Laminin activates NF-κB in Schwann cells to enhance neurite outgrowth

Neuroscience Letters 439 (2008) 42–46

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Laminin activates NF-␬B in Schwann cells to enhance neurite outgrowth Stephanie J. Armstrong a , Mikael Wiberg b , Giorgio Terenghi a , Paul J. Kingham a,∗ a b

Blond McIndoe Laboratories, The University of Manchester, Manchester, UK Departments of Surgical & Perioperative Science (Handsurgery) and Integrative Medical Biology (Anatomy), Umea University, Sweden

a r t i c l e

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Article history: Received 3 January 2008 Received in revised form 12 April 2008 Accepted 22 April 2008 Keywords: Axon Extraceullar matrix Glia NF-␬B Schwann cell

a b s t r a c t Extracellular matrix (ECM) molecules and Schwann cells (SCs) are important components of peripheral nerve regeneration. In this study, the role of the transcription factor nuclear factor kappa B (NF-␬B) in SC activation in response to laminin and the subsequent effect on in vitro neurite outgrowth was investigated. Immunocytochemistry and Western blot analysis showed that compared with poly-d-lysine (PDL), laminin enhanced the phosphorylation of I␬B and p65 NF-␬B signalling proteins in SCs. Phospho NF␬B-p65 was localised to the nucleus indicating activation of NF-␬B. To assess the functional effect of NF-␬B activation, SCs plated on PDL or laminin were pre-treated with NF-␬B inhibitors, 6-amino-4-(4phenoxyphenylethylamino)quinazoline (QNZ) or Z-leu-leu-leu-CHO (MG-132) before NG108-15 neuronal cells were seeded on the SC monolayer. After 24 h co-culture in the absence of inhibitors, SCs seeded on laminin enhanced the mean number and length of neurites extended by NG108-15 cells (1.87 ± 0.13 neurites; 238.74 ± 8.53 ␮m) compared with those cultured in the presence of SCs and PDL (1.26 ± 0.07 neurites; 157.57 ± 9.80 ␮m). At 72 h, neurite length had further increased to 321.83 ± 6.60 ␮m in the presence of SCs and laminin. Inhibition of NF-␬B completely abolished the effect of laminin on SC evoked neurite outgrowth at 24 h and reduced the enhancement of neurite length by over 60% at 72 h. SC proliferation was unaffected by NF-␬B inhibition suggesting that the NF-␬B signalling pathway plays a discrete role in the activation of SCs and their neurotrophic potential. © 2008 Elsevier Ireland Ltd. All rights reserved.

Nuclear Factor kappa B (NF-␬B) is a transcription factor best characterised for its involvement in the cellular response to external stimuli such as stress, cytokines, free radicals and its role in adaptive and immune responses. Recent studies have also shown that the NF-␬B pathway is active in the nervous system in both neuronal and glial cells [8,12,13]. NF-␬B is comprised of homo or hetero dimers derived from a family of five structurally related proteins, p65 (Rel A), p50, p52, Rel B and cRel. The p50/p65 heterodimer is the predominant combination in many cell types. Under normal circumstances NF-␬B is sequestered in the cytoplasm by an inhibitory protein I␬B rendering it inactive. In response to an external stimulus I␬B becomes phosphorylated by an I␬B kinase. The I␬B protein is then ubiquitinated and degraded allowing nuclear translocation of NF-␬B and activation of target genes [10]. Many genes are up-regulated in response to NF-␬B activation including cytokines such as interleukins (IL)-1,2,3,6,8,12, tumour necrosis factor (TNF)-␣, and various cell adhesion molecules

∗ Corresponding author at: Blond McIndoe Laboratories, Tissue Injury & Repair Research Group, School of Clinical and Laboratory Sciences, The University of Manchester, Room 3.106 Stopford Building, Oxford Road, Manchester M13 9PT, UK. Tel.: +44 161 275 1596; fax: +44 161 275 1814. E-mail address: [email protected] (P.J. Kingham). 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.04.091

(CAMs) including ICAM1, VCAM1 and E-selectin [10]. These molecules have been well characterised in the immune system but they are also important regulators of the nervous system. NF-␬B has been shown to regulate neurite outgrowth from neurons both in the developing central and peripheral nervous systems [7,13]. BAY 11-7082, a known inhibitor of the NF-␬B signalling pathway, decreases the size and complexity of neurite branching in sensory neurons [7]. The NF-␬B signalling pathway has also been identified in SCs and its activation is required for peripheral myelin formation. Critical differentiation signals are sent to the SCs allowing them to differentiate into a pro-myelinating phenotype and up-regulate their expression of the transcription factor Oct 6, which is essential for correct myelin formation [12]. In a previous study by our group [1] we showed that neurite outgrowth from the NG108-15 neuronal cell line was increased in response to SCs seeded on extracellular matrix (ECM) molecules. We found that laminin produced a significantly greater effect than collagen or fibronectin [1]. In this current study we have investigated the signalling mechanisms mediating this process, hypothesising that NF-kB plays an important role in laminininduced activation of SCs. Sciatic nerves were removed aseptically from 1 to 2 days old neonatal rat pups and SCs isolated and cultured as previously described [1].

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Tissue culture plastic flasks used for Western blot lysate preparation or slide flasks for immunostaining were pre-coated with either PDL (100 ␮g/cm2 ) or laminin from Engelbreth-Holm-Swarm (EHS) murine sarcoma basement membrane (2 ␮g/cm2 ) according to the manufacturer’s guidelines (Sigma, UK). SCs (1.5 × 103 ) were seeded on either PDL or laminin coated slide flasks. After 24 h SCs were fixed with 4% (w/v) paraformaldehyde and immunostained with rabbit anti-p-NF-␬B p65 or mouse pI␬B (1:500, Autogen Bioclear, UK). p-NF-␬B p65 stained slides were additionally treated with ice cold methanol for 3 min. FITC labelled goat anti-rabbit (1:200, Vector Labs, UK) or Cy3-labelled goat antimouse (1:200, Amersham Bioscience, UK) secondary antibodies were used. SCs (4 × 106 ) were seeded on PDL or laminin coated 75 cm2 tissue culture plastic flasks and cultured for 24 h. Cells were washed twice with PBS, suspended in lysis buffer (pH 7.9) containing 10 mM HEPES, 1.5 mM MgCl2 , 10 mM KCl, 0.5 mM DTT, 0.05% NP40, 1 mM Na3 VO4 , 1 mM EGTA, 100 mM NaF plus protease inhibitors (Sigma, Poole, UK). Proteins were separated on 10% (v/v) acrylamide gels using SDS-PAGE (120 V). The proteins were then transferred to nitrocellulose membrane and incubated overnight at 4 ◦ C with mouse anti-pI␬B (1:1000, Autogen Bioclear, UK) or rabbit anti-p65 (1:1500, Autogen Bioclear, UK). HRP-conjugated secondary antibodies (mouse 1:1000, rabbit 1:2000, Cell Signalling, USA) were applied and the membranes developed by the addition of electrochemiluminescent substrate and exposure to Kodak light sensitive film (Sigma, UK). An in vitro co-culture model was employed to assess the effect of NF-␬B inhibitors, 6-amino-4-(4-phenoxyphenylethylamino)quinazoline (QNZ; Tebu-bio, UK) and Z-leu-

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leu-leu-CHO (MG-132, Calbiochem, UK) on neurite outgrowth from NG108-15 cells. SCs were seeded at a density of 5 × 104 for 24 h on laminin and PDL coated slide flasks with or without the addition of 40 nm QNZ or 1 h pre-treatment with 5 ␮m MG-132. After 24 h in culture the cells were washed extensively to remove any inhibitor. 1 × 104 NG108-15 cells were added to the flask and cultured for a further 24 h or 72 h. Neurite outgrowth from the NG108-15 cells was determined in the co-culture system by immunostaining with a monoclonal pan neurofilament antibody (dilution 1:500, BioMol, UK) and measurements were made using Image ProPlus software (MediaCybernetics, USA) as previously described [1]. Statistical analysis was performed using Kruskal–Wallis one way ANOVA with Dunns comparison test to determine the statistical significance between data sets, * p < 0.05. SC proliferation was determined using an Alamar blue assay as previously described [1]. SCs were seeded at a density of 2 × 103 on PDL or laminin coated tissue culture plastic wells either with or without addition of 40 nm QNZ. Measurements were taken every 24 h for 4 days. To determine if NF-␬B was activated in SCs in response to laminin, the presence and localisation of pI␬B and p-NF-␬B p65 proteins were investigated. Immunocytochemistry showed pI␬B staining was localised to the cytoplasm in the SCs and was increased in the presence of laminin (Fig. 1A). Constitutive faint immunoreactivity of phosphorylated NF-␬B p65 was observed in SCs seeded on PDL, this was increased in the nucleus of the SCs in the presence of laminin (Fig. 1B). There was also some immunoreactivity in the cytoplasm of the SCs seeded on laminin (Fig. 1B) most likely as the result of continuous production and degradation of NF-␬B p65. Western blotting was used to further confirm NF-

Fig. 1. Phosphorylation of I␬B and NF␬B p65 in response to laminin demonstrates activation of the NF␬B signalling pathway. Immunofluorescence staining of (A) pI␬B and (B) p-NF␬B p65 in SCs seeded on laminin compared with PDL.Scale bar = 40 ␮m. (C) Western blot analysis of p-NF␬B p65 and pI␬B proteins for SCs seeded on laminin and PDL for 24 h. Blots were analysed by densitometry and values normalised to the ␤ tubulin loading control.

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␬B activation. SCs seeded on laminin showed an increase in the phosphorylation of both I␬B and NF-␬B p65 proteins compared with SCs seeded on PDL (Fig. 1C). Densitometry indicated there was an approximate two-fold increase in pI␬B and p-NF-␬B p65 protein in SCs seeded on laminin compared with SCs seeded on PDL. To assess the functional effects of inhibiting NF-␬B, SCs and NG108-15 cells were co-cultured together on slide flasks pre coated with either laminin or PDL. QNZ, an inhibitor of NF-␬B transcriptional activation [4,16,17] was added to a group of cultures to assess its effect on neurite outgrowth. NG108-15 cells grown alone on PDL or laminin showed minimal neurite outgrowth for both the number of neurites per cell body and neurite length (Figs. 2 and 3). The number of neurites per cell body increased when NG108-15 cells

were seeded on SCs and laminin (1.87 ± 0.13) compared with SCs and PDL (1.26 ± 0.07). NG108-15 cells also extended longer neurites in the laminin co-culture system than in the PDL co-culture system (238.74 ± 8.53 ␮m versus 157.57 ± 9.80 ␮m). Addition of QNZ to SCs for 24 h prior to seeding of the NG108-15 cells, completely abolished (significance p < 0.05) the enhancement of neurite number and length evoked by laminin treatment of the SCs. In contrast, QNZ produced only a negligible reduction in neurite outgrowth evoked by SCs seeded on PDL. The effects of QNZ on neurite outgrowth were replicated when an alternative NF-␬B inhibitor, MG-132, was incubated with the SCs (Fig. 3). Neurite outgrowth was also determined after 72 h of co-culture. SCs seeded on laminin further enhanced neurite length (321.83 ± 6.60 ␮m compared with 238.74 ± 8.53 ␮m after 24 h) but the number of neurites per NG108-15 cell did not

Fig. 2. Inhibition of the NF␬B signalling pathway abolishes the increase in neurite outgrowth in response to laminin activated SCs. NG108-15 cells were grown in the absence (control) or presence of SCs seeded on slide flasks pre-coated with PDL or laminin. 40 nM QNZ was added to both the PDL and laminin co-culture systems for 24 h prior to addition of NG108-15 cells (+SC + QNZ). Neurites were visualised using neurofilament immunofluorescence staining (red). Background DAPI staining (blue) shows the nuclei of the Schwann cell monolayer. Scale bar = 40 ␮m. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

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Fig. 4. Effect of QNZ inhibitor on SC proliferation. SCs were seeded on tissue culture plastic pre-coated with (A) PDL or (B) laminin. Proliferation of SCs was quantified using the Alamar blue assay. SCs were seeded at a density of 2 × 103 with or without the addition of 40 nM QNZ. Measurements were recorded over a 96 h period and data plotted as mean ± S.E.M.

Fig. 3. Quantification of neurite outgrowth. Using Image ProPlus software the number of neurites per cell body (A) and the average median neurite length (B) were calculated in NG108-15 cells cultured on PDL or laminin (lam) in the absence or presence of SCs which were either untreated or had been pre-incubated with the NF-␬B inhibitors, QNZ and MG-132. Measurements were made after 24 h and 72 h. Statistical analysis was performed using Kruskal-Wallis one way ANOVA with Dunn’s comparison test to determine the statistical significance between data, * p < 0.05 significantly reduced in the presence of QNZ or MG-132.

increase with time. Treatment of SCs with QNZ reduced the neurite length by 61.36 ± 18.82% (Fig. 3). QNZ was added to cultures of SCs to determine if SC proliferation was mediated by NF-␬B signalling. An Alamar blue assay was carried out to measure SC growth on PDL and laminin in the presence of the inhibitor. Addition of 40 nm QNZ did not significantly decrease SC proliferation over 4 days in culture for SCs seeded on PDL (Fig. 4A) or laminin (Fig. 4B). NF-␬B is a transcription factor that is part of a cascade pathway comprised of many different proteins (reviewed in [10]). In this study we have analysed the phosphorylation of two key proteins, p65 and I␬B, in SCs. Phosphorylation of I␬B occurs on two N terminal serine residues, at positions 32 and 36, this then triggers rapid degradation of the protein [5]. Immunocytochemistry showed strong immunoreactivity for the phosphorylated form of

I␬B in the cytoplasm of SCs seeded on laminin, but only faint immunoreactivity in the SCs seeded on PDL. Upon degradation of I␬B, NF-␬B becomes phosphorylated and is released into the cytoplasm before it translocates to the nucleus [14]. Constitutive faint immunoreactivity of phosphorylated NF-␬B p65 was observed in SCs seeded on PDL whereas much stronger phosphorylated NF-␬B p65 immunoreactivity was observed in the nucleus of SCs seeded on laminin. To a lesser extent immunoreactivity was observed in the cytoplasm of SCs seeded on laminin due to the continuous production and degradation of NF-␬B p65 in response to a stimulus. These results indicate that the NF-␬B pathway was activated in SCs in response to the ECM molecule laminin. NF-␬B activation has been shown to be involved in the proliferation of various cell types including T and B cells and neural stem cells [9,18]. When QNZ, an inhibitor of NF-␬B transcriptional activation, was added to SC cultures there was no effect on cell proliferation rates in response to either PDL or laminin. A previous study by Nikols et al. also showed that SN50, another inhibitor of NF-␬B activation, had no detrimental effect on the growth of SC [12]. We previously showed that SCs seeded on laminin enhanced neurite outgrowth from NG108-15 cells [1]. Treatment of SCs with QNZ or MG-132 prior to seeding on laminin significantly inhibited this enhanced neurite outgrowth. Neurite length was reduced to a similar level to that of SCs seeded on PDL. Furthermore, there was a negligible decrease in the neurite length for NG108-15 cells

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seeded on SCs and PDL. Addition of the inhibitors reduced the effect on neurite outgrowth but only the increased effect due to laminin, reducing it to a constitutive basal level after 24 h of culture. QNZ also greatly diminished the neurite elongation after 72 h culture implying that both initial sprouting and longer term growth and extension seen in response to SCs seeded on laminin is mediated by NF-␬B. A mechanism by which SCs might enhance neurite outgrowth is via cell–cell interaction. Remodelling of the ECM occurs after injury and the ECM molecules which are found in the basal laminae can be synthesised by SCs [3]. Individual components of the ECM are able to interact with integrin receptors on SCs and activate protein tyrosine kinases including focal adhesion kinase (FAK) and Shc [6]. These kinases then activate signalling pathways including the Ras-extracellular signal-regulated kinase (Erk)-1/MAP kinase pathway leading to alteration in gene expression and subsequently changes in cellular function [6,11]. Chen and Strickland hypothesised that laminin interacts with SCs via the ␤1 integrin receptor, activating NF-␬B, leading to interaction between SCs and axons resulting in the formation of peripheral myelin [2]. Taylor et al. also demonstrated that ␤1 integrin and FAK proteins cluster in SC and sensory neuron co-cultures [15]. It is conceivable that a similar process occurs in our co-culture system in response to laminin. In our study we used laminin derived from EHS sarcoma basement membrane and it is therefore unknown if a specific laminin isoform mediates the NF␬B activation. NF␬B activation within the SC could lead to increased expression of CAMs. Homophilic interactions occur between CAMs so it is possible that the NG108-15 cells CAMs could interact with the SC CAMs [6]. Thus it is conceivable that laminin activation of NF␬B may be responsible for an up regulation of the expression. In conclusion we have shown that the NF-␬B signalling pathway is activated in SCs in response to laminin and that this is beneficial for neurite outgrowth. Further understanding of the downstream mechanisms of NF-␬B activation will be needed to fully understand how this functional effect is achieved. Acknowledgements This work was funded by a University of Manchester strategic studentship award (SJA), the Swedish Medical Research Council, the County of Vasterbotten and the Aners Foundation. The authors also wish to thank Acorda Therapeutics for their generous gift of GGF-2.

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