Trk receptors need neutral sphingomyelinase activity to promote cell viability

FEBS Letters 588 (2014) 167–174

journal homepage: www.FEBSLetters.org

Trk receptors need neutral sphingomyelinase activity to promote cell viability Ana Candalija a,1, Roger Cubí a,1, Arturo Ortega c, José Aguilera a,b,⇑, Carles Gil a,⇑ a

Department of Biochemistry and Molecular Biology, Institute of Neurosciences, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Catalunya, Spain Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Spain c Departamento de Genética y Biología Molecular, Cinvestav-IPN, México DF, Mexico b

a r t i c l e

i n f o

Article history: Received 12 July 2013 Revised 19 November 2013 Accepted 25 November 2013 Available online 5 December 2013 Edited by Sandro Sonnino Keywords: Neurotrophin Sphingomyelinase Ceramide Phosphorylation Granule neuron PC12

a b s t r a c t Neurotrophins are a group of secreted polypeptides, which comprises Nerve Growth Factor (NGF) and Brain-Derived Neurotrophic Factor (BDNF). Each neurotrophin can bind specifically to a tyrosine kinase Trk receptor (TrkA, TrkB or TrkC), while all of the neurotrophins can bind, with similar affinity, to the p75 neurotrophin receptor (p75NTR). Experiments on cell viability promotion by BDNF in granule neurons or by NGF in PC12 cells show that neurotrophin-exerted cell viability is neutral sphingomyelinase (nSMase)-dependent, since GW4869 or siRNA knockdown abrogates the protective effects, as well as neurotrophin-induced Akt phosphorylation. Finally, the assessment of nSMase activity promotion drives to the conclusion that neurotrophins can promote cell viability through Trk receptors in a manner depending on basal nSMase but not through SMase activity enhancement. Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction Mammalian neurotrophins (NTs) are a group of secreted polypeptides, which comprises Nerve Growth Factor (NGF), Brain-Derived Neurotrophic Factor (BDNF), Neurotrophin-3 (NT-3) and Neurotrophin-4/5 (NT-4/5). NTs act as dimmers on target cells by means of their interaction with transmembrane receptors, activating a set of signaling cascades and modulating the development and maintenance of the vertebrate nervous system. Each NT binds specifically to a tyrosine kinase Trk (tropomyosin-related kinase) receptor (NGF binds to TrkA, BDNF to TrkB, NT-3 mainly to TrkC and NT-4/5 also to TrkB), while all of them can bind, with similar affinity, to the p75 Neurotrophin Receptor (p75NTR). The role of Trk receptors in neuronal survival, differentiation and function is well-defined, including activation of signaling pathways (PI3K-

Abbreviations: BDNF, Brain-Derived Neurotrophic Factor; Cer, ceramide; CGN, cultured granule neurons; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NGF, Nerve Growth Factor; nSMase, neutral sphingomyelinase; NT, neurotrophins; SM, sphingomyelin; SphK, sphingosine kinase ⇑ Corresponding authors at: Department of Biochemistry and Molecular Biology, School of Medicine, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, 08193 Barcelona, Spain. Fax: +34 93 581 1573. E-mail addresses: [email protected] (J. Aguilera), [email protected] (C. Gil). 1 Contributed equally to the work.

Akt, ras-ERK-1/2 and PLCc-PKC) [See [1] for a review]. In contrast, the function mode of p75NTR has not been clearly defined. p75NTR is included in the Tumor Necrosis Factor Receptor (TNFR) superfamily [2], whose members lack intrinsic enzymatic activity, and signaling occurs through association with adaptor proteins to mediate diverse cellular functions, such as apoptosis, survival or axonal growth, depending on the cellular context. Thus, the p75NTR intracellular domain (ICD) contains several regions likely to mediate interactions with downstream signaling elements [3]. These proteins have been largely isolated using yeast two-hybrid screening and include NRAGE (neurotrophin-interacting MAGE homolog) [4], NADE (p75NTR associated cell-death executor), TRAF (TNF receptor-associated factor) proteins [5] and NRIF 1 and 2 (Neurotrophin receptor interacting factor) [6]. Additionally, the extracellular domain of p75NTR can interact with other membrane proteins, as is the case of the aforementioned Trk receptors [7], altering Trk docking subdomains [8]. An ability of p75NTR, which is shared with some members of the TNFR family, is the induction of sphingomyelin hydrolysis and the increase of cellular ceramide levels, after the binding of its ligands [9], the aforementioned hydrolysis being a consequence of sphingomyelinase activity promotion [10]. It has been demonstrated that ceramide, and its related metabolites, acts as a second messenger, being involved in apoptosis [11] as well as in cell survival

0014-5793/$36.00 Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2013.11.032

168

A. Candalija et al. / FEBS Letters 588 (2014) 167–174

[12], through the activation, on the one hand, of kinases such as JNK [13] or ERK-1/2 [14] or, on the other hand, of phosphatases PP1 and PP2A [15]. Several enzymes with SMase activity have been described, the neutral (nSMase) and the acid sphingomyelinases (aSMase) being the most studied [16]. Despite extensive studies, the precise cellular function of each of these sphingomyelinases in sphingomyelin turnover and in the regulation of ceramide-mediated responses is not well understood. In mammals, three Mg2+dependent neutral SMases have been identified (nSM1, nSM2 and nSM3). Among the three enzymes, nSMase2 is the most studied and has been involved in some physiological responses including apoptosis, development and inflammation [17]. In addition, recent studies have also supported a role for SMase activity in membrane trafficking, suggesting that nSMase2 may act in the endosomal compartments giving rise to multivesicular endosomes through formation of intravesicular membranes [18]. Recently, the existence if a ‘‘sphingolipid rheostat’’ has been postulated [19], a mechanism based on the fact that sphingosine-1-phosphate and ceramide are biologically interconvertible lipids [20], determining their relative levels the cell fate (life or death) [21]. Moreover, a prominent role of SMase activity in endocytosis has also been extensively postulated, in physiological conditions [22] as well as in pathogen invasion [23]. Another p75NTR-dependent signaling event is Akt phosphorylation, which occurs in a variety of cell types in a TrkA-independent manner [24]. Moreover, p75NTR expression increased survival in cells exposed to stress, indicating that p75NTR facilitates cell survival through novel signaling cascades involving Akt activation [24]. Apart from the synergic action of Trk and p75NTR, published evidences also suggest an inhibitory activity of TrkA on p75NTRmediated signaling, in some cases. An example supporting this hypothesis is the cross-talk between p75NTR and the TrkA signaling pathway demonstrated in the case of the sphingomyelin hydrolysis induced by NGF [25]. Thus, while co-expression of TrkA with p75NTR abolished p75NTR-dependent sphingomyelin hydrolysis, inhibition of TrkA activity with K252a enhanced p75NTR-mediated sphingomyelin hydrolysis in PC12 cells, thus further supporting the modulatory role of TrkA on p75NTR signaling [25]. Additionally, p75NTR can activate NFkB signaling, the time-course of NF-kB activation being very slow in PC12 cells (approx. 1 h), and probably due to the modulatory activity of TrkA on p75NTR signaling [26]. In accordance with this, it has been demonstrated that in rat Schwanoma cells, which express p75NTR but not TrkA, activation of NFkB occurs within 30 min [27], more rapidly than in PC12 cells [26]. Also, in the case of the proneurotrophin proNGF, a cross-talk mechanism between neurotrophin receptors has been described. ProNGF induces an inhibitory effect of p75NTR on TrkB signaling through the activation of PTEN (phosphatase and tensin homolog delete on chromosome 10) phosphatase, causing abrogation of the Akt pathway [28]. In accordance with the aforementioned literature, we have studied the use of nSMase by both TrkA and TrkB receptors to yield cell survival.

addition of NGF 50 ng/ml (Sigma–Aldrich) for seven days, with change of medium at DIV3. BDNF was also purchased from Sigma–Aldrich, while the nSMase inhibitor GW4869 was obtained from Calbiochem. For siRNA knockdown assays, PC12 cells were cultured for 24 h, and siRNA was transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cells were transfected with functional, non-targeting control siRNA (All Stars Negative Control from Qiagen) or a mix of four pre-designed nSMase2-specific Flexitube siRNAs against the smpd3 gen (Qiagen). For each well, 20 pmol of each siRNA was added. After 15 min at room temperature, lipid and the siRNA mixture was added to each well containing cells. Cells were then incubated for an additional 72 h prior to the experiments. Decrease of the nSMase-2 protein expression was assessed by means of Western blot using a monoclonal antibody raised against amino acids 461–655 mapping at the C-terminus of nSM2 of human origin (Santa Cruz, antibody G-6). This region shows very little homology with nSM1 and nSM3. 2.2. Measurement of cell viability Cell viability was determined by using the conventional 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. In the MTT assay, the viable cells convert cell-permeable soluble dye MTT to insoluble blue formazan crystals and this reaction is catalyzed by the succinate dehydrogenase, a mitochondrial respiratory-chain enzyme easily inactivated by oxidative stress. After incubation with the indicated chemicals, cells were treated with MTT solution (1 mg/ml final concentration) for 2 h at 37 °C. The dark blue formazan crystals formed inside the intact mitochondria were solubilized with dimethylsulfoxide, and the absorbance of the blue color was measured at 570 nm using a microplate reader (TECAN GmbH; Salzburg, Austria). 2.3. Hoechst staining assay PC12 cells were seeded in 24-well plates at 5
104 cells/well and treated with hoechst fluorochrome (100 lg/mL) for 5 min at 37 °C in dark. Hoechst-stained nuclei were observed by using a fluorescence microscope (Nikon, Japan) at an excitation wavelength of 350 nm and an emission wavelength of 460 nm. A total of 100 cells from 30 random high power fields were counted. The percentage of apoptosis was expressed as ratio of apoptotic cells to total cells. 2.4. Measurement of caspase-3 activity The activity of caspase-3 like protease in the lysate was measured using a colorimetric caspase-3 assay. In brief, the reaction mixture (total volume, 100 lL) contained 25 lL of cell lysate and 50 lL of caspase-3 substrate acetyl-Asp-Glu-Val-Asp-AMC (final concentration, 200 lM) in assay buffer, and the assay was carried out in 96-well plate. Mixtures were incubated for 5 h at 37 °C and the absorbance was read at 405 nm. Each caspase-3 activity was expressed as percentage respect to non-deprived cells.

2. Materials and methods 2.5. Sphingomyelinase activity analysis 2.1. Cellular cultures Cerebellar granule neuron (CGN) cultures were prepared as described [42]. PC12 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% horse serum, 5% fetal calf serum, 50 U/ml penicillin and 50 lg/ml streptomycin, at 37 °C in a humidified 5% CO2 atmosphere. For experiments, cells were seeded at a density of 7  104 cells/cm2 in plates coated with 10 lg/ml poly-L-lysine. Cells were left overnight before starting experimental treatments. PC12 differentiation was achieved by

After treatment, cells were harvested with 500 ll of 1  reaction buffer (Tris–HCl 0.1 M, pH 7.4, 10 mM MgCl2, 1% Triton X-100 and protease/phosphatase inhibitors), sonicated and centrifuged at 9000g for 30 min at 4 °C. The supernatant fraction was used for neutral sphingomyelinase (nSMase) activity determination using the Amplex Red Sphingomyelinase Assay Kit, according to the manufacturer’s instructions (Molecular Probes, Invitrogen). Fluorescence was determined with a Series BioAssay Reader (Perkin Elmer Instruments; Waltham, MA, USA).

169

A. Candalija et al. / FEBS Letters 588 (2014) 167–174

3. Results 3.1. nSMase activity is essential for the prosurvival effect of neurotrophins in CGN and in PC12 cells CGN survival is dependent on the membrane potential influenced by the potassium concentration in the medium. Thus, a high potassium concentration in the medium (25 mM, indicated as K25) is necessary to maintain the cultures, but neuronal death occurs in the medium containing low potassium concentrations (5 mM, indicated as K5), even in the presence of serum. Thus, the previous results showing the nSMase dependence of prosurvival signaling, such as Akt, were corroborated by measuring neuronal viability by means of the MTT reduction assay (Fig. 1A). Potassium withdrawal for 24 h induces the loss of CGN viability, which is highly restored by BDNF, but not by NGF. The prosurvival effect exerted by BDNF is abolished when cells are pretreated with GW4869, a nSMase inhibitor. Moreover, the slight but significant additional decrease observed in MTT reduction due to GW4869 alone is interesting, pointing to a role of basal nSMase activity in cellular viability. Additionally, experiments of cell survival in PC12 inducing cell death by means of serum deprivation show an absence of prosurvival activity of BDNF, as expected due to the lack of TrkB receptors, but a clear effect of NGF, which is also totally dependent on nSMase activity (Fig. 1B). Again, an additional decrease observed in the MTT reduction due to GW4869 alone is observed, as in CGN (Fig. 1A). In order to corroborate the role of nSMase activity in neurotrophin-caused cell protection, the expression of nSMase-2 (nSM2) in PC12 cells was abrogated by means of a siRNA protocol. Western blots of extracts from cells transfected with siRNA

200 ***

MTT % vs v K5

After treatment, cells were resuspended in 0.5 ml of homogenization buffer containing 20 mM Tris/HCl (pH 7.5), 2 mM EDTA, 0.5 mM EGTA, 2 mM DTT, 1 mM Na3VO4, 50 mM NaF, 2 mM PMSF, 10 lg/ml leupeptin and 25 lg/ml aprotinin and disrupted by sonication in a Dynatech Sonic Dismembrator. Each sample was analyzed by SDS/PAGE and Western blotting. The separated proteins were transferred to Protran nitrocellulose membrane (Schleicher and Schuell; Dassel, Germany), using a Mini TransBlot Cell 3 (Bio-Rad; Hercules, CA, USA) at 100 V for 1 h. The blotting buffer contained 25 mM Tris, 200 mM glycine and 10% (v/v) methanol. The membrane filters were blocked for 1 h with Tris-buffered saline, supplemented with 0.1% Tween 20 and 5% (w/v) defatted powdered milk. Then the membranes were incubated overnight with the corresponding antibody diluted in blocking buffer. Antibodies against actin and tubulin were purchased by Sigma–Aldrich. Antibodies against phS473Akt, phT308Akt, total Akt, phT202/Y204 ERK-1/2, total ERK-1/2 and php38 were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibody against TrkA was from Upstate Biotechnology (Lake Placid, NY, USA). Antibodies against TrkB and against phY705 Trk were from Abcam (Cambridge, UK). Next, the membrane filters were incubated for 1 h with a secondary antibody conjugated with horseradish peroxidase diluted in blocking buffer. Several washes with Tris-buffered saline/0.1% Tween 20 were performed between all of the steps. The Western blots were developed using detection reagents from Amersham Pharmacia Biotech (Little Chalfont, Buckinghamshire, UK) and visualized using a GeneGnome HR chemiluminescence detection system coupled to a CCD camera (Syngene; Cambridge, UK). When required, quantification of the bands was performed with the GeneTools program (Syngene; Cambridge, UK), which is able to detect saturated signals. Only non-saturated signals were used for quantification purposes.

(A) CGN

**

150

*

100 50 0

-

K25

BDNF GW BDNF NGF +GW

K5

(B) PC12 MTT % vss deprived

2.6. Western blot analysis

***

200 *** 150 100

**

**

50 0

C

-

BD BDNF NGF NGF GW NF +GW +GW

Serum deprived Fig. 1. Neurotrophin-dependent protection of CGN and of PC12 in front of cell death depends on nSMase activity. (A) CGN cells were grown for seven DIV in 25 mM KCl medium. BDNF or NGF (50 ng/ml both) was added 30 min before switching the medium from a medium with 25 mM KCl (K25) to a medium with 5 mM KCl (K5), and maintained for 24 h. The nSMase activity was inhibited, where indicated, by adding GW4869 10 lM 1 h before BDNF. After this time, cell viability was estimated by the MTT assay, as stated in Section 2. ⁄P < 0.05, ⁄⁄P < 0.01 and ⁄⁄⁄P < 0.001, when compared with neurons switched to K5 medium, using one-way ANOVA, followed by Bonferroni’s post hoc test. (B) PC12 cells were seeded overnight, and BDNF or NGF (50 ng/ml both) was added 30 min before switching the medium to a serumfree medium, and maintained for 24 h. The nSMase activity was inhibited by adding GW4869 10 lM 1 h before the addition of each neurotrophin. Cell viability was estimated by the MTT assay. ⁄⁄P < 0.01 and ⁄⁄⁄P < 0.001, when compared with cells deprived and without any additional treatment, using one-way ANOVA, followed by the Bonferroni’s post hoc test.

targeting the smpd3 gene show a clear decrease in nSM2 protein (Fig. 2A). A faint band of slightly higher molecular weight remains in the siRNA treated cells, which probably corresponds to an unspecific detection (Fig. 2A). Then, the same kinds of experiments shown in the Fig. 1B were conducted. Results show that abrogation of nSM2 expression in PC12 cells impairs the induction of viability under deprivation stress induced by NGF (Fig. 2B), thus confirming the important role of nSMase activity in NGF-induced viability of PC12 cells. In order to corroborate the results shown in Fig. 2, caspase 3 activity and nuclei condensation were assessed, since serum deprivation is described to cause apoptotic death. The silencing of nSM2 protein with siRNA renders NGF unable to reverse the caspase 3 activity induced by serum withdrawal (Fig. 3A). Moreover, the determination of nuclear condensation with Hoechst staining reinforces the results obtained in the section A, since the results show that NGF action on the prevention of apoptotic nuclei is abolished by nSM2 silencing (Fig. 3B and C). In summary, nSM2 is essential in the NGF-induced protection against serum deprivation. 3.2. TrkB-induced Akt signaling in CGN strongly depends on nSMase In order to further study the role of nSMase in prosurvival events, cultures of primary cultures of granule neurons (CGN) were

A. Candalija et al. / FEBS Letters 588 (2014) 167–174

Transfected

(A)

NT Csi siRNA

Caspase 3 act. % vs ND C

170

(A) 72 kDa

nSM2 acn

(B) MTT % vs ND

125

**

**

300

150 100 50

ND

75 50 25

+ + + Control nSM2 Non siRNA transfected siRNA Serum deprived

+

-

+

Control Non transfected siRNA

-

+

NGF

nSM2 siRNA

Serum deprived Fig. 2. The effect of siRNA knockdown of nSM2 expression on NGF-induced viability of PC12 cells under serum deprivation stress. (A) Short interfering RNAs were used to reduce cellular nSM2 levels (siRNA), while a non-silencing siRNA was used as control (Control siRNA, Csi). Western blots were used to assess the reduction of nSM2 levels. Non-transfected cells were also analysed (NT). Actin was assessed as loading control. (B) Treatment of PC12 cells with siRNA against nSM2 markedly decreased NGF-induced viability of the cells that were deprived of serum, as determined by MTT assay (ND, non-deprived). Each histogram is the average ± S.D. from three independent experiments. ⁄⁄P < 0.01, using one-way ANOVA, followed by the Bonferroni’s post hoc test.

% apoptocc nuclei

(B) -

*** 25

3.3. NGF induces ceramide production only when Trk receptor activity is not present The p75NTR receptor exerts its functions by the binding of adaptor proteins and subsequent enhancement of enzymatic activities, SMase activity being one of them, leading to the increase of the ceramide/sphingomyelin (Cer/SM) ratio [9]. In consequence, after the observation that neurotrophin-promoted survival depends on nSMase, the activation of nSMase activity by NGF was assessed. Treatment of CGN (a TrkA /TrkB+/p75NTR+ system) with NGF induces nSMase activity, but not with BDNF (Fig. 5A). Treatment with GW4869 alone induces a decrease in the basal nSMase activity.

NGF

*** ***

20 15 10 5

ND

+ + Control Non transfected siRNA

+ nSM2 siRNA

NGF

Serum deprived (C)

used, which express p75NTR, as well as TrkB and, to a lesser extent, TrkC, but not TrkA [29]. When phosphorylation of Akt in Ser 473 and in Thr 308 in CGN is detected in CGN by Western blot, no enhancing after NGF treatment (50 ng/mL, 15 min) is observed, but rather a strong increase after BDNF (50 ng/mL, 15 min) is detected. This increase is highly dependent on basal nSMase activity, since GW4869 abolishes Akt phosphorylation in both residues induced by BDNF (Fig. 4A and B). This result is in strong agreement with the nSMase dependence of the BDNF-induced cell viability shown in Fig. 1A. In parallel, it is observed that NGF does not induce ERK-1/2 phosphorylation in residues Thr202 and Tyr204, but GW4869 alone induces a significant increase in the phosphoERK-1/2 signal, indicating that the basal nSMase activity, or nSMase-dependent mechanisms, silences the ERK-1/2 pathway. On the contrary, BDNF triggers ERK-1/2 phosphorylation, which is not significantly impaired by GW4869 treatment (Fig. 4C and D). Regarding phosphorylation of p38 in Thr180 and Tyr182, NGF has no effect, but BDNF does induce a moderate but consistent increase, which is nSMase-independent, since GW4869 has no effect on p38 phosphorylation (Fig. 4E and F).

ns

200

100

ND

*

250

NT-

Csi-

Nsi-

NT NT+

Csi+

Nsi Nsi+

ND

Fig. 3. The effect of siRNA knockdown of nSM2 expression on NGF-induced prevention of apoptotic events in PC12 cells under serum deprivation stress. (A) nSM2 protein has a role in the NGF-induced negative control of caspase 3 activity induced by serum deprivation. ⁄P < 0.05, ⁄⁄P < 0.01, ns: non-significant, using oneway ANOVA, followed by the Bonferroni’s post hoc test. (ND, non-deprived). (B) Condensed nuclei were visualized by means of Hoechst staining and the percentages of apoptotic nuclei were measured. The results show that nSM2 clearly participates in the NGF-dependent prevention of the apoptotic death caused by serum deprivation. ⁄⁄⁄P < 0.001, using one-way ANOVA, followed by the Bonferroni’s post hoc test. (C) Representative images of the results shown in B. White arrowheads show condensed nuclei. (ND, non-deprived; NT , non-transfected, without NGF; NT+, non-transfected, with NGF; CSi , control siRNA, without NGF; CSi+, control siRNA, with NGF; Nsi , nSM2 siRNA, without NGF; Nsi+, nSM2 siRNA, with NGF).

Action of BDNF on TrkB is monitored by means of detection of Trk receptors phosphorylated in tyrosine 705 (Fig. 5A). On the other hand, the Cer/SM ratio after acute NGF treatment was determined in NGF-differentiated PC12 cells (a TrkA+/TrkB /p75NTR+ system) for seven DIV or in an alternative condition without NGF differentiation. The results show that cells respond to acute NGF treatment, in terms of an increase in the Cer/SM ratio, only when PC12 cells are NGF-differentiated. The absence of functional TrkA receptor in the NGF-differentiated cells is corroborated by means of Western blot against Trk phosphorylated in tyrosine 705, which is related to Trk receptor activation (Fig. 5B). Thus, neurotrophins induce cell viability through Trk receptors in a manner depending on basal nSMase activity but not through promotion of nSMase. On

171

(B)

phS473 hS473 Akt

phT308 Akt Akt

(C) phERK-1/2 ERK-1/2

(E)

(F)

php38 Tubulin NGF

-

+

+

-

-

-

BDNF

-

-

-

+

+

-

GW

-

-

+

-

+

+

4

hAkt phAkt 473 308

2

6 phERK 4 2

Phospho (a.u.)

(D)

Phospho (a.u..)

(A)

Phospho (a.u u.)

A. Candalija et al. / FEBS Letters 588 (2014) 167–174

4 3 2 1

php38

NGF

-

+

+

-

-

-

BDNF

-

-

-

+

+

-

GW

-

-

+

-

+

+

Fig. 4. Neurotrophin-dependent Akt and ERK-1/2 signaling in cultured granule neurons (CGN) depend on nSMase. CGN cells were grown for seven DIV in 25 mM KCl medium. To decrease signaling, cells were switched from a medium with 25 mM KCl to a medium with 5 mM KCl without serum for 1 h. When indicated, BDNF or NGF (50 ng/ml both) were added for 15 min. When indicated, GW4869 10 lM was added 1 h before neurotrophin. (A) After treatment, cell homogenates were resolved in SDS–PAGE (15 lg of protein per lane) and phosphorylated Akt in Ser 473 and Thr 308 was detected by Western blot, in parallel with total Akt. (B) Phosphorylation levels were assessed by image densitometry and shown as bar graphics (a.u., arbitrary units). The means ± S.D. of three experiments are represented. Phospho ERK-1/2 (C and D) and phospho-p38 (E and F) were also detected using phosphospecific antibodies, in parallel with total ERK-1/2 and tubulin as loading control.

(A)

(B)

3.4. nSM2 knockdown impairs basal Akt phosphorylation, but not ERK-1/2 phosphorylation As demonstrated above, neurotrophins need nSMase activity to induce prosurvival events, but without any enhancing of this enzymatic activity. In consequence, we aimed to determine whether basal nSMase activity in PC12 is implicated in Akt phosphorylation. Western blots of extracts from naive, i.e. non-deprived, PC12 cells transfected with siRNA targeting the smpd3 gene show a clear decrease in nSM2 protein (Fig. 6A, siRNA lanes), in comparison with non-transfected (Fig. 6A, NT lanes) or transfected cells with control siRNA (Fig. 6A, Csi lanes). Results show that abrogation of nSM2 expression in PC12 cells impairs basal phosphorylation of Akt in Ser473 and Thr308, but has no effect on ERK-1/2 phosphorylation, as can be also observed in the quantification of the signals (Fig. 6B). These results demonstrate that, in unstimulated PC12 cells, Akt phosphorylation highly depends on basal nSM2.

Fig. 5. Absence of Trk activity correlates with an increase of ceramide levels promoted by NGF. (A) CGNs were incubated with 50 ng/ml NGF, with or without cotreatment with GW4869, a specific nSMase inhibitor. Alternatively, CGN were also treated with 50 ng/ml BDNF. SMase activity was measured at pH 7.5 using the Amplex Red Sphingomyelinase Assay Kit in each case. Each point represents the mean ± S.D. of three independent experiments. Values were analyzed using oneway ANOVA, followed by Dunnett’s post hoc test. ⁄⁄⁄P < 0.001. In parallel, the activity of Trk receptors was monitored in the different treatments, using a phosphospecific antibody against phosphoTyr 705, as well as total levels of TrkB protein. (B) The ceramide/sphingomyelin ratio (Cer/SM) was assessed, as stated in the Section 2, after acute NGF treatment for 1 h, in both NGF-differentiated and in non-differentiated PC12 cells. An increase was detected only in NGF-differentiated cells. The data shown are the mean ± S.D. of three experiments. The values were analyzed by one-way ANOVA followed by the Bonferroni post hoc test to compare NGF-treated cells, with respect to control. ⁄⁄P < 0.01. In parallel, the activity of Trk receptors was monitored in the different treatments, using a phosphospecific antibody against phosphoTyr 705, as well as total levels of TrkA protein.

the other hand, we observe that activated Trk receptor correlates with prevention of the p75NTR-dependent nSMase activity (Fig. 5B).

4. Discussion The differential effects of neurotrophins on their target cells rely on a complex array of events, including the balance between mature and immature neurotrophins and the proportion of Trk to p75NTR or other co-receptors. In accordance with this, published evidences suggest an inhibitory activity of Trk receptors on p75NTR-mediated signaling. A relevant example supporting this scenario is the NGF-mediated induction of sphingomyelin hydrolysis in a p75NTR+/TrkA system, which is abolished by co-expression of TrkA [25]. On the contrary, in the same work, it is shown that the inhibition of TrkA activity with K252a enhances p75NTRmediated sphingomyelin hydrolysis [25]. Another result supporting the inhibition of p75NTR signaling by TrkA is the observation that TrkA mediates developmental sympathetic neuron survival in vivo by silencing an ongoing p75NTR-mediated death signal

172

A. Candalija et al. / FEBS Letters 588 (2014) 167–174

(A) PC12

Transfected NT Csi siRNA

nSM2 phT308 Akt phS473 Akt Akt phERK-1/2 ERK-1/2

Phospho (% of o NT)

(B) 125

***

ns

***

ns

ns

100 75 50 25

phS473 Akt

phERK 1/2 phT308 phERK-1/2 Akt

Fig. 6. Basal Akt signaling, but not ERK-1/2 signaling, depend on nSMase in PC12 cells. (A) Short interfering RNAs were used to reduce cellular nSM2 levels (siRNA), while a non-silencing siRNA was used as control (Control siRNA, Csi). Western blots were used to assess the reduction of nSM2 levels. Non-transfected cells were also analysed (NT). After knockdown treatment, cell homogenates were resolved in SDS–PAGE (15 lg of protein per lane) and phosphorylated Akt in Ser 473 and Thr 308, as well as phosphorylated ERK-1/2, were detected by Western blot, in parallel with total Akt and ERK-1/2. (B) Phosphorylation levels were assessed by image densitometry and shown as bar graphics (showing percentage respect to NT cells). Each point represents the mean ± S.D. of three independent experiments. Values were analyzed using one-way ANOVA, followed by Dunnett’s post hoc test. ⁄⁄⁄ P < 0.001.

[29]. Thanks to studies in PC12nnr5, cells devoid of Trk receptors, a differential cross-regulation of TrkA and of TrkC receptors with p75NTR has been described, since, in the absence of Trk activation, the expression of TrkC is permissive of trophic and differentiation signals induced by p75NTR ligands, which are abolished by the expression of TrkA [30]. This is in accordance with the observation that, in contrast to TrkA signaling, p75NTR had a negative effect on TrkB tyrosine autophosphorylation in response to BDNF or NT-4/5. On the other hand, p75NTR did not influence the Ras/mitogen-activated protein kinase signaling pathway in BDNF, NT-3, or NT-4/5 treatments [31]. These results also point to differential signaling pathways, and thus to a different array of signaling proteins, used by each Trk receptor, in spite of the fact that all of the Trk receptors share a high homology in sequence and the structure of the intracellular domain. Another signaling event involved in NT receptor cross-talk which can be p75NTR-dependent is Akt phosphorylation, as has been described in human Niemann-Pick fibroblasts, which lack acidic SMase activity, but not nSMase activity. Authors found p75NTR-dependent Akt phosphorylation as being independent of TrkA signaling and depending on PI3-kinase. Moreover, p75NTR expression increased survival in cells exposed to staurosporine or subjected to serum withdrawal. These findings indicate that p75NTR can facilitate cell survival through signaling cascades that result in Akt activation [24], although no

implication of SMase activity in this facilitation was described by the authors. Regarding the role of PI3-kinase in NT receptor cross-talk, it has been described that this enzyme regulates the inhibitory signaling between TrkA and p75NTR-dependent sphingolipid signaling pathways, and the authors demonstrated the presence of NGF-dependent interaction between PI3K and acid SMase localized in caveolae [32]. The same authors described that caveolae-located TrkA inhibition exists, which is in agreement with the interaction between TrkA and caveolin and correlates with an increased ability of NGF to induce sphingomyelin hydrolysis through p75NTR [33]. The association of nSM2 with the plasma membrane, as well as with Golgi and recycling compartments, correlates with the functional link between p75NTR and SMase activity [34]. The mechanism of SMase activation by p75NTR is unknown, although a protein related to SMase activation by means of TNFRI has been described, called FAN, from factor associated with nSMase activation. FAN is responsible for the stimulation of nSMase through its binding to a TNFRI region designated NSD (neutral SMase activation domain) [35]. FAN is also responsible for TNF-induced caspase-3-dependent apoptosis [36], but if this protein is involved in NT-induced nSMase activity is unknown. Another protein that is involved in the interaction between nSM2 and the activating receptor is EED (embryonic ectodermal development) [37], while calcineurin binds constitutively to nSM2, acting as a sensor of the basal activity of nSM2 [38]. Regarding the mechanism that could explain the functional antagonism between TrkA and p75NTR, the competition by these two receptors for the downstream signaling protein p62, also known as sequestosome-1 or SQSTM1, has been proposed. The presence or association of TrkA with p75NTR may compete for p62, a protein that interacts with PKC members of the atypical family, which is a critical signaling scaffold for the NF-kB activation pathway [39]. Adaptor protein p62 has been linked to the extrinsic apoptosis pathway, placing p62 at critical decision points that control cell death and survival [40]. Pointing also to NT receptors cross-talk, but this time in the contrary sense, it has been reported that prolonged treatment (3 h) of PC12 cells with C2-ceramide induces dimerization and tyrosine phosphorylation of TrkA receptors in the absence of NGF [41]. The increase in ceramide levels is supposed to change membrane configuration, increasing the content in ceramide platforms, which are considered to be a subset of the so-called ‘lipid rafts’, which are membrane microdomains rich in cholesterol and sphingolipids. Recently, it has been demonstrated that the fragment Hc from tetanus toxin induces the formation of ceramide platforms and protects cells from oxidative stress in a nSMase-dependent manner [42]. Regarding the role of SMase activity in cytoprotective outcome, some metabolites derived from sphingolipid processing have been involved in Akt signaling, pointing to S1P as a crucial effector molecule in some events leading to Akt activation [43]. Moreover, studies in hepatocytes showed that TNFa-induced activations of PI3K and Akt were inhibited by N,N-dimethylsphingosine, an inhibitor of sphingosine kinase (SphK), indicating that NF-kB, S1P and PI3K/Akt are involved in the signaling that leads to protection of human hepatocytes from the apoptotic action of TNFa [44]. In any case, it is clear that, in our system, TrkBenhanced Akt phosphorylation is highly dependent on basal nSMase activity, since no nSMase enhancement is observed in CGN by BDNF, as seen in Fig. 4B. On the other hand, nSMase blocks the basal ERK-1/2 pathway but has little effect on the BDNF-enhanced ERK-1/2 pathway (Fig. 5). The regulation of neurotrophin-triggered Akt and ERK-1/2 pathways has been related to the protease TACE (TNFa-converting enzyme) action on p75NTR, since the generation of a proteolysis fragment from p75NTR due to TACE has been described as necessary for Akt and ERK-1/2 activation [45]. The breakdown of plasma membrane sphingomyelin

A. Candalija et al. / FEBS Letters 588 (2014) 167–174

or enrichment of the plasma membrane with ceramide increases proteolysis (in a mechanism also known as ‘ectodomain shedding’) of another TACE substrate, such as the interleukin-6 receptor (IL-6R) [46]. Thus, according to published data, a model in which the nSMase-activated TACE contributes to Akt activation arises, which is in agreement with the nSMase-dependent phosphorylation of Akt exerted by the BDNF action in CGN, shown in the present work, as well as in previous work from our group [42]. Acknowledgments The excellent technical assistance of Cristina Gutierrez in cell culture is gratefully acknowledged. We also thank to C. Simmons for help with the English language. This work was partially supported by SAF2009-13626 from the Ministerio de Ciencia e Innovación from the Spanish Government. References [1] Skaper, S.D. (2012) The neurotrophin family of neurotrophic factors: an overview. Methods Mol Biol 846, 1–12. [2] Reichardt, L.F. (2006) Neurotrophin-regulated signalling pathways. Philos Trans R Soc Lond B Biol Sci 361, 1545–1564. [3] Roux, P.P. and Barker, P.A. (2002) Neurotrophin signaling through the p75 neurotrophin receptor. Prog Neurobiol 67, 203–233. [4] Bertrand, J.M., Kenchappa, R.S., Andrieu, D., Leclercq-Smekens, M., Nguyen, H.N.T., Carter, B.D., Muscatelli, F., Barker, P.A. and De Backer, O. (2008) NRAGE, a p75NTR adaptor protein, is required for developmental apoptosis in vivo. Cell Death Differ 15, 1921–1929. [5] Mukai, J., Hachiya, T., Shoji-Hoshino, S., Kimura, M.T., Nadano, D., Suvanto, P., Hanaoka, T., Li, Y., Irie, S., Greene, L.A. and Sato, T.A. (2000) NADE, a p75NTRassociated cell death executor, is involved in signal transduction mediated by the common neurotrophin receptor p75NTR. J Biol Chem 275, 17566–17570. [6] Linggi, M.S., Burke, T.L., Williams, B.B., Harrington, A., Kraemer, R., Hempstead, B.L., Yoon, S.O. and Carter, B.D. (2005) Neurotrophin receptor interacting factor (NRIF) is an essential mediator of apoptotic signaling by the p75 neurotrophin receptor. J Biol Chem 280, 13801–13808. [7] Bibel, M., Hoppe, E. and Barde, Y. (1999) Biochemical and functional interactions between the neurotrophin receptors trk and p75NTR. EMBO J 18, 616–622. [8] Zaccaro, M.C., Ivanisevic, L., Perez, P., Meakin, S.O. and Saragovi, H.U. (2001) P75 co-receptors regulate ligand-dependent and ligand-independent Trk receptor activation, in part by altering Trk docking subdomains. J Biol Chem 276, 31023–31029. [9] Dobrowsky, R.T. and Carter, B.D. (1998) Coupling of the p75 neurotrophin receptor to sphingolipid signaling. Ann NY Acad Sci 845, 32–45. [10] Brann, A.B., Tcherpakov, M., Williams, I.M., Futerman, A.H. and Fainzilber, M. (2002) Nerve growth factor-induced p75-mediated death of cultured hippocampal neurons is age-dependent and transduced through ceramide generated by neutral sphingomyelinase. J Biol Chem 277, 9812–9818. [11] Ruvolo, P.P. (2003) Intracellular signal transduction pathways activated by ceramide and its metabolites. Pharm Res 47, 383–392. [12] Plummer, G., Perreault, K.R., Holmes, C.F.B. and Posse de Chaves, E.I. (2005) Activation of serine/threonine protein phosphatase-1 is required for ceramide-induced survival of sympathetic neurons. Biochem J 385, 685–693. [13] Brenner, B., Koppenhoefer, U., Weinstock, C., Linderkamp, O., Lang, F. and Gulbins, E. (1997) Fas- or ceramide-induced apoptosis is mediated by a Rac1regulated activation of Jun N-terminal kinase/p38 kinases and GADD153. J Biol Chem 272, 22173–22181. [14] Zhang, Y., Yao, B., Delikat, S., Bayoumy, S., Lin, X., Basu, S., McGinley, M., ChanHui, P., Lichenstein, H. and Kolesnick, R. (1997) Kinase suppressor of Ras Is ceramide-activated protein kinase. Cell 89, 63–72. [15] Mukhopadhyay, A., Saddoughi, S.A., Song, P., Sultan, I., Ponnusamy, S., Senkal, C.E., Snook, C.F., Arnold, H.K., Sears, R.C., Hannun, Y.A. and Ogretmen, B. (2009) Direct interaction between the inhibitor 2 and ceramide via sphingolipidprotein binding is involved in the regulation of protein phosphatase 2A activity and signaling. FASEB J 23, 751–763. [16] Samet, D. and Barenholz, Y. (1999) Characterization of acidic and neutral sphingomyelinase activities in crude extracts of HL-60 cells. Chem Phys Lipids 102, 65–77. [17] Wu, B.X., Clarke, C.J. and Hannun, Y.A. (2010) Mammalian neutral sphingomyelinases: regulation and roles in cell signaling responses. NeuroMol Med 12, 320–330. [18] Trajkovic, K., Hsu, C., Chiantia, S., Rajendran, L., Wenzel, D., Wieland, F., Schwille, P., Brügger, B. and Simons, M. (2008) Ceramide triggers budding of exosome vesicles into multivesicular endosomes. Science 319, 1244–1247. [19] Taniguchi, M., Kitatani, K., Kondo, T., Hashimoto-Nishimura, M., Asano, S., Hayashi, A., Mitsutake, S., Igarashi, Y., Umehara, H., Takeya, H., Kigawa, J. and

[20] [21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

173

Okazak, T. (2012) Regulation of autophagy and its associated cell death by ‘‘sphingolipid rheostat’’. J Biol Chem 287, 39898–39910. Hannun, Y.A. and Obeid, L.M. (2008) Principles of bioactive lipid signalling. Lessons from sphingolipids. Nat Rev Mol Cell Biol 9, 139–150. Bektas, M., Jolly, P.S., Müller, C., Eberle, J., Spiegel, S. and Geilen, C.C. (2005) Sphingosine kinase activity counteracts ceramide-mediated cell death in human melanoma cells. Role of Bcl-2 expression. Oncogene 24, 178–187. Serrano, D., Bhowmick, T., Chadha, R., Garnacho, C. and Muro, S. (2012) Intercellular adhesion molecule 1 engagement modulates sphingomyelinase and ceramide, supporting uptake of drug carriers by the vascular endothelium. Arterioscler Thromb Vasc Biol 32, 1178–1185. Grassmé, H., Riehle, A., Wilker, B. and Gulbins, E. (2005) Rhinoviruses infect human epithelial cells via ceramide-enriched membrane platforms. J Biol Chem 280, 26256–26262. Roux, P.P., Bhakar, A.L., Kennedy, T.E. and Barkeri, P.A. (2001) The p75 neurotrophin receptor activates Akt (protein kinase B) through a phosphatidylinositol 3-kinase-dependent pathway. J Biol Chem 276, 23097– 23104. Dobrowsky, R.T., Jenkins, G.M. and Hannun, Y.A. (1995) Neurotrophins induce sphingomyelin hydrolysis. Modulation by co-expression of p75NTR with Trk receptors. J Biol Chem 270, 22135–22142. Wooten, M.W., Seibenhener, M.L., Zhou, G., Vandenplas, M.L. and Tan, T.H. (1999) Overexpression of atypical PKC in PC12 cells enhances NGFresponsiveness and survival through an NF-kappaB dependent pathway. Cell Death Differ 6, 753–764. Carter, B.D., Kaltschmidt, C., Kaltschmidt, B., Offenhauser, N., Bohm-Matthaei, R., Baeuerle, P.A. and Barde, Y. (1996) Selective activation of NF-kappa B by nerve growth factor through the neurotrophin receptor p75. Science 272, 542– 545. Song, W., Volosin, M., Cragnolini, A.B., Hempstead, B.L. and Friedman, W.J. (2010) ProNGF induces PTEN via p75NTR to suppress Trk-mediated survival signaling in brain neurons. J Neurosci 30, 15608–15615. Majdan, M., Walsh, G.S., Aloyz, R. and Miller, F.D. (2001) TrkA mediates developmental sympathetic neuron survival in vivo by silencing an ongoing p75NTR-mediated death signal. J Cell Biol 155, 1275–1285. Ivanisevic, L., Banerjee, K. and Saragovi, H.U. (2003) Differential crossregulation of TrkA and TrkC tyrosine kinase receptors with p75. Oncogene 22, 5677–5685. Vesa, J., Kruttgen, A. and Shooter, E.M. (2000) P75 reduces TrkB tyrosine autophosphorylation in response to brain-derived neurotrophic factor and neurotrophin 4/5. J Biol Chem 275, 24414–24420. Bilderback, T.R., Gazula, V.R. and Dobrowsky, R.T. (2001) Phosphoinositide 3kinase regulates crosstalk between Trk A tyrosine kinase and p75NTRdependent sphingolipid signaling pathways. J Neurochem 76, 1540–1551. Bilderback, T.R., Gazula, V.R., Lisanti, M.P. and Dobrowsky, R.T. (1999) Caveolin interacts with Trk A and p75NTR and regulates neurotrophin signaling pathways. J Biol Chem 274, 257–263. Milhas, D., Clarke, C.J. and Hannun, Y.A. (2010) Sphingomyelin metabolism at the plasma membrane: implications for bioactive sphingolipids. FEBS Lett 584, 1887–1894. Adam-Klages, S., Adam, D., Wiegmann, K., Struve, S., Kolanus, W., SchneiderMergener, J. and Krönke, M. (1996) FAN, a novel WD-repeat protein, couples the p55 TNF-receptor to neutral sphingomyelinase. Cell 86, 937–947. Ségui, B., Cuvillier, O., Adam-Klages, S., Garcia, V., Malagarie-Cazenave, S., Lévêque, S., Caspar-Bauguil, S., Coudert, J., Salvayre, R., Krönke, M. and Levade, T. (2001) Involvement of FAN in TNF-induced apoptosis. J Clin Invest 108, 143–151. Philipp, S., Puchert, M., Adam-Klages, S., Tchikov, V., Winoto-Morbach, S., Mathieu, S., Deerberg, A., Kolker, L., Marchesini, N., Kabelitz, D., Hannun, Y.A., Schütze, S. and Adam, D. (2010) The polycomb group protein EED couples TNF receptor 1 to neutral sphingomyelinase. Proc Natl Acad Sci USA 107, 1112– 1117. Filosto, S., Fry, W., Knowlton, A.A. and Goldkorn, T. (2010) Neutral sphingomyelinase-2 (nSMase2) is a phosphoprotein regulated by calcineurin (PP2B). J Biol Chem 285, 10213–10222. Wooten, M.W., Seibenhener, M.L., Mamidipudi, V., Diaz-Meco, M.T., Barker, P.A. and Moscat, J. (2001) The atypical protein kinase C-interacting protein p62 is a scaffold for NF-kappaB activation by nerve growth factor. J Biol Chem 276, 7709–7712. Mathew, R., Karp, C.M., Beaudoin, B., Vuong, N., Chen, G., Chen, H., Bray, K., Reddy, A., Bhanot, G., Gelinas, C., DiPaola, R.S., Karantza-Wadsworth, V. and White, E. (2009) Autophagy suppresses tumorigenesis through elimination of p62. Cell 137, 1062–1075. MacPhee, I. and Barker, P.A. (1999) Extended ceramide exposure activates the trkA receptor by increasing receptor homodimer formation. J Neurochem 72, 1423–1430. Cubí, R., Candalija, A., Ortega, A., Gil, C. and Aguilera, J. (2013) Tetanus toxin Hc fragment induces the formation of ceramide platforms and protects neuronal cells against oxidative stress. PLoS ONE 8 (6), e68055. Osawa, Y., Seki, E., Kodama, Y., Suetsugu, A., Miura, K., Adachi, M., Ito, H., Shiratori, Y., Banno, Y., Olefsky, J.M., Nagaki, M., Moriwaki, H., Brenner, D.A. and Seishima, M. (2011) Acid sphingomyelinase regulates glucose and lipid metabolism in hepatocytes through AKT activation and AMP-activated protein kinase suppression. FASEB J 25, 1133–1144. Osawa, Y., Banno, Y., Nagaki, M., Brenner, D.A., Naiki, T., Nozawa, Y., Nakashima, S. and Moriwaki, H. (2001) TNF-a-Induced sphingosine

174

A. Candalija et al. / FEBS Letters 588 (2014) 167–174

1-phosphate inhibits apoptosis through a phosphatidylinositol 3-kinase/Akt pathway in human hepatocytes. J Immunol 167, 173–180. [45] Kommaddi, R.P., Thomas, R., Ceni, C., Daigneault, K. and Barker, P.A. (2011) Trk-dependent ADAM17 activation facilitates neurotrophin survival signaling. FASEB J 25, 2061–2070.

[46] Matthews, V., Schuster, B., Schütze, S., Bussmeyer, I., Ludwig, A., Hundhausen, C., Sadowski, T., Saftig, P., Hartmann, D., Kallen, K.J. and Rose-John, S. (2003) Cellular cholesterol depletion triggers shedding of the human interleukin-6 receptor by ADAM10 and ADAM17 (TACE). J Biol Chem 278, 38829–38839.