Ca2+involvement in activation of extracellular-signalregulated-kinase 1/2 and m-calpain after axotomy of the sciatic nerve

Lisa B. M?rtensson, Charlotta Lindwall Blom, Lars B. Dahlin,

1 Department of Translational Medicine - Hand Surgery, Lund University, Malm?, Sweden

2 Cellectricon AB, M?lndal, Sweden

3 Department of Hand Surgery, Sk?ne University Hospital, Malm?, Sweden

Ca2+involvement in activation of extracellular-signalregulated-kinase 1/2 and m-calpain after axotomy of the sciatic nerve

Lisa B. M?rtensson1, Charlotta Lindwall Blom2, Lars B. Dahlin1,3,*

1 Department of Translational Medicine - Hand Surgery, Lund University, Malm?, Sweden

2 Cellectricon AB, M?lndal, Sweden

3 Department of Hand Surgery, Sk?ne University Hospital, Malm?, Sweden

How to cite this article:M?rtensson LB, Blom CL, Dahlin LB (2017) Ca2+involvement in activation of extracellular-signal-regulated-kinase 1/2 and m-calpain after axotomy of the sciatic nerve. Neural Regen Res 12(4):623-628.

Open access statement:Tis is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.

Funding:Tis study was supported by the Research School in Pharmaceutical Science in Lund, Te Royal Physiographic Society in Lund, Te Swedish Research Council (Medicine), the Craaford’s and Ture Nilsson’s Funds for Medical Research, Funds for diabetic research, Lund University and Region Sk?ne.

Detailed mechanisms behind regeneration aer nerve injury, in particular signal transduction and the fate of Schwann cells (SCs), are poorly understood. Here, we investigated axotomy-induced activation of extracellular-signal-regulated kinase-1/2 (ERK1/2; important for proliferation) and m-calpainin vitro, and the relation to Ca2+deletion and Schwann cell proliferation and death aer rat sciatic nerve axotomy. Nerve segments were cultured for up to 72 hours with and without ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA). In some experiments, 5-bromo-2′-deoxyuridine (BrdU) was added during the last 24 hours to detect proliferating cells and propidium iodide (PI) was added at the last hour to detect dead and/or dying cells. Immunohistochemistry of sections of the cultured nerve segments was performed to label m-calpain and the phosphorylated and activated form of ERK1/2.e experiments revealed that immunoreactivity for p-ERK1/2 increased with time in organotypically cultured SCs. p-ERK1/2 and m-calpain were also observed in axons. A signif i cant increase in the number of dead or dying SCs was observed in nerve segments cultured for 24 hours. When deprived of Ca2+, activation of axonal m-calpain was reduced, whereas p-ERK1/2 was increased in SCs. Ca2+deprivation also signif i cantly reduced the number of proliferating SCs, and instead increased the number of dead or dying SCs. Ca2+seems to play an important role in activation of ERK1/2 in SCs and in SC survival and proliferation. In addition, extracellular Ca2+levels are also required for m-calpain activation and up-regulation in axons.us, regulation of Ca2+levels is likely to be a useful method to promote SC proliferation.

nerve regeneration; p-ERK1/2; m-calpain; nerve injury; signal transduction; cell proliferation; cell death; activation; axotomy; sciatic nerve; neural regeneration

Introduction

When a peripheral nerve is injured, the supporting Schwann cells (SCs) near the site of injury are also damaged, and the SCs in the distal nerve segment become activated and start to proliferate in order to support the regeneration process in the specif i c microenvironment,i.e., tissue niche where cells can be modified by the milieu (Andersson-Sjoland et al., 2011). Even if many of the damaged SCs survive, some may, however, die as a result of the injury.

We, and others, have previously found that activation of the MAP-kinase ERK1/2 by phosphorylation may be part of the injury-induced response in SCs, ultimately leading to de-differentiation, survival, proliferation and regeneration of peripheral nerve (Martensson et al., 2007; Tsuda et al., 2011; Napoli et al., 2012). Furthermore, injury to the peripheral nerve induces a massive influx of Ca2+into the damaged SCs and axons due to the difference between extracellular and intracellular Ca2+levels (Finkbeiner and Greenberg, 1996; Agell et al., 2002; Cook and Lockyer, 2006; Soletti et al., 2010).e Ca2+inf l ux leads to an activation and up-regulation of calpains, a family of Ca2+-dependent proteases, and the subsequent degradation of neurof i laments distal to the injury (Cheng and Zochodne, 2002; Glass et al., 2002; Raf f et al., 2002; Stoll et al., 2002). However, the result of the Ca2+increase following cellular damage appears to differ between cell types and situations. Importantly, following a nerve injury, Ca2+has been demonstrated to be involved in pathological events in neurons, such as apoptosis and autophagy (Gerschenson and Rotello, 1992; Cook and Lockyer, 2006; Knoferle et al., 2010), and in physiological regenerative processes, such as proliferation, and regulation of differentiation, regeneration and guidance (Shivakumar and Kumaran, 2001; Cook and Lockyer, 2006; Jacques-Fricke et al., 2006; Blackiston et al., 2009; Capiod, 2011). Less is, however,known about the outcome of injury-induced Ca2+inf l ux in SCs, although it has been demonstrated that ERK1/2 can become either activated or inhibited by the presence of Ca2+in injured neurons and neuroblastoma cells (Finkbeiner and Greenberg, 1996; Agell et al., 2002; Schmitt et al., 2004; Cook and Lockyer, 2006; Soletti et al., 2010). Also, high levels of activated ERK1/2 leads to SC cell death instead of survival, which is an interesting contradictionary aspect of this kinase (Finkbeiner and Greenberg, 1996; Cook and Lockyer, 2006).

In the present study, we investigated the activation of ERK1/2 and m-calpain (calpain II) in explanted rat sciatic nerve pieces and how such events were related to Ca2+changes. To determine the ef f ects of calcium on the organotypically cultured SCs, we chelated extracellular Ca2+by adding ethylene glycol tetra-acetic acid (EGTA) to the culture medium.

Materials and Methods

Animals

Adult female Sprague-Dawley rats (M?llegaard Breeding Center, Copenhagen, Denmark), weighing 200 g, were used in all experiments. The experimental procedures were approved by the ethical committee on animal welfare in Lund, Sweden (approval No. M131-14).e animals were kept on a 12-hour light/dark cycle with water and foodad libitum. Totally nine rats were used.

Organotypic culture of sciatic nerve segments

All animals were sacrif i ced by an intraperitoneal overdose of sodium pentobarbital (60 mg/mL; Apoteksbolaget, Sweden) followed by heart puncture.e sciatic nerves on both sides were exposed, dissected and then cut into 4 mm-long segments.e pieces of sciatic nerve were incubated free-f l oating in serum-free RPMI-1640 (Roswell Park Memorial Institute 1640) medium supplemented with penicillin/streptomycin (Invitrogen Life Technologies, Carlsbad, CA, USA) at 37°C for 24–72 hours (Martensson et al., 2007; Blom et al., 2014; Park et al., 2015). In our experimental setup, some nerve segments were cultured for 2, 24 or 48 hours in medium supplemented with ethylene glycol bis(beta-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA; Sigma-Aldrich, St. Louis, MO, USA) at a fi nal concentration of 2 mM, in order to inhibit the ef f ect of Ca2+. For the proliferation studies, 5-bromo-2′-deoxyuridine (BrdU) (Molecular Probes,ermo-Fisher, Waltham, MA, USA) was added to the medium for the fi nal 24 hour culture at a final concentration of 55 μM. In some cases, propidium iodide (PI; Sigma-Aldrich) was added to the culture medium during the last hour of tissue culture at a fi nal concentration of 10 μg/mL to assess cell viability.

Immunohistochemistry

For immunohistochemical detection, the frozen sciatic nerve segments were cut into longitudinal 10 μm-thick sections on a cryostat.e sections were immediately mounted on objective slides and leto dry. Sections were then washed with phosphate buf f ered saline (PBS) and incubated overnight at 4°C with primary antibodies (Table 1) diluted in PBS with 0.25% bovine serum albumin (BSA) and 0.25% Triton X-100.e sections were then washed again with PBS, followed by incubation with a secondary antibody for 1 hour at room temperature (Table 1) diluted as described for primary antibodies. Finally, the sections were washed and the nuclei were counterstained with bisbenzimide (1:10,000 in PBS).e sections were washed again and coverslipped with PBS/ glycerol (1:1, v:v).

Photography and image analysis

Images were captured using an Olympus AX70 fluorescence microscope (Olympus, Japan) equipped with a Nikon DS-Ri1 camera and the NIS-Elements BR3.0 image acquisition program (Nikon, Japan). The images were converted to 8-bit greyscale TIFF using Adobe Photoshop 9.0.2 (Adobe Systems Incorporated, San Jose, CA, USA) and imported into ImageJ 1.40g (a public domain image analysis program developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info. nih.gov/nih-image). In ImageJ, the tool threshold was used to determine the immunostained area of the nerve. The image analyses were performed on images captured at 10× objective magnif i cation.

A region of interest (ROI) (100 × 100 pixels) was selected in the endoneurial area furthest away from the transection site in order to estimate the immunof l ourescent intensity of the background. To determine the level of immunolabeling, the threshold was set to ±3 standard deviations of the background.e immunostained area was then measured on the entire 10 μm sections and expressed in percentage of the total area of the nerve section (a method previously described (Martensson et al., 2007)). The number of immunostained and bizbenzimide stained nuclei was counted using the ImageJ tool “particle analysis”, where the minimum and maximum particle sizes were set to 20 and 200 pixels, respectively, using the10× objective. The number of immunostained nuclei was expressed as a percentage of the total number of nuclei.

Statistical analysis

Data were expressed as the mean ± SEM. One way analysis of variance (ANOVA) was used to determine the signif i cance of the fluctuations in PI and m-calpain immunoreactivity.e two-tailedt-test was used to evaluate if there were any signif i cant changes in immunoreactivity. AP-value of ＜ 0.05 was considered statistically signif i cant.e soware used for the statistical analyses was StatView 5.0.1 (SAS Institute Inc., Cary, NC, USA) and MicrosoExcel 2010 (MicrosoCooperation, Redmond, WA, USA).

Results

p-ERK1/2

The activation of ERK1/2 along the length of the cultured nerve segments was investigated. As expected from our previous work (Martensson et al., 2007), p-ERK1/2 labeling was increased at the end of the nerve segments already at 2 hours (6.0%;P= 0.0144) and still at 24 hours (10.7%;P＜ 0.0001) as compared to the 0 hour control (2.8%) (Figure 1A).

BrdU

The proliferation of SCs was quantified by analyzing the BrdU incorporation. After 24 hour culture, the amount of BrdU positive nuclei was 0.9% and after 48 hours this amount had increased to 10.5% (P= 0.0041) of the total number of nuclei in the nerve section (Figure 1B).

PI

When PI was added to the culture medium, the number of labeled SCs signif i cantly increased from 0.7% in control sections to 9.2% (P= 0.0132) at the edge of the cultured sciatic nerve segments (i.e., the “site of injury”) after 24 hour culture. A further increase to 15.5% (P= 0.0002) was found after 48 hours, and at this time point PI stained cells were also found further in the nerve segment. At 72 hours, there was still a signif i cantly higher number of PI stained SCs in the damaged nerve segment as compared to the numbers in control (i.e., freshly dissected; 0 hour) segments (11.5%;P= 0.0027), although slightly less than at 48 hours (Figure 2A).

M-calpain

The m-calpain immunostained area significantly increased from 1.9% in the 0 hour control to 10.6% (P＜ 0.0001) aer 24 hour culture, but at 48 and 72 hours, the m-calpain immunoreactivity was again reduced to the levels in control nerves (Figure 2B). The m-calpain immunoreactivity was localized to axons at the site of transection.

Efects of Ca2+deprivation

p-ERK1/2

When the nerve segments were treated with EGTA, the number of p-ERK1/2 labeled cells had increased, as compared to the segments cultured in regular medium, with a statistically signif i cant increase at 2 hours (10.7%; P = 0.009) as compared to 6.0%, but not later, indicating that this ef f ect was immediate (Figure 3).

BrdU

When the nerve segments were cultured in the presence of EGTA, the number of BrdU immunostained cells was signif i cantly reduced both at 24 hours (0.6%;P= 0.009) and at 48 hours (0.6%;P= 0.003) as compared to nerve segments cultured in Ca2+containing medium (24 hours: 0.9% and 48 hours: 10.5%) (Figure 4).

PI

When deprived of extracellular Ca2+, the SCs showed no signif i cant increase in PI incorporation aer 24 hours, but the ef f ect of EGTA caused a signif i cant increase in dead or dying SCs aer 48 hours of culture (18.3%;P＜ 0.001) as compared to those cultured with Ca2+(9.1%) (Figure 5).

M-calpain

Discussion

The levels of Ca2+-related enzyme m-calpain increased significantly after axotomy, but only in axons and not in SCs, which is in accordance with the findings of Glass et al. (2002). In this study, m-calpain increased dramatically in neurites at 24 hours after injury, and then the levels decrease again, before the onset of axonal degeneration. M-calpain is activated and upregulated by high concentrations of Ca2+, and can only be attained intracellularly if the cell membrane is disrupted.e axons in the cultured nervepieces are destined to degenerate since they have lost contact with their cell bodies; thus, similar to the events in the distal nerve segment aer a nerve injury.erefore, if such an increase in m-calpainis related to SC death, it would be via the neuron-SC communication. However, this issue needs to be investigated more thoroughly in order to establish a connection between injury-induced neurite Ca2+fl uctuations to SC death, although Ca2+regulated Schwann cell death may very well be an event separated from the Ca2+dependent cytoskeletal reconstruction in regenerating neurons.

Table 1 Primary and secondary antibodies

Figure 1 Amount of p-ERK1/2 and BrdU labeling over time in cultured sciatic nerve segments.

Figure 2 Amount of propidium iodide (PI) and m-calpain labeling over time in cultured sciatic nerve segments.

Figure 3 Efect of Ca2+deprivation on the activation of ERK1/2 in cultured sciatic nerve segments.

Figure 4 Efect of Ca2+deprivation on SC proliferation in cultured sciatic nerve segments.

Figure 5 Efect of Ca2+deprivation on Schwann cell death in cultured sciatic nerve segments.

Figure 6 Efect of Ca2+deprivation on the activation of m-calpain in cultured sciatic nerve segments.

A main fi nding in the present study was that the levels of activated ERK1/2 increased in the nerve pieces cultured with reduced levels of calcium as compared to the pieces cultured in normal medium, which is in accordance with previous studies (Finkbeiner and Greenberg, 1996; Agell et al., 2002; Schmitt et al., 2004; Cook and Lockyer, 2006; Soletti et al., 2010). Although we did not measure intracellular Ca2+, it is reasonable to assume that a decrease in intracellular Ca2+is responsible for the present effect. The results are in accordance with studies in fi broblasts, where Ca2+has been shown to have an inhibitory effect on ERK1/2; thus, keeping the ERK1/2 at levels appropriate for cell survival and proliferation (Ji and Carpenter, 2000; Agell et al., 2002). When Ca2+levels are lowered, the ERK1/2 pathway is on the other hand over-stimulated leading to cell death instead of proliferation and survival.is ef f ect has also been demonstrated in experiments by Widerberg et al. (1997), where a slight decrease in systemic Ca2+levels increased nerve regeneration, while inhibition of Ca2+uptake signif i cantly reduced nerve regeneration.is fi nding of a detrimental increase in p-ERK1/2 is supported by the decrease of proliferating SCs and increase of SC death that we observed when nerve segments were cultured in Ca2+free medium.

EGTA treatment of cultured nerve segments also attenuated the levels of axonal m-calpain aer culture for 24 hours. Again, this illustrates a possible link between Ca2+and the calpains. However, since the increase of m-calpain, as a response to a nerve injury, only occurred in axons that were degenerating and not in SCs, we cannot draw any conclusions on what impact this has on the SCs.

As a response to cellular damage, there is an activation of both ERK1/2 and m-calpain in cultured nerve pieces. Here, we show that Ca2+regulates the activation of ERK1/2 in SCs and the activation and upregulation of m-calpain in degenerating axons. Depletion of Ca2+increases ERK1/2 activation in SCs to a point where it increases the number of dead or dying SCs and reduces SC proliferation. Removal of extracellular Ca2+decreases the activation of m-calpain in degenerating axons. We conclude that the Ca2+fl ux into the damaged SCs is important for survival and proliferation andthat gaining control over this fl ux can be vital for successful nerve regeneration.

Author contributions:LBM, CLB and LBD conceived and designed the study, defned intellectual content, were responsible for literature retrieval, edited and reviewed the paper. LBM was also responsible for lieratire retrieval, experimental studies, data acquisition and analysis, and paper prepration. LBM and LBD were the guarantors of the program of the study. All authors approved the fnal version of this paper.

Conficts of interest:None declared.

Plagiarism check:This paper was screened twice using CrossCheck to verify originality before publication.

Peer review:Tis paper was double-blinded and stringently reviewed by international expert reviewers.

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Agell N, Bachs O, Rocamora N, Villalonga P (2002) Modulation of the Ras/Raf/MEK/ERK pathway by Ca(2+), and calmodulin. Cell Signal 14:649-654.

Andersson-Sjoland A, Nihlberg K, Eriksson L, Bjermer L, Westergren-orsson G (2011) Fibrocytes and the tissue niche in lung repair. Respir Res 12:76.

Blackiston DJ, McLaughlin KA, Levin M (2009) Bioelectric controls of cell proliferation: ion channels, membrane voltage and the cell cycle. Cell Cycle 8:3527-3536.

Blom CL, Martensson LB, Dahlin LB (2014) Nerve injury-induced c-Jun activation in Schwann cells is JNK independent. Biomed Res Int 2014:392971.

Capiod T (2011) Cell proliferation, calcium inf l ux and calcium channels. Biochimie 93:2075-2079.

Cheng C, Zochodne DW (2002) In vivo proliferation, migration and phenotypic changes of Schwann cells in the presence of myelinated fi bers. Neuroscience 115:321-329.

Cook SJ, Lockyer PJ (2006) Recent advances in Ca(2+)-dependent Ras regulation and cell proliferation. Cell Calcium 39:101-112.

Finkbeiner S, Greenberg ME (1996) Ca(2+)-dependent routes to Ras: mechanisms for neuronal survival, differentiation, and plasticity? Neuron 16:233-236.

Gerschenson LE, Rotello RJ (1992) Apoptosis: a different type of cell death. FASEB J 6:2450-2455.

Glass JD, Culver DG, Levey AI, Nash NR (2002) Very early activation of m-calpain in peripheral nerve during Wallerian degeneration. J Neurol Sci 196:9-20.

Jacques-Fricke BT, Seow Y, Gottlieb PA, Sachs F, Gomez TM (2006) Ca2+ influx through mechanosensitive channels inhibits neurite outgrowth in opposition to other inf l ux pathways and release from intracellular stores. J Neurosci 26:5656-5664.

Ji QS, Carpenter G (2000) Role of basal calcium in the EGF activation of MAP kinases. Oncogene 19:1853-1856.

Knoferle J, Koch JC, Ostendorf T, Michel U, Planchamp V, Vutova P, Tonges L, Stadelmann C, Bruck W, Bahr M, Lingor P (2010) Mechanisms of acute axonal degeneration in the optic nerve in vivo. Proc Natl Acad Sci U S A 107:6064-6069.

Martensson L, Gustavsson P, Dahlin LB, Kanje M (2007) Activation of extracellular-signal-regulated kinase-1/2 precedes and is required for injury-induced Schwann cell proliferation. Neuroreport 18:957-961.

Napoli I, Noon LA, Ribeiro S, Kerai AP, Parrinello S, Rosenberg LH, Collins MJ, Harrisingh MC, White IJ, Woodhoo A, Lloyd AC (2012) A central role for the ERK-signaling pathway in controlling Schwann cell plasticity and peripheral nerve regeneration in vivo. Neuron 73:729-742.

Park BS, Kim HW, Rhyu IJ, Park C, Yeo SG, Huh Y, Jeong NY, Jung J (2015) Hydrogen sulfide is essential for Schwann cell responses to peripheral nerve injury. J Neurochem 132:230-242.

Raff MC, Whitmore AV, Finn JT (2002) Axonal self-destruction and neurodegeneration. Science 296:868-871.

Saito H, Dahlin LB (2008) Expression of ATF3 and axonal outgrowth are impaired aer delayed nerve repair. BMC Neurosci 9:88.

Saito H, Kanje M, Dahlin LB (2009) Delayed nerve repair increases number of caspase 3 stained Schwann cells. Neurosci Lett 456:30-33.

Schmitt JM, Wayman GA, Nozaki N, Soderling TR (2004) Calcium activation of ERK mediated by calmodulin kinase I. J Biol Chem 279:24064-24072.

Shivakumar K, Kumaran C (2001) L-type calcium channel blockers and EGTA enhance superoxide production in cardiac fi broblasts. J Mol Cell Cardiol 33:373-377.

Soletti RC, del Barrio L, Daf f re S, Miranda A, Borges HL, Moura-Neto V, Lopez MG, Gabilan NH (2010) Peptide gomesin triggers cell death through L-type channel calcium inf l ux, MAPK/ERK, PKC and PI3K signaling and generation of reactive oxygen species. Chem Biol Interact 186:135-143.

Stoll G, Jander S, Myers RR (2002) Degeneration and regeneration of the peripheral nervous system: from Augustus Waller’s observations to neuroinf l ammation. J Peripher Nerv Syst 7:13-27.

Tsuda Y, Kanje M, Dahlin LB (2011) Axonal outgrowth is associated with increased ERK 1/2 activation but decreased caspase 3 linked cell death in Schwann cells after immediate nerve repair in rats. BMC Neurosci 12:12.

Widerberg A, Bergman S, Danielsen N, Lundborg G, Dahlin LB (1997) Nerve injury induced by vibration: prevention of the ef f ect of a conditioning lesion by D600, a Ca2+ channel blocker. Occup Environ Med 54:312-315.

Copyedited by Li CH, Song LP, Zhao M

*< class="emphasis_italic">Correspondence to: Lars B. Dahlin, M.D., Ph.D, Lars.Dahlin@med.lu.se.

Lars B. Dahlin, M.D., Ph.D, Lars.Dahlin@med.lu.se.

orcid: 0000-0003-1334-3099 (Lars B. Dahlin)

10.4103/1673-5374.205103

Accepted: 2017-03-18