGF-β1 mRNA as 1.5-1.7 kb by Northern blot hybridization and determine that the ovine TGF-β1 gene occupies a single locus in the sheep genome by chromosomal in situ hybridization.     

Overexpression of HuD, but not of its truncated form HuD I+II, promotes GAP-43 gene expression and neurite outgrowth in PC12 cells in the absence of nerve growth factor

We have previously shown that the RNA-binding protein HuD binds to a regulatory element in the growth-associated protein (GAP)-43 mRNA and that this interaction involves its first two RNA recognition motifs (RRMs). In this study, we investigated the functional significance of this interaction by overexpression of human HuD protein (pcHuD) or its truncated form lacking the third RRM (pcHuD I+II) in PC12 cells. Morphological analysis revealed that pcHuD cells extended short neurites containing GAP-43-positive growth cones in the absence of nerve growth factor (NGF). These processes also contained tubulin and F-actin filaments but were not stained with antibodies against neurofilament M protein. In correlation with this phenotype, pcHuD cells contained higher levels of GAP-43 without changes in levels of other NGF-induced proteins, such as SNAP-25 and tau. In mRNA decay studies, HuD stabilized the GAP-43 mRNA, whereas HuD I+II did not have any effect either on GAP-43 mRNA stability or on the levels of GAP-43 protein. Likewise, pcHuD I+II cells showed no spontaneous neurite outgrowth and deficient outgrowth in response to NGF. Our results indicate that HuD is sufficient to increase GAP-43 gene expression and neurite outgrowth in the absence of NGF and that the third RRM in the protein is critical for this function.     

Activation of the mitogen-activated protein kinase cascade through nitric oxide synthesis as a mechanism of neuritogenic effect of genipin in PC12h cells

Prominent neurite outgrowth induced by genipin, a plant-derived iridoid, was substantially inhibited by addition of NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide (NO) synthase (NOS) inhibitor, and carboxy-PTIO, an NO scavenger, in PC12h cells. Increases of the NADPH-diaphorase activity and neuronal and inducible NOS proteins in cells preceded the neurite outgrowth after addition of genipin to medium. NO donors could induce the neurite outgrowth dose-dependently in the cells. On the other hand, an inhibitor of soluble guanylate cyclase (SGC), which is known to be a stimulatory target of NO, abolished greatly the genipin-induced neurite outgrowth. Addition of extracellular signal-regulated kinase (ERK) kinase inhibitors could almost completely abolish the neurite induction. L-NAME remarkably depressed genipin-stimulated phosphorylation of ERK-1 and -2. A neuritogenic effect of nerve growth factor (NGF) in PC12h cells was also remarkably inhibited by the NOS inhibitor, NO scavenger and SGC inhibitor. These findings suggest that induced NO production followed by cyclic GMP-mediated stimulation of the mitogen-activated protein kinase (MAPK) cascade is implicated in the neuritogenesis by genipin and NGF in PC12h cells.     

Chromatographic Resolution and Characterization of a Nerve Growth Factor-Dependent Kinase That Phosphorylates Microtubule-Associated Proteins 1 and 2 in PC12 Cells

When the supernatant fractions from extracts of control and nerve growth factor (NGF)- or dibutyryl cyclic AMP-treated PC12D cells were applied to DEAE-Sepharose columns and proteins were eluted with a gradient of NaCl, three separate peaks of kinase activity that phosphorylated microtubule-associated proteins (MAPs) were recovered. Enhancement of the kinase activity in peak 1 was noted in the case of dibutyryl cyclic AMP-treated cells. In contrast, the kinase activity in the third peak was markedly elevated, in terms of the ability to phosphorylate MAP1 and MAP2, in the case of the extract from NGF-treated cells. This activity was designated previously as NGF-dependent MAP kinase. The apparent molecular mass of the active kinase was 45-50 kDa. The apparent Km value was 35 microM for ATP with either MAP1 or MAP2 as substrate. When the kinase activity in the fractions from the DEAE-Sepharose column was assayed in the presence of Mn2+ instead of Mg2+, another NGF-stimulated kinase activity was detected in the fractions eluted by a lower concentration of NaCl than that which eluted the Mg(2+)-activated kinase. Other growth factors, namely, epidermal growth factor and basic fibroblast growth factor, also stimulated the activity of NGF-dependent MAP kinase. Possible involvement of the kinase in the outgrowth of neurites has been suggested. The NGF-induced activation of NGF-dependent MAP kinase was blocked by the presence of K-252a. In contrast, the activation of NGF-dependent MAP kinase by basic fibroblast growth factor and by epidermal growth factor was not blocked, but actually stimulated by K-252a, a result that correlates well with the analogous actions of the drug on the outgrowth of neurites that is induced by these growth factors. The latter observation strengthens the possibility of a close relationship between the outgrowth of neurites and the activation of NGF-dependent MAP kinase.     

Impaired integrin α5/β1‐mediated hepatocyte growth factor release by stellate cells of the aged liver

Hepatic blood flow and sinusoidal endothelial fenestration decrease during aging. Consequently, fluid mechanical forces are reduced in the space of Disse where hepatic stellate cells (HSC) have their niche. We provide evidence that integrin α₅/β₁ is an important mechanosensor in HSC involved in shear stress‐induced release of hepatocyte growth factor (HGF), an essential inductor of liver regeneration which is impaired during aging. The expression of the integrin subunits α₅ and β₁ decreases in liver and HSC from aged rats. CRISPR/Cas9‐mediated integrin α₅ and β₁ knockouts in isolated HSC lead to lowered HGF release and impaired cellular adhesion. Fluid mechanical forces increase integrin α₅ and laminin gene expression whereas integrin β₁ remains unaffected. In the aged liver, laminin β2 and γ1 protein chains as components of laminin‐521 are lowered. The integrin α₅ knockout in HSC reduces laminin expression via mechanosensory mechanisms. Culture of HSC on nanostructured surfaces functionalized with laminin‐521 enhances Hgf expression in HSC, demonstrating that these ECM proteins are critically involved in HSC function. During aging, HSC acquire a senescence‐associated secretory phenotype and lower their growth factor expression essential for tissue repair. Our findings suggest that impaired mechanosensing via integrin α₅/β₁ in HSC contributes to age‐related reduction of ECM and HGF release that could affect liver regeneration.     

Degradation of amyloid β peptide by neprilysin expressed from Borna disease virus vector

Accumulation of amyloid β (Aβ40 and Aβ42) in the brain is a characteristic of Alzheimer disease (AD). Because neprilysin (NEP) is a major Aβ‐degrading enzyme, NEP delivery in the brain is a promising gene therapy for AD. Borna disease virus (BoDV) vector enables long‐term transduction of foreign genes in the central nerve system. Here, the proteolytic ability of NEP transduced by the BoDV vector was evaluated and it was found that the amounts of Aβ40 and Aβ42 decreased significantly, suggesting that NEP expressed from the BoDV vector is functional in that it degrades Aβ.

     

Production of transforming growth factor (TGF) β by fetal lung cells

Summary— TGF β is supposed to play an important role in the process of epithelial maturation in the developing fetal lung. Using an immunofluorescence approach, we showed that fetal rat lung fibroblasts elaborate the three TGF β isoforms known in mammals (TGF β1, β2 and β3) whereas epithelial cells appear to synthesize only TGF β1 and β3. Isolated fibroblasts secrete the three isoforms. Biological assay of TGF β activity in fibroblast-conditioned media did not reveal significant changes according to the stage when fibroblasts were isolated.     

Nerve growth factor stimulates the phosphorylation of a 250 kDa cytoskeletal protein in cell-free extracts of PC12 cells

Nerve growth factor (NGF) rapidly stimulates the phosphorylation of a 250 kDa cytoskeletally-associated protein (pp250) by a protein kinase which is also associated with structural elements of the cell. We have solubilized these proteins and demonstrated that NGF-stimulated phosphorylation can be observed in cell free extracts of cytoskeletons from NGF-treated PC12 cells. The pp250 substrate and the 250-kinase were solubilized from PC12 cytoskeletons by treatment with 2 M urea. Phosphorylation of pp250 was maximally stimulated following treatment of the cells for 5 min with NGF. This effect was transient, diminishing with longer exposure of the cells to hormone. The 250-kinase preferred Mn²⁺ over Mg²⁺ and was inhibited by both Na⁺ and K⁺. The phosphorylation of pp250 was not affected by Ca²⁺. Upon fractionation of the urea-soluble cytoskeletal proteins by gel filtration, the 250-kinase eluted in two peaks; one peak of enzyme activity coeluting with the pp250 substrate, and a second peak of enzyme activity eluting with an apparent Mr of approximately 60 kDa. Treatment of the PC12 cells with the phorbol ester TPA also stimulated the phosphorylation of pp250, although this effect was not as great as that produced by NGF. This cell free system should be a valuable tool in the investigation of the mechanisms of NGF action.Special issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.     

The effect of extrinsic Wnt/β‐catenin signaling in Muller glia on retinal ganglion cell neurite growth

Muller glia are the predominant glial cell type in the retina, and they structurally and metabolically support retinal neurons. Wnt/β‐catenin signaling pathways play essential roles in the central nervous system, including glial and neuronal differentiation, axonal growth, and neuronal regeneration. We previously demonstrated that Wnt signaling activation in retinal ganglion cells (RGC) induces axonal regeneration after injury. However, whether Wnt signaling within the adjacent Muller glia plays an axongenic role is not known. In this study, we characterized the effect of Wnt signaling in Muller glia on RGC neurite growth. Primary Muller glia and RGC cells were grown in transwell co‐cultures and adenoviral constructs driving Wnt regulatory genes were used to activate and inhibit Wnt signaling specifically in primary Muller glia. Our results demonstrated that activation of Wnt signaling in Muller glia significantly increased RGC average neurite length and branch site number. In addition, the secretome of Muller glia after induction or inhibition of Wnt signaling was characterized using protein profiling of conditioned media by Q Exactive mass spectrometry. The Muller glia secretome after activation of Wnt signaling had distinct and more numerous proteins involved in regulation of axon extension, axon projection and cell adhesion. Furthermore, we showed highly redundant expression of Wnt signaling ligands in Muller glia and Frizzled receptors in RGCs and Muller glia. Therefore, this study provides new information about potential neurite growth promoting molecules in the Muller glia secretome, and identified Wnt‐dependent target proteins that may mediate the axonal growth.     

Transforming growth factor type β can act as a potent competence factor for AKR-2B cells

Transforming growth factor type β (TGFβ) is a pleiotropic regulator of cell growth with specific high-affinity cell-surface receptors on a large number of cells; its mechanism of action, however, is poorly defined. In this report, we utilized the mouse fibroblast line AKR-2B to explore the question of the temporal requirements during the cell cycle in regard to both the growth inhibitory and the growth stimulatory action of TGFβ. The results indicate that AKR-2B cells are most sensitive to the inhibitory action of TGFβ during early to mid-G₁. In addition, TGFβ need be present only briefly (as little as l min) in order to exert its inhibitory effect on EGF-induced DNA synthesis. Likewise, the stimulatory effect of TGFβ in the absence of EGF requires only an equally brief exposure to TGFβ. Use of homogeneous ¹²⁵I-labeled TGFβ in a cell-binding assay demonstrates that TGFβ bound to cell-surface receptors can readily exchange into the culture medium T_1/2 = 120 min), helping to rule out the possibility that persistent receptor-bound TGFβ is the source of a continuous stimulus. The data indicate that TGFβ exposure induces a stable state in the cell (T_1/2 = 20 h) similar to but distinct from the state of “competence” induced by platelet-derived growth factor (PDGF).     

Ischemia-induced phosphorylation of initiation factor 2 in differentiated PC12 cells: role for initiation factor 2 phosphatase

An in vitro model of ischemia was obtained by subjecting PC12 cells differentiated with nerve growth factor to a combination of glucose deprivation plus anoxia. Immediately after the ischemic period, the protein synthesis rate was significantly inhibited (80%) and western blots of cell extracts revealed a significant accumulation of phosphorylated eukaryotic initiation factor 2, alpha subunit, eIF2(alphaP) (42%). Upon recovery, eIF2(alphaP) levels returned to control values after 30 min, whereas protein synthesis was still partially inhibited (33%) and reached almost control values within 2 h. The activities of the mammalian eIF2alpha kinases, double-stranded RNA-activated protein kinase, mammalian GCN2 homologue, and endoplasmic reticulum-resident kinase, were determined. None of the eIF2alpha kinases studied showed increased activity in ischemic cells as compared with controls. Exposure of cells to cell-permeable inhibitors of protein phosphatases 1 and 2A, calyculin A or tautomycin, induced dose- and time-dependent accumulation of eIF2(alphaP), mimicking an ischemic effect. Protein phosphatase activity, as measured with [(32)P]phosphorylase a as a substrate, diminished during ischemia and returned to control levels upon 30-min recovery. In addition, the rate of eIF2(alphaP) dephosphorylation was significantly lower in ischemic cells, paralleling both the greatest translational inhibition and the highest eIF2(alphaP) levels. The endogenous phosphatase activity from control and ischemic extracts showed different sensitivity to inhibitor 2 and fostriecin in in vitro assays, inhibitor-2 effect in ischemic cells being lower than in control cells. Together these results indicate that an eIF2alpha phosphatase, probably protein phosphatase 1, is implicated in the ischemia-induced eIF2(alphaP) accumulation in PC12 cells.     

Phospholipase C‐η2 is required for retinoic acid‐stimulated neurite growth

Phospholipase C‐η2 is a recently identified phospholipase C (PLC) implicated in the regulation of neuronal differentiation/maturation. PLCη2 activity is triggered by intracellular calcium mobilization and likely serves to amplify Ca²⁺ signals by stimulating further Ca²⁺ release from Ins(1,4,5)P₃‐sensitive stores. The role of PLCη2 in neuritogenesis was assessed during retinoic acid (RA)‐induced Neuro2A cell differentiation. PLCη2 expression increased two‐fold during a 4‐day differentiation period. Stable expression of PLCη2‐targetted shRNA led to a decrease in the number of differentiated cells and total length of neurites following RA‐treatment. Furthermore, RA response element activation was perturbed by PLCη2 knockdown. Using a bacterial two‐hybrid screen, we identified LIM domain kinase 1 (LIMK1) as a putative interaction partner of PLCη2. Immunostaining of PLCη2 revealed significant co‐localization with LIMK1 in the nucleus and growing neurites in Neuro2A cells. RA‐induced phosphorylation of LIMK1 and cAMP‐responsive element‐binding protein was reduced in PLCη2 knock‐down cells. The phosphoinositide‐binding properties of the PLCη2 PH domain, assessed using a FRET‐based assay, revealed this domain to possess a high affinity toward PtdIns(3,4,5)P₃. Immunostaining of PLCη2 together with PtdIns(3,4,5)P₃ in the Neuro2A cells revealed a high degree of co‐localization, indicating that PtdIns(3,4,5)P₃ levels in cellular compartments are likely to be important for the spatial control of PLCη2 signaling.     

Expression and regulation of α1β1 integrin in Schwann cells

The interaction of cells with the extracellular matrix plays a critical role in morphogenesis and cell differentiation. To define how Schwann cells might interact with the extracellular matrix, we chose to study the expression of the laminin/collagen receptor α1β1 integrin during nerve development in the rat from embryonic day 14 to maturity. We found that this integrin is expressed predominantly on mature non-myelin-forming cells and only at very low levels on myelin-forming cells. Significant levels of this integrin were not detected on Schwann cell precursors or embryonic Schwann cells in vivo. Experiments using transected and crushed sciatic nerve showed that α1β1 integrin expression is regulated at least in part by axonal contact. Furthermore, Schwann cell culture experiments showed that α1β1 integrin levels are strongly upregulated by transforming growth factor-βs and phorbol esters. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 914–928, 1997