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Tissue kallikrein mediates neurite outgrowth through epidermal growth factor receptor and flotillin-2 pathway in vitro
Cellular Signalling 26 (2014) 220–232
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Tissue kallikrein mediates neurite outgrowth through epidermal growth factor receptor and flotillin-2 pathway in vitro Zhengyu Lu a, Mei Cui a, Hong Zhao b, Tao Wang b, Yan Shen c, Qiang Dong a,⁎ a
Department of Neurology, Huashan Hospital, State Key Laboratory of Medical Neurobiology, Fudan University, No.12 Mid. Wulumuqi Road, Shanghai 200040, PR China Department of Neurology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, No.110 Ganhe Road, Shanghai 200437, PR China c Department of Cardiology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, No.110 Ganhe Road, Shanghai 200437, PR China b
a r t i c l e
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Article history: Received 29 July 2013 Received in revised form 8 October 2013 Accepted 31 October 2013 Available online 6 November 2013 Keywords: Tissue kallikrein Neurite outgrowth Epidermal growth factor receptor Flotillin-2 Extracellular signal-regulated kinase
a b s t r a c t Tissue kallikrein (TK) was previously shown to take most of its biological effects through bradykinin receptors. In this study, we assumed that TK mediated neurite outgrowth was independent of bradykinin receptors. To test the hypothesis, we investigated TK-induced neurite outgrowth and its signaling mechanisms in cultured primary neurons and human SH-SY5Y cells. We found that TK stimulation could increase the number of processes and mean process length of primary neurons, which were blocked by epidermal growth factor receptor (EGFR) inhibitor or down-regulation, small interfering RNA for flotillin-2 and extracellular signal-regulated kinase (ERK) 1/2 inhibitor. Moreover, TK-induced neurite outgrowth was associated with EGFR and ERK1/2 activation, which were inhibited by EGFR antagonist or RNA interference and flotillin-2 knockdown. Interestingly, inhibition of bradykinin receptors had no significant effects on EGFR and ERK1/2 phosphorylation. In the present research, our data also suggested that EGFR and flotillin-2 formed constitutive complex that translocated to around the nuclei in the TK stimulation. In sum, our findings provided evidence that TK could promote neurite outgrowth via EGFR, flotillin-2 and ERK1/2 signaling pathway in vitro. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Tissue kallikrein (TK), an important component of the kallikrein– kinin system (KKS), belongs to a subgroup of serine proteinases and processes low molecular weight (LMW) kininogen to release kinin peptide [1,2]. Most of the cellular effects of TK are thought to be mediated by bradykinin (BK), which acts via G protein-coupled B1 and B2 bradykinin receptors (B1R and B2R). However, recent findings revealed that TK could directly activate B2R independent of kininogen and kinin release in cultured CHO cells [3,4]. Moreover, TK was found to trigger the proteinase-activated receptor 1 (PAR1) signaling pathway and epidermal growth factor receptor (EGFR) transactivation in HaCaT keratinocyte cells [5]. These combined information indicated that actions of TK might contribute to other signaling events in addition to BK pathway. EGFR is a transmembrane receptor comprising a family of classical receptor tyrosine kinases that trigger a rich network of signaling
Abbreviations: TK, tissue kallikrein; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; KKS, kallikrein–kinin system; BK, bradykinin; B1R, B1 bradykinin receptor; B2R, B2 bradykinin receptor; PAR, proteinase-activated receptor; Flot, flotillin; DIV, days in vitro. ⁎ Corresponding author. Tel.: +86 21 6248 99991401; fax: +86 21 6248 1401. E-mail address: [email protected] (Q. Dong). 0898-6568/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cellsig.2013.10.010
pathways to control cell survival and growth [6,7]. There are at least 11 endogenous EGFR ligands, each of which is synthesized as precursors and cleaved to generate the mature one. Ligand binding results in multiple phosphorylation events of EGFR, which in many cases are mediated by tyrosine kinases. Simultaneous activation of downstream cascades, such as the mitogen-activated protein kinase (MAPK) pathway, translates in the nucleus into distinct transcriptional programs. Furthermore, previous research showed that lipid rafts were involved in the signaling and/or trafficking of EGFR [8,9]. Reggie-1/flotillin-2 (Flot2) and reggie-2/flotillin-1 (Flot1) are two highly conserved lipid-raft-associated proteins, which were originally discovered as proteins up-regulated in retinal ganglion cells during axon regeneration [10,11]. Flotillins share an SPFH (Stomatin/ Prohibitin/Flotillin/HflK/C) domain at their N-terminus containing residues for myristoylation and palmitoylation and thus membrane anchorage [12,13]. The C-terminus of reggies harbors a unique flotillin domain that is predicted to adopt an alpha-helical coiled-coil structure [10,11]. Even though the function of flotillins has remained elusive and controversial, their widespread expression and conservation imply that these proteins regulate basic cellular processes. Evidence is accumulating that flotillins are involved in the signaling processes of several membrane receptors, phagocytosis, non-clathrin endocytosis, and organization of the actin cytoskeleton [14–20]. They have also been suggested to function as key proteins for neurite outgrowth [21,22]. In
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addition, recent studies have demonstrated that flotillins were modified by EGFR signaling, and that Flot2 became phosphorylated at several tyrosine residues and translocated from plasma membrane into endosomes in the stimulation of EGFR [15]. Although such molecular effects of flotillins have been reported, the role of Flot2 in TK-induced neurite outgrowth has received relatively little attention. Here, we describe a novel neurite growth mechanism whereby TK activates the extracellular signal-regulated kinase (ERK) 1/2 pathway through EGFR and Flot2 independent of BK receptors. 2. Materials and methods 2.1. Materials
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Invitrogen with non-targeting sequence was used as a non-specific control. SH-SY5Y cells were transiently transfected with Lipofectamine 2000 according to the manufacturer's instructions, and experiments were carried out at 48 h after transfection. 2.3. Cell experiment treatment For drug treatment, primary neurons were exposed to TK at 1 μM for 48 h, and SH-SY5Y cells were serum-starved overnight before various concentration of TK (0.25 to 1 μM) were given for 5, 15 or 30 min. To study the involvement of BK receptors on TK effects, B1R antagonist DALBK (1 μM) or B2R antagonist HOE140 (10 μM) was added 1 h prior to TK treatment. TK was inactivated by pre-incubation with fivefold molar excess of aprotinin for 1 h at 37 °C [23]. In some experiments cells were treated with EGFR inhibitor AG1478 (300 nM) or ERK kinase inhibitor PD98059 (10 μM) for 1 h previous to TK stimulation.
TK was from Techpool Bio-Pharma Co. (Guangzhou, China). EGFR, phospho-EGFR (Tyr1068), ERK1/2 (Thr202/Tyr204), phospho-ERK1/2 (Thr202/Tyr204), Flot2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibodies were from Cell Signaling Technology (Beverly, MA, USA). Affinity-purified rabbit anti-microtubuleassociated protein 2 (MAP2) antibody was from Sigma-Aldrich (St. Louis, MO, USA). B1R polyclonal antibody and B2R monoclonal antibody were from Abcam (Cambridge, UK). Secondary antibodies coupled to HRP were from Cell Signaling Technology (Beverly, MA, USA). Alexa Fluor 488- and 555-conjugated secondary antibodies were from Invitrogen (Grand Island, NY, USA). Lys-(des-Arg9, Leu8)BK (DALBK), HOE140, aprotinin, AG1478, and PD98059 were from Sigma-Aldrich (St. Louis, MO, USA). The Lipofectamine 2000 transfection reagent and protein G Dynabeads were from Invitrogen (Grand Island, NY, USA).
Starved SH-SY5Y cells were either stimulated with 1 μM TK for 5 min or left untreated and then lysed in NP-40 lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40 and protease inhibitor mixture) supplemented with 1 mM PMSF. Antibody against Flot2 (diluted 1:100) or EGFR (diluted 1:100) was coupled on protein G Dynabeads. The lysates and beads were rolled overnight at 4 °C, and then the beads were washed 3 times in washing buffer. The beads and lysates were supplemented with SDS sample buffer and heated at 94 °C for 5 min.
2.2. Cell cultures and transfections
2.5. Western blotting
All of the procedures were performed according to the NIH Guide for the Care and Use of Laboratory Animals as well as Fudan University experimental standards. Primary neurons were prepared from embryonic BALB/c mice. Briefly, cortex was isolated, digested, and homogenized. Cells were plated on poly-L-lysine-coated coverslips in Neurobasal-A medium containing B-27 supplement, 0.5 mM L-glutamine and 1% penicillin–streptomycin (Invitrogen). Cultured primary neurons from 3 days in vitro (DIV) were transfected as described previously [22]. Mouse Flot2 and EGFR RNA interference target sequences for primary neurons were shown in Table 1. The negative control (NC) siRNA with non-targeting sequence for mouse was from Invitrogen. Experiments were performed at 48 h after transfection. Human SH-SY5Y cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen) at 37 °C and 5% CO2. RNA interference target sequences against human Flot2, B1R, B2R and EGFR for SH-SY5Y cells were shown in Table 1. The commercial siRNA from
The cells were lysed in RIPA lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS containing phosphatase and protease inhibitor mixture) supplemented with 1 mM PMSF on ice for 30 min. Protein concentration was determined using the BCA kit. Equal amounts of protein were analyzed by 8% or 10% SDS-PAGE and were transferred to PVDF membrane (Millipore). Immunoblotting was performed with the appropriate primary antibody followed by HRP-conjugated secondary antibody. Antibody against p-EGFR (diluted 1:1000), EGFR (diluted 1:1000), p-ERK1/2 (diluted 1:2000), ERK1/2 (diluted 1:1000), Flot2 (diluted 1:1000), B1R (diluted 1:500), B2R (diluted 1:1000) or GAPDH (diluted 1:10000) was used in the corresponding experiments. Blotted proteins were visualized using an enhanced chemiluminescence assay. Western blot bands of phosphorylated proteins were quantified by scanning densitometry using Scion Image software and normalized against the total amount of the respective protein unless stated, otherwise GAPDH was used as an equal loading control.
2.4. Co-immunoprecipitation
Table 1 RNA interference target sequences. siRNA
Sense
Antisense
Vendor
Mouse Flot2
GUUCAUGGCAGACACCAAGTT GGUGAAGAUCAUGACGGAGTT GGUUUAUAGGCCUUCUUCCTT GGGAACUGCCCAUGCGGAATT CCAUCAAGGAGUUAAGAGATT GAAUAUUAAGCAGCAUUUATT GUUCAUGGCAGACACCAAGTT GGUGAAGAUCAUGACGGAGTT CUGCCAACAUUUAUCAUCUTT CAAGGAUUGUGGAGUUAAATT CUGCGAUCGUCUUCUUCAATT UGCCAUUAUCUCCAUGAACTT GGGAAGUGUUCACCAACAUTT GGCUCUGGAGGAAAAGAAATT
CUUGGUGUCUGCCAUGAACTT CUCCGUCAUGAUCUUCACCTT GGAAGAAGGCCUAUAAACCTT UUCCGCAUGGGCAGUUCCCTT UCUCUUAACUCCUUGAUGGTT UAAAUGCUGCUUAAUAUUCTT CUUGGUGUCUGCCAUGAACTT CUCCGUCAUGAUCUUCACCTT AGAUGAUAAAUGUUGGCAGTT UUUAACUCCACAAUCCUUGTT UUGAAGAAGACGAUCGCAGTT GUUCAUGGAGAUAAUGGCATT AUGUUGGUGAACACUUCCCTT UUUCUUUUCCUCCAGAGCCTT
Invitrogen Invitrogen Invitrogen Invitrogen Invitrogen Invitrogen Invitrogen Invitrogen Santa Cruz Santa Cruz Santa Cruz GenePharma GenePharma GenePharma
Mouse EGFR
Human Flot2 Human B1R
Human B2R Human EGFR
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2.6. Immunocytochemistry
3.3. Exogenous active TK mediates neurite outgrowth
Treated with TK, primary neurons or SH-SY5Y cells grown on glass coverslips were fixed with 4% paraformaldehyde and were blocked in TBS-T containing 5% bovine serum albumin (BSA) at room temperature, and then were exposed to primary antibody in TBS-T containing 5% BSA overnight at 4 °C. Antibody against MAP2 (diluted 1:200), EGFR (diluted 1:50) or Flot2 (diluted 1:50) was used in the corresponding experiments. Afterwards, cells were incubated with fluorescent secondary antibody (diluted 1:1000) for 1 h at room temperature in the dark. Cell nuclei were stained by Hoechst 33258. Fluorescent labeling was then visualized under a fluorescence microscope (Olympus, Japan) or a confocal microscopy (Olympus, Japan).
To explore whether the active or inactive form of TK played a role in the process of neurite outgrowth, TK or TK pre-incubated with aprotinin was tested in primary neurons and SH-SY5Y cells. Primary neurons exposed to active TK for 48 h showed increased number of processes and mean process length compared to neurons with inactive TK (Fig. 4A and B). TK pre-incubated with aprotinin did not stimulate EGFR and ERK1/2 phosphorylation in SH-SY5Y cells (Fig. 4C and D).
2.7. Neurite outgrowth quantification Neurite outgrowth was quantitated using ImageJ software. Data were collected from between 25 and 30 neurons with each treatment. For statistical analyses, mean process length (total length of neurites divided by the number of cells) and number of processes (total number of neurites divided by number of cells) were calculated. 2.8. Statistical analysis In general, all experiments presented in this paper were performed at least three times. Data were shown as mean ± S.D. Differences between groups were considered statistically significant according to one-way ANOVA followed by the Bonferroni post hoc tests for parametric data or the Kruskal–Wallis for non-parametric data with GraphPad Prism 5 software. Values of P b 0.05 were considered significant, whereas values of P b 0.01 were defined very significant. 3. Results 3.1. TK activates the EGFR and ERK1/2 signaling pathway in a concentration-dependent manner To determine the possible neurite outgrowth mechanism of TK, we investigated the ERK1/2 signaling through EGFR which was reported to have a close relationship with the biological functions of kallikreins [2,5]. As shown in Fig. 1, serum-starved SH-SY5Y cells exposed to TK at 0.25 to 1 μM for 5 min exhibited a concentration-dependent increase in EGFR and ERK1/2 phosphorylation. The cells were stimulated with 1 μM TK for 5, 15 or 30 min, the time course of ERK1/2 response paralleled with EGFR activation. Interestingly, the EGFR and ERK1/2 phosphorylation peaked in 5 min of TK stimulation. In accordance with the protein level of p-EGFR and p-ERK1/2 varying with time and dose, SH-SY5Y cells were treated with 1 μM TK for 5 min in the following experiments. 3.2. TK activates the EGFR and ERK1/2 signaling pathway independent of BK receptors In this experiment, we focused on the TK effects in the absence of an exogenous source of kininogen, and we hypothesized that TK activated the EGFR and ERK1/2 signaling pathway independent of BK receptors. To exclude the influence of BK receptors, we tested whether B1R antagonist DALBK or B2R antagonist HOE140 affected TK-stimulated EGFR and ERK1/2 activation. As shown in Fig. 2, both DALBK and HOE140 had no response on TK-induced EGFR and ERK1/2 phosphorylation effects in SH-SY5Y cells. Also we confirmed the role of BK receptors using siRNA knockdown of B1R and B2R. Consistent with the data above, down-regulation of BK receptors had no significant effects on TK-induced phosphorylation of EGFR and ERK1/2 (Fig. 3).
3.4. TK-induced neurite outgrowth is dependent on EGFR phosphorylation To test if the phosphorylation of EGFR was involved in TK-mediated neurite outgrowth, primary neurons or SH-SY5Y cells were preincubated with EGFR inhibitor AG1478 before TK stimulus. The number of processes and mean process length of neurons were reduced by AG1478 (Fig. 5A and B). The phosphorylation level of EGFR in SHSY5Y cells was totally blocked by EGFR inhibitor, while TK-stimulated ERK1/2 activation was partially inhibited (Fig. 5C and D). The incomplete effect of AG1478 revealed that there might be some other TKinduced pathways contributing to ERK1/2 activation. Then we verified the role of EGFR by using siRNA knockdown of EGFR. Similarly, TKinduced neurite extension was inhibited by EGFR down-regulation in primary neurons (Fig. 6A and B); and TK-mediated ERK1/2 phosphorylation significantly declined in EGFR-knockdown SH-SY5Y cells (Fig. 6C and D).
3.5. Flot2 is required for TK-induced neurite outgrowth Flot2 has previously been suggested to take on key role in axon growth and regeneration [21,22], we therefore next verified whether Flot2 was essential for TK-induced neurite outgrowth by using siRNAinduced knockdown of Flot2. To circumvent sequence-specific offtarget effects of the Flot2 siRNA in cultured primary neurons, a mixture of three different sequences was used according to the previous study [22]. Neurons transfected with Flot2 siRNAs showed decreased number of processes and mean process length compared to those transfected with control siRNA (Fig. 7A and B). Depletion of Flot2 inhibited the number of processes and mean process length of neurons with TK stimulation (Fig. 7A and B). In human SH-SY5Y cells, a mixture of two different sequences was applied and led to approximately 74% reduction of Flot2 protein level (data not shown). When the TK-induced phosphorylation level of EGFR and ERK1/2 was measured from control or Flot2 siRNA-transfected SH-SY5Y cells, we found a remarkable downregulation of phosphorylated EGFR and ERK1/2 in Flot2-knockdown cells (Fig. 7C and D).
3.6. TK-induced neurite outgrowth is dependent on ERK1/2 phosphorylation Our previous work has reported that TK could trigger a series of biological effects through the ERK1/2 pathway [24]. Also newly studies demonstrated that both EGFR and flotillin effects resulted in activation of ERK1/2 signaling events [15,22]. In this study, we carried out to see whether TK could affect neurite outgrowth via ERK1/2 phosphorylation signaling. Primary neurons or SH-SY5Y cells were treated with ERK kinase inhibitor for 1 h previous to TK stimulus. The number of processes and mean process length of neurons were reduced by PD98059 (Fig. 8A and B). The level of phospho-ERK in SH-SY5Y cells was substantially blocked by ERK1/2 inhibitor, but there was no significant change in TK-stimulated EGFR phosphorylation (Fig. 8C and D). These data implied that TK gave rise to phosphorylation of ERK1/2, which was the downstream event of EGFR signaling, in the course of neurite outgrowth.
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Fig. 1. TK results in the activation of EGFR and ERK1/2 in SH-SY5Y cells. (A, C) SH-SY5Y cells were treated with TK at increasing concentrations for 5 min, and EGFR or ERK1/2 phosphorylation was determined by Western blot. (B, D) SH-SY5Y cells were stimulated or not with 1 μM TK for 5, 15, or 30 min, and phosphorylated EGFR or ERK1/2 was detected by Western blot. Total cell EGFR or ERK1/2 was used as an equal loading control. Bars represent the mean ± S.D. of at least three individual experiments. **, P b 0.01 compared to unstimulated control.
3.7. EGFR and Flot2 form constitutive complex in the TK stimulation Because knockdown of Flot2 affected EGFR phosphorylation in the TK stimulation, we carried out co-immunoprecipitation experiments to prove if EGFR and Flot2 form a functional complex.
Immunoprecipitation was performed for either EGFR (Fig. 9A) or Flot2 (Fig. 9B) from unstimulated and TK-stimulated SH-SY5Y cells. Flot2 was coprecipitated with EGFR with or without TK stimulation, which demonstrated that the complex was constitutive. In the TK-stimulated samples, two bands against EGFR or p-EGFR antibody
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Fig. 2. Activation of EGFR and ERK1/2 in SH-SY5Y cells by TK is independent of BK receptors. (A, C) SH-SY5Y cells were pre-incubated for 1 h in the presence or absence of the B1R antagonist, DALBK (1 μM), prior to treatment with 1 μM TK for 5 min. (B, D) SH-SY5Y cells were pre-incubated for 1 h in the presence or absence of the B2R antagonist, HOE140 (10 μM), prior to treatment with 1 μM TK for 5 min. Phosphorylated EGFR or ERK1/2 was detected from whole cell lysates as described. Total cell EGFR or ERK1/2 was blotted as an equal loading control. The bar graph depicts the mean ± S.D. for at least three individual experiments. **, P b 0.01 compared to control group.
were detected in the immunoprecipitates that were precipitated with Flot2 (Fig. 9B).
remarkably promoted the green and the red fluorescence to accumulate around the nuclei. As shown by confocal microscopy, the EGFR and Flot2 staining colocalized with yellow in overlay after exposure to 1 μM TK for 5 min (Fig. 10, lower part).
3.8. TK facilitates the translocation of EGFR and Flot2 to around the nuclei To observe the TK-triggered effects on intracellular localization of EGFR and Flot2, immunofluorescent labels were performed using EGFR and Flot2 antibodies. We found that EGFR and Flot2 resided in the cytoplasm and plasma membrane under normal condition in SH-SY5Y cells (Fig. 10, upper part). However, the addition of TK
4. Discussion TK has been reported to play an important part in various physiological and pathological processes. It is well elucidated that human TK gene transfer could promote neurogenesis to protect against neurological
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Fig. 3. TK results in the activation of EGFR and ERK1/2 in SH-SY5Y cells independent of BK receptors. (A, B) SH-SY5Y cells were transiently transfected with B1R siRNAs. After transfection 48 h cells were treated with or without 1 μM TK for 5 min, the level of EGFR or ERK1/2 was detected by Western blot. The p-EGFR or p-ERK1/2 was normalized to total cell EGFR or ERK respectively. At least three individual experiments were performed. Values are represented as mean ± S.D. **, P b 0.01 compared to NC group. (C, D) SH-SY5Y cells were transiently transfected with B2R siRNAs. After transfection 48 h cells were treated with or without 1 μM TK for 5 min, the level of p-EGFR or p-ERK1/2 was detected as described. Total cell EGFR or ERK1/2 was blotted as a control for equal protein loading. Bars represent the mean ± S.D. of at least three individual experiments. **, P b 0.01 compared to NC group.
dysfunction [25]. However, limited information is available on TKinduced actions of nerve growth. The complex function of TK in health and disease and its potential as a therapeutic target underscore the importance of finding out its molecular mechanism. In the present study, we have provided convincing evidence for the role of TK in neurite
outgrowth signaling through EGFR, Flot2 and ERK1/2. We also discovered a positive correlation between EGFR and Flot2 in the TK stimulation. In the initial experiment, our data suggested that TK facilitated the activation of EGFR and ERK1/2 cascade in a concentration-dependent
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Fig. 4. Exogenous active TK mediates neurite outgrowth and stimulates EGFR and ERK1/2 phosphorylation. (A) Cultured primary neurons from 5 DIV were treated with active TK (1 μM) or inactive TK (1 μM) for 48 h and then stained with MAP2. Images captured at 20×. Scale bar = 10 μm. (B) Neurons exposed to active TK, but not inactive form, for 48 h showed increased number of processes and mean process length compared to neurons in control group. N = 25-30 neurons each. **, P b 0.01 compared to control group. ##, P b 0.01 compared to active TK stimulated neurons. (C, D) SH-SY5Y cells were stimulated with active TK (1 μM) or inactive TK (1 μM) for 5 min, and phosphorylated EGFR or ERK1/2 was detected by Western blot. Total cell EGFR or ERK1/2 was blotted as a control for equal protein loading. Bars represent the mean ± S.D. of at least three individual experiments. **, P b 0.01 compared to unstimulated control. ##, P b 0.01 compared to active TK stimulated cells.
manner, which consisted with the previous reports that kallikreins lead to EGFR transactivation followed by ERK1/2 phosphorylation [2,5]. In our in vitro study, we learned that the level of p-EGFR and p-ERK1/2 peaked in serum-starved SH-SY5Y cells exposed to TK at 1 μM for 5 min, which involved neither B1R nor B2R. Therefore, we tried using the concentration on primary neurons and SH-SY5Y cells for further
research. And then we observed that exogenous active TK was able to increase the phosphorylation of EGFR and ERK1/2, as well as the number of processes and mean process length of neurons, which were block by EGFR inhibitor or RNA interference. However, we did not coprecipitate TK with EGFR (data not shown). Correlative experiments showed that members of the TK gene family could directly active G
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Fig. 5. TK-induced neurite outgrowth is dependent on EGFR phosphorylation. (A) Cultured primary neurons from 5 DIV were pre-incubated for 1 h in the presence or absence of the EGFR antagonist, AG1478 (300 nM), prior to treatment with 1 μM TK for 48 h, and then neurons were stained with MAP2 in 7 DIV. Images captured at 20×. Scale bar = 10 μm. (B) Neurons exposed to TK for 48 h showed increased number of processes and mean process length compared to control neurons, which were reduced by AG1478. N = 25–30 neurons each. **, P b 0.01 compared to control group. ##, P b 0.01 compared to TK stimulated group. (C, D) SH-SY5Y cells were pre-incubated for 1 h with or without the EGFR antagonist, AG1478 (300 nM), prior to treatment with 1 μM TK for 5 min, and EGFR or ERK1/2 phosphorylation was detected by Western blot. The p-EGFR or p-ERK1/2 was normalized to total amount of EGFR or ERK respectively. At least three individual experiments were performed. Values are represented as mean ± S.D. **, P b 0.01 compared to control group. ##, P b 0.01 compared to TK stimulated group.
protein-coupled PAR1, PAR2 and PAR4 [26,27]. TK might stimulate the PAR1-mediated protein kinase C and Src kinase pathway, leading to EGFR transactivation resulting from metalloproteinase-induced EGFR ligand shedding [5]. These combined results revealed a novel BK
receptors-independent mechanism of TK action which might contribute to the process of neurite growth via EGFR. The flotillin proteins were reported to function as key regulators of the cell-intrinsic program which was required for axon growth and
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Fig. 6. TK-induced neurite outgrowth is dependent on EGFR activation. (A) Cultured primary neurons from 3 DIV were performed RNA interference experiments. After transfection 48 h neurons were treated with or without 1 μM TK for 48 h, and were stained with MAP2 in 7 DIV. Images captured at 20×. Scale bar = 10 μm. (B) NC transfected neurons exposed to TK for 48 h showed increased number of processes and mean process length compared to NC group, which were blocked by knockdown of EGFR. N = 25–30 neurons each. **, P b 0.01 compared to NC group. ##, P b 0.01 compared to NC/TK group. (C, D) SH-SY5Y cells were transiently transfected with EGFR siRNA. After transfection 48 h cells were treated with or without 1 μM TK for 5 min, the level of EGFR or ERK1/2 was detected by Western blot. The p-EGFR or p-ERK1/2 was normalized to GAPDH or total ERK respectively. At least three individual experiments were performed. Values are represented as mean ± S.D. **, P b 0.01 compared to NC group. ##, P b 0.01 compared to NC/TK group.
regeneration in neurons [21,22]. In support of this theory, we proposed that Flot2 played a fundamental role in the TK-induced neurite outgrowth. Consistent with the published information, we found that Flot2 down-regulation by siRNAs would impair the process of neurite growth in primary neurons. In the current study, the formation and development of neurite processes induced by TK were inhibited in Flot2 knockdown neurons. Our evidence also suggested that there was a reduced phosphorylation of EGFR and ERK1/2 after TK stimulation in
the absence of Flot2, indicating that Flot2 was necessary for TK downstream signaling pathways. These research findings raised the question whether Flot2 was directly involved in the EGFR signaling of TK. Recent published data indicated that one significant function of flotillins might be to regulate the formation of macromolecular complexes/clusters at the plasma membrane which was necessary for initiating cellular processes such as signal transduction, endocytosis and proteolytic processing [28,29].
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Fig. 7. SiFlot2 blocks TK-induced neurite outgrowth through EGFR and ERK1/2 phosphorylation. (A) Cultured primary neurons from 3 DIV were performed RNA interference experiments. After transfection 48 h neurons were treated with or without 1 μM TK for 48 h, and were stained with MAP2 in 7 DIV. Images captured at 20×. Scale bar = 10 μm. (B) NC transfected neurons exposed to TK for 48 h showed increased number of processes and mean process length compared to NC group, which were blocked by knockdown of Flot2. N = 25–30 neurons each. **, P b 0.01 compared to NC group. ##, P b 0.01 compared to NC/TK group. (C, D) SH-SY5Y cells were transiently transfected with Flot2 siRNAs. After transfection 48 h cells were treated with or without 1 μM TK for 5 min, the level of EGFR or ERK1/2 was detected as described. The p-EGFR or p-ERK1/2 was normalized to total cell EGFR or ERK respectively. At least three individual experiments were performed. Values are represented as mean ± S.D. **, P b 0.01 compared to NC group. ##, P b 0.01 compared to NC/TK group.
In line with this, we were able to coimmunoprecipitate EGFR together with Flot2 and vice versa. We detected p-EGFR as well in the TK-stimulated samples which were precipitated with Flot2. Flotillins and EGFR were reported to colocalize after endocytosis [30], therefore it could be reasonable to hypothesize that Flot2 was involved in the TK-mediated activation of EGFR. By means of immunofluorescence
staining, we observed that TK stimulation resulted in translocation of EGFR and Flot2 from plasma and cytoplasm membrane to around the nuclei. Taken together, our experiments indicated that EGFR and Flot2 formed constitutive complex in the TK stimulation, which might act as a molecular mediator to activate the downstream signaling toward ERK1/2.
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Fig. 8. TK-induced neurite outgrowth is dependent on ERK1/2 phosphorylation. (A) Cultured primary neurons from 5 DIV were pre-incubated for 1 h with or without the ERK1/2 antagonist, PD98059 (10 μM), prior to treatment with 1 μM TK for 48 h, and then neurons were stained with MAP2 in 7 DIV. Images captured at 20×. Scale bar = 10 μm. (B) Neurons exposed to TK for 48 h showed increased number of processes and mean process length compared to control neurons, which were reduced by PD98059. N = 25–30 neurons each. *, P b 0.05 and **, P b 0.01 compared to control group. ##, P b 0.01 compared to TK stimulated group. (C, D) SH-SY5Y cells were pre-incubated for 1 h with or without the ERK1/2 antagonist, PD98059 (10 μM), prior to treatment with 1 μM TK for 5 min, and EGFR or ERK1/2 phosphorylation was detected as described. The p-EGFR or p-ERK1/2 was normalized to total cell EGFR or ERK respectively. At least three individual experiments were performed. Values are represented as mean ± S.D. **, P b 0.01 compared to control group. ##, P b 0.01 compared to TK stimulated group.
It has been noted that ERKs, members of the classical MAPK family, were crucial in regulating cellular survival, cell growth and proliferation [31]. Previous information suggested that TK promoted the activation of ERK1/2, which was needed for axon growth and regeneration [21,22], with the involvement of EGFR phosphatase activity [5,32]. Consistent
with these studies, we verified that the effect of TK in neurite outgrowth was blocked by ERK inhibitor, which was also reduced by EGFR antagonist or RNA interference and Flot2 down-regulation. Focus on ERK1/2 activity, we perform our tests by means of Western blot and immunocytochemistry (data not shown), and found that TK could promote the
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Fig. 9. EGFR and Flot2 form constitutive complex in the TK stimulation. (A) SH-SY5Y cells (unstimulated or 1 μM TK stimulated for 5 min) were lysed and immunoprecipitated with antibody against EGFR. Co-immunoprecipitation of EGFR with Flot2 was determined in all samples. (B) SH-SY5Y cells (unstimulated or 1 μM TK stimulated for 5 min) were lysed and immunoprecipitated with antibody against Flot2. Co-immunoprecipitation of Flot2 with EGFR was determined in all samples. In the TK-stimulated samples, two bands against EGFR or p-EGFR antibody were detected by immunoprecipitate that were precipitated with Flot2. Equal loading was verified with GAPDH.
phosphorylation of ERK1/2 which significantly decreased after the intervention with AG1478 or EGFR down-regulation, Flot2 siRNAs and PD98059 respectively. All experimental evidence that we have collected agreed with our hypothesis that TK promoted neurite outgrowth through ERK1/2 activation which occurred downstream of EGFR and Flot2 complex. In conclusion, we demonstrated that TK provided beneficial effects of neurite outgrowth through EGFR, Flot2 and ERK1/2 pathway, which was a novel transmembrane signaling mechanism independent of BK receptors. Combining our previous work [24,33,34] with updated findings here, we indicated the potential of TK as a therapeutic target for
patients with neural injury especially in stroke. However, future studies should explore the neurogenesis role of TK in stroke models. And it will be valuable to exhibit the exact molecular chain of events which takes place during the course of neurological functional reconstruction in vivo.
Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (Nos. 81271295, 81000487).
Fig. 10. TK facilitates the translocation of EGFR and Flot2 to around the nuclei. Starved SH-SY5Y cells were treated with 1 μM TK for 5 min, and then were stained with EGFR (in green), Flot2 (in red) or nuclei (in blue). The addition of TK remarkably promoted the green and the red fluorescence to accumulate around the nuclei, which colocalized with yellow fluorescence in overlay. Images captured at 20×.
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Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig
Tissue kallikrein mediates neurite outgrowth through epidermal growth factor receptor and flotillin-2 pathway in vitro Zhengyu Lu a, Mei Cui a, Hong Zhao b, Tao Wang b, Yan Shen c, Qiang Dong a,⁎ a
Department of Neurology, Huashan Hospital, State Key Laboratory of Medical Neurobiology, Fudan University, No.12 Mid. Wulumuqi Road, Shanghai 200040, PR China Department of Neurology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, No.110 Ganhe Road, Shanghai 200437, PR China c Department of Cardiology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, No.110 Ganhe Road, Shanghai 200437, PR China b
a r t i c l e
i n f o
Article history: Received 29 July 2013 Received in revised form 8 October 2013 Accepted 31 October 2013 Available online 6 November 2013 Keywords: Tissue kallikrein Neurite outgrowth Epidermal growth factor receptor Flotillin-2 Extracellular signal-regulated kinase
a b s t r a c t Tissue kallikrein (TK) was previously shown to take most of its biological effects through bradykinin receptors. In this study, we assumed that TK mediated neurite outgrowth was independent of bradykinin receptors. To test the hypothesis, we investigated TK-induced neurite outgrowth and its signaling mechanisms in cultured primary neurons and human SH-SY5Y cells. We found that TK stimulation could increase the number of processes and mean process length of primary neurons, which were blocked by epidermal growth factor receptor (EGFR) inhibitor or down-regulation, small interfering RNA for flotillin-2 and extracellular signal-regulated kinase (ERK) 1/2 inhibitor. Moreover, TK-induced neurite outgrowth was associated with EGFR and ERK1/2 activation, which were inhibited by EGFR antagonist or RNA interference and flotillin-2 knockdown. Interestingly, inhibition of bradykinin receptors had no significant effects on EGFR and ERK1/2 phosphorylation. In the present research, our data also suggested that EGFR and flotillin-2 formed constitutive complex that translocated to around the nuclei in the TK stimulation. In sum, our findings provided evidence that TK could promote neurite outgrowth via EGFR, flotillin-2 and ERK1/2 signaling pathway in vitro. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Tissue kallikrein (TK), an important component of the kallikrein– kinin system (KKS), belongs to a subgroup of serine proteinases and processes low molecular weight (LMW) kininogen to release kinin peptide [1,2]. Most of the cellular effects of TK are thought to be mediated by bradykinin (BK), which acts via G protein-coupled B1 and B2 bradykinin receptors (B1R and B2R). However, recent findings revealed that TK could directly activate B2R independent of kininogen and kinin release in cultured CHO cells [3,4]. Moreover, TK was found to trigger the proteinase-activated receptor 1 (PAR1) signaling pathway and epidermal growth factor receptor (EGFR) transactivation in HaCaT keratinocyte cells [5]. These combined information indicated that actions of TK might contribute to other signaling events in addition to BK pathway. EGFR is a transmembrane receptor comprising a family of classical receptor tyrosine kinases that trigger a rich network of signaling
Abbreviations: TK, tissue kallikrein; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; KKS, kallikrein–kinin system; BK, bradykinin; B1R, B1 bradykinin receptor; B2R, B2 bradykinin receptor; PAR, proteinase-activated receptor; Flot, flotillin; DIV, days in vitro. ⁎ Corresponding author. Tel.: +86 21 6248 99991401; fax: +86 21 6248 1401. E-mail address: [email protected] (Q. Dong). 0898-6568/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cellsig.2013.10.010
pathways to control cell survival and growth [6,7]. There are at least 11 endogenous EGFR ligands, each of which is synthesized as precursors and cleaved to generate the mature one. Ligand binding results in multiple phosphorylation events of EGFR, which in many cases are mediated by tyrosine kinases. Simultaneous activation of downstream cascades, such as the mitogen-activated protein kinase (MAPK) pathway, translates in the nucleus into distinct transcriptional programs. Furthermore, previous research showed that lipid rafts were involved in the signaling and/or trafficking of EGFR [8,9]. Reggie-1/flotillin-2 (Flot2) and reggie-2/flotillin-1 (Flot1) are two highly conserved lipid-raft-associated proteins, which were originally discovered as proteins up-regulated in retinal ganglion cells during axon regeneration [10,11]. Flotillins share an SPFH (Stomatin/ Prohibitin/Flotillin/HflK/C) domain at their N-terminus containing residues for myristoylation and palmitoylation and thus membrane anchorage [12,13]. The C-terminus of reggies harbors a unique flotillin domain that is predicted to adopt an alpha-helical coiled-coil structure [10,11]. Even though the function of flotillins has remained elusive and controversial, their widespread expression and conservation imply that these proteins regulate basic cellular processes. Evidence is accumulating that flotillins are involved in the signaling processes of several membrane receptors, phagocytosis, non-clathrin endocytosis, and organization of the actin cytoskeleton [14–20]. They have also been suggested to function as key proteins for neurite outgrowth [21,22]. In
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addition, recent studies have demonstrated that flotillins were modified by EGFR signaling, and that Flot2 became phosphorylated at several tyrosine residues and translocated from plasma membrane into endosomes in the stimulation of EGFR [15]. Although such molecular effects of flotillins have been reported, the role of Flot2 in TK-induced neurite outgrowth has received relatively little attention. Here, we describe a novel neurite growth mechanism whereby TK activates the extracellular signal-regulated kinase (ERK) 1/2 pathway through EGFR and Flot2 independent of BK receptors. 2. Materials and methods 2.1. Materials
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Invitrogen with non-targeting sequence was used as a non-specific control. SH-SY5Y cells were transiently transfected with Lipofectamine 2000 according to the manufacturer's instructions, and experiments were carried out at 48 h after transfection. 2.3. Cell experiment treatment For drug treatment, primary neurons were exposed to TK at 1 μM for 48 h, and SH-SY5Y cells were serum-starved overnight before various concentration of TK (0.25 to 1 μM) were given for 5, 15 or 30 min. To study the involvement of BK receptors on TK effects, B1R antagonist DALBK (1 μM) or B2R antagonist HOE140 (10 μM) was added 1 h prior to TK treatment. TK was inactivated by pre-incubation with fivefold molar excess of aprotinin for 1 h at 37 °C [23]. In some experiments cells were treated with EGFR inhibitor AG1478 (300 nM) or ERK kinase inhibitor PD98059 (10 μM) for 1 h previous to TK stimulation.
TK was from Techpool Bio-Pharma Co. (Guangzhou, China). EGFR, phospho-EGFR (Tyr1068), ERK1/2 (Thr202/Tyr204), phospho-ERK1/2 (Thr202/Tyr204), Flot2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibodies were from Cell Signaling Technology (Beverly, MA, USA). Affinity-purified rabbit anti-microtubuleassociated protein 2 (MAP2) antibody was from Sigma-Aldrich (St. Louis, MO, USA). B1R polyclonal antibody and B2R monoclonal antibody were from Abcam (Cambridge, UK). Secondary antibodies coupled to HRP were from Cell Signaling Technology (Beverly, MA, USA). Alexa Fluor 488- and 555-conjugated secondary antibodies were from Invitrogen (Grand Island, NY, USA). Lys-(des-Arg9, Leu8)BK (DALBK), HOE140, aprotinin, AG1478, and PD98059 were from Sigma-Aldrich (St. Louis, MO, USA). The Lipofectamine 2000 transfection reagent and protein G Dynabeads were from Invitrogen (Grand Island, NY, USA).
Starved SH-SY5Y cells were either stimulated with 1 μM TK for 5 min or left untreated and then lysed in NP-40 lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40 and protease inhibitor mixture) supplemented with 1 mM PMSF. Antibody against Flot2 (diluted 1:100) or EGFR (diluted 1:100) was coupled on protein G Dynabeads. The lysates and beads were rolled overnight at 4 °C, and then the beads were washed 3 times in washing buffer. The beads and lysates were supplemented with SDS sample buffer and heated at 94 °C for 5 min.
2.2. Cell cultures and transfections
2.5. Western blotting
All of the procedures were performed according to the NIH Guide for the Care and Use of Laboratory Animals as well as Fudan University experimental standards. Primary neurons were prepared from embryonic BALB/c mice. Briefly, cortex was isolated, digested, and homogenized. Cells were plated on poly-L-lysine-coated coverslips in Neurobasal-A medium containing B-27 supplement, 0.5 mM L-glutamine and 1% penicillin–streptomycin (Invitrogen). Cultured primary neurons from 3 days in vitro (DIV) were transfected as described previously [22]. Mouse Flot2 and EGFR RNA interference target sequences for primary neurons were shown in Table 1. The negative control (NC) siRNA with non-targeting sequence for mouse was from Invitrogen. Experiments were performed at 48 h after transfection. Human SH-SY5Y cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen) at 37 °C and 5% CO2. RNA interference target sequences against human Flot2, B1R, B2R and EGFR for SH-SY5Y cells were shown in Table 1. The commercial siRNA from
The cells were lysed in RIPA lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS containing phosphatase and protease inhibitor mixture) supplemented with 1 mM PMSF on ice for 30 min. Protein concentration was determined using the BCA kit. Equal amounts of protein were analyzed by 8% or 10% SDS-PAGE and were transferred to PVDF membrane (Millipore). Immunoblotting was performed with the appropriate primary antibody followed by HRP-conjugated secondary antibody. Antibody against p-EGFR (diluted 1:1000), EGFR (diluted 1:1000), p-ERK1/2 (diluted 1:2000), ERK1/2 (diluted 1:1000), Flot2 (diluted 1:1000), B1R (diluted 1:500), B2R (diluted 1:1000) or GAPDH (diluted 1:10000) was used in the corresponding experiments. Blotted proteins were visualized using an enhanced chemiluminescence assay. Western blot bands of phosphorylated proteins were quantified by scanning densitometry using Scion Image software and normalized against the total amount of the respective protein unless stated, otherwise GAPDH was used as an equal loading control.
2.4. Co-immunoprecipitation
Table 1 RNA interference target sequences. siRNA
Sense
Antisense
Vendor
Mouse Flot2
GUUCAUGGCAGACACCAAGTT GGUGAAGAUCAUGACGGAGTT GGUUUAUAGGCCUUCUUCCTT GGGAACUGCCCAUGCGGAATT CCAUCAAGGAGUUAAGAGATT GAAUAUUAAGCAGCAUUUATT GUUCAUGGCAGACACCAAGTT GGUGAAGAUCAUGACGGAGTT CUGCCAACAUUUAUCAUCUTT CAAGGAUUGUGGAGUUAAATT CUGCGAUCGUCUUCUUCAATT UGCCAUUAUCUCCAUGAACTT GGGAAGUGUUCACCAACAUTT GGCUCUGGAGGAAAAGAAATT
CUUGGUGUCUGCCAUGAACTT CUCCGUCAUGAUCUUCACCTT GGAAGAAGGCCUAUAAACCTT UUCCGCAUGGGCAGUUCCCTT UCUCUUAACUCCUUGAUGGTT UAAAUGCUGCUUAAUAUUCTT CUUGGUGUCUGCCAUGAACTT CUCCGUCAUGAUCUUCACCTT AGAUGAUAAAUGUUGGCAGTT UUUAACUCCACAAUCCUUGTT UUGAAGAAGACGAUCGCAGTT GUUCAUGGAGAUAAUGGCATT AUGUUGGUGAACACUUCCCTT UUUCUUUUCCUCCAGAGCCTT
Invitrogen Invitrogen Invitrogen Invitrogen Invitrogen Invitrogen Invitrogen Invitrogen Santa Cruz Santa Cruz Santa Cruz GenePharma GenePharma GenePharma
Mouse EGFR
Human Flot2 Human B1R
Human B2R Human EGFR
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2.6. Immunocytochemistry
3.3. Exogenous active TK mediates neurite outgrowth
Treated with TK, primary neurons or SH-SY5Y cells grown on glass coverslips were fixed with 4% paraformaldehyde and were blocked in TBS-T containing 5% bovine serum albumin (BSA) at room temperature, and then were exposed to primary antibody in TBS-T containing 5% BSA overnight at 4 °C. Antibody against MAP2 (diluted 1:200), EGFR (diluted 1:50) or Flot2 (diluted 1:50) was used in the corresponding experiments. Afterwards, cells were incubated with fluorescent secondary antibody (diluted 1:1000) for 1 h at room temperature in the dark. Cell nuclei were stained by Hoechst 33258. Fluorescent labeling was then visualized under a fluorescence microscope (Olympus, Japan) or a confocal microscopy (Olympus, Japan).
To explore whether the active or inactive form of TK played a role in the process of neurite outgrowth, TK or TK pre-incubated with aprotinin was tested in primary neurons and SH-SY5Y cells. Primary neurons exposed to active TK for 48 h showed increased number of processes and mean process length compared to neurons with inactive TK (Fig. 4A and B). TK pre-incubated with aprotinin did not stimulate EGFR and ERK1/2 phosphorylation in SH-SY5Y cells (Fig. 4C and D).
2.7. Neurite outgrowth quantification Neurite outgrowth was quantitated using ImageJ software. Data were collected from between 25 and 30 neurons with each treatment. For statistical analyses, mean process length (total length of neurites divided by the number of cells) and number of processes (total number of neurites divided by number of cells) were calculated. 2.8. Statistical analysis In general, all experiments presented in this paper were performed at least three times. Data were shown as mean ± S.D. Differences between groups were considered statistically significant according to one-way ANOVA followed by the Bonferroni post hoc tests for parametric data or the Kruskal–Wallis for non-parametric data with GraphPad Prism 5 software. Values of P b 0.05 were considered significant, whereas values of P b 0.01 were defined very significant. 3. Results 3.1. TK activates the EGFR and ERK1/2 signaling pathway in a concentration-dependent manner To determine the possible neurite outgrowth mechanism of TK, we investigated the ERK1/2 signaling through EGFR which was reported to have a close relationship with the biological functions of kallikreins [2,5]. As shown in Fig. 1, serum-starved SH-SY5Y cells exposed to TK at 0.25 to 1 μM for 5 min exhibited a concentration-dependent increase in EGFR and ERK1/2 phosphorylation. The cells were stimulated with 1 μM TK for 5, 15 or 30 min, the time course of ERK1/2 response paralleled with EGFR activation. Interestingly, the EGFR and ERK1/2 phosphorylation peaked in 5 min of TK stimulation. In accordance with the protein level of p-EGFR and p-ERK1/2 varying with time and dose, SH-SY5Y cells were treated with 1 μM TK for 5 min in the following experiments. 3.2. TK activates the EGFR and ERK1/2 signaling pathway independent of BK receptors In this experiment, we focused on the TK effects in the absence of an exogenous source of kininogen, and we hypothesized that TK activated the EGFR and ERK1/2 signaling pathway independent of BK receptors. To exclude the influence of BK receptors, we tested whether B1R antagonist DALBK or B2R antagonist HOE140 affected TK-stimulated EGFR and ERK1/2 activation. As shown in Fig. 2, both DALBK and HOE140 had no response on TK-induced EGFR and ERK1/2 phosphorylation effects in SH-SY5Y cells. Also we confirmed the role of BK receptors using siRNA knockdown of B1R and B2R. Consistent with the data above, down-regulation of BK receptors had no significant effects on TK-induced phosphorylation of EGFR and ERK1/2 (Fig. 3).
3.4. TK-induced neurite outgrowth is dependent on EGFR phosphorylation To test if the phosphorylation of EGFR was involved in TK-mediated neurite outgrowth, primary neurons or SH-SY5Y cells were preincubated with EGFR inhibitor AG1478 before TK stimulus. The number of processes and mean process length of neurons were reduced by AG1478 (Fig. 5A and B). The phosphorylation level of EGFR in SHSY5Y cells was totally blocked by EGFR inhibitor, while TK-stimulated ERK1/2 activation was partially inhibited (Fig. 5C and D). The incomplete effect of AG1478 revealed that there might be some other TKinduced pathways contributing to ERK1/2 activation. Then we verified the role of EGFR by using siRNA knockdown of EGFR. Similarly, TKinduced neurite extension was inhibited by EGFR down-regulation in primary neurons (Fig. 6A and B); and TK-mediated ERK1/2 phosphorylation significantly declined in EGFR-knockdown SH-SY5Y cells (Fig. 6C and D).
3.5. Flot2 is required for TK-induced neurite outgrowth Flot2 has previously been suggested to take on key role in axon growth and regeneration [21,22], we therefore next verified whether Flot2 was essential for TK-induced neurite outgrowth by using siRNAinduced knockdown of Flot2. To circumvent sequence-specific offtarget effects of the Flot2 siRNA in cultured primary neurons, a mixture of three different sequences was used according to the previous study [22]. Neurons transfected with Flot2 siRNAs showed decreased number of processes and mean process length compared to those transfected with control siRNA (Fig. 7A and B). Depletion of Flot2 inhibited the number of processes and mean process length of neurons with TK stimulation (Fig. 7A and B). In human SH-SY5Y cells, a mixture of two different sequences was applied and led to approximately 74% reduction of Flot2 protein level (data not shown). When the TK-induced phosphorylation level of EGFR and ERK1/2 was measured from control or Flot2 siRNA-transfected SH-SY5Y cells, we found a remarkable downregulation of phosphorylated EGFR and ERK1/2 in Flot2-knockdown cells (Fig. 7C and D).
3.6. TK-induced neurite outgrowth is dependent on ERK1/2 phosphorylation Our previous work has reported that TK could trigger a series of biological effects through the ERK1/2 pathway [24]. Also newly studies demonstrated that both EGFR and flotillin effects resulted in activation of ERK1/2 signaling events [15,22]. In this study, we carried out to see whether TK could affect neurite outgrowth via ERK1/2 phosphorylation signaling. Primary neurons or SH-SY5Y cells were treated with ERK kinase inhibitor for 1 h previous to TK stimulus. The number of processes and mean process length of neurons were reduced by PD98059 (Fig. 8A and B). The level of phospho-ERK in SH-SY5Y cells was substantially blocked by ERK1/2 inhibitor, but there was no significant change in TK-stimulated EGFR phosphorylation (Fig. 8C and D). These data implied that TK gave rise to phosphorylation of ERK1/2, which was the downstream event of EGFR signaling, in the course of neurite outgrowth.
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Fig. 1. TK results in the activation of EGFR and ERK1/2 in SH-SY5Y cells. (A, C) SH-SY5Y cells were treated with TK at increasing concentrations for 5 min, and EGFR or ERK1/2 phosphorylation was determined by Western blot. (B, D) SH-SY5Y cells were stimulated or not with 1 μM TK for 5, 15, or 30 min, and phosphorylated EGFR or ERK1/2 was detected by Western blot. Total cell EGFR or ERK1/2 was used as an equal loading control. Bars represent the mean ± S.D. of at least three individual experiments. **, P b 0.01 compared to unstimulated control.
3.7. EGFR and Flot2 form constitutive complex in the TK stimulation Because knockdown of Flot2 affected EGFR phosphorylation in the TK stimulation, we carried out co-immunoprecipitation experiments to prove if EGFR and Flot2 form a functional complex.
Immunoprecipitation was performed for either EGFR (Fig. 9A) or Flot2 (Fig. 9B) from unstimulated and TK-stimulated SH-SY5Y cells. Flot2 was coprecipitated with EGFR with or without TK stimulation, which demonstrated that the complex was constitutive. In the TK-stimulated samples, two bands against EGFR or p-EGFR antibody
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Fig. 2. Activation of EGFR and ERK1/2 in SH-SY5Y cells by TK is independent of BK receptors. (A, C) SH-SY5Y cells were pre-incubated for 1 h in the presence or absence of the B1R antagonist, DALBK (1 μM), prior to treatment with 1 μM TK for 5 min. (B, D) SH-SY5Y cells were pre-incubated for 1 h in the presence or absence of the B2R antagonist, HOE140 (10 μM), prior to treatment with 1 μM TK for 5 min. Phosphorylated EGFR or ERK1/2 was detected from whole cell lysates as described. Total cell EGFR or ERK1/2 was blotted as an equal loading control. The bar graph depicts the mean ± S.D. for at least three individual experiments. **, P b 0.01 compared to control group.
were detected in the immunoprecipitates that were precipitated with Flot2 (Fig. 9B).
remarkably promoted the green and the red fluorescence to accumulate around the nuclei. As shown by confocal microscopy, the EGFR and Flot2 staining colocalized with yellow in overlay after exposure to 1 μM TK for 5 min (Fig. 10, lower part).
3.8. TK facilitates the translocation of EGFR and Flot2 to around the nuclei To observe the TK-triggered effects on intracellular localization of EGFR and Flot2, immunofluorescent labels were performed using EGFR and Flot2 antibodies. We found that EGFR and Flot2 resided in the cytoplasm and plasma membrane under normal condition in SH-SY5Y cells (Fig. 10, upper part). However, the addition of TK
4. Discussion TK has been reported to play an important part in various physiological and pathological processes. It is well elucidated that human TK gene transfer could promote neurogenesis to protect against neurological
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Fig. 3. TK results in the activation of EGFR and ERK1/2 in SH-SY5Y cells independent of BK receptors. (A, B) SH-SY5Y cells were transiently transfected with B1R siRNAs. After transfection 48 h cells were treated with or without 1 μM TK for 5 min, the level of EGFR or ERK1/2 was detected by Western blot. The p-EGFR or p-ERK1/2 was normalized to total cell EGFR or ERK respectively. At least three individual experiments were performed. Values are represented as mean ± S.D. **, P b 0.01 compared to NC group. (C, D) SH-SY5Y cells were transiently transfected with B2R siRNAs. After transfection 48 h cells were treated with or without 1 μM TK for 5 min, the level of p-EGFR or p-ERK1/2 was detected as described. Total cell EGFR or ERK1/2 was blotted as a control for equal protein loading. Bars represent the mean ± S.D. of at least three individual experiments. **, P b 0.01 compared to NC group.
dysfunction [25]. However, limited information is available on TKinduced actions of nerve growth. The complex function of TK in health and disease and its potential as a therapeutic target underscore the importance of finding out its molecular mechanism. In the present study, we have provided convincing evidence for the role of TK in neurite
outgrowth signaling through EGFR, Flot2 and ERK1/2. We also discovered a positive correlation between EGFR and Flot2 in the TK stimulation. In the initial experiment, our data suggested that TK facilitated the activation of EGFR and ERK1/2 cascade in a concentration-dependent
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Fig. 4. Exogenous active TK mediates neurite outgrowth and stimulates EGFR and ERK1/2 phosphorylation. (A) Cultured primary neurons from 5 DIV were treated with active TK (1 μM) or inactive TK (1 μM) for 48 h and then stained with MAP2. Images captured at 20×. Scale bar = 10 μm. (B) Neurons exposed to active TK, but not inactive form, for 48 h showed increased number of processes and mean process length compared to neurons in control group. N = 25-30 neurons each. **, P b 0.01 compared to control group. ##, P b 0.01 compared to active TK stimulated neurons. (C, D) SH-SY5Y cells were stimulated with active TK (1 μM) or inactive TK (1 μM) for 5 min, and phosphorylated EGFR or ERK1/2 was detected by Western blot. Total cell EGFR or ERK1/2 was blotted as a control for equal protein loading. Bars represent the mean ± S.D. of at least three individual experiments. **, P b 0.01 compared to unstimulated control. ##, P b 0.01 compared to active TK stimulated cells.
manner, which consisted with the previous reports that kallikreins lead to EGFR transactivation followed by ERK1/2 phosphorylation [2,5]. In our in vitro study, we learned that the level of p-EGFR and p-ERK1/2 peaked in serum-starved SH-SY5Y cells exposed to TK at 1 μM for 5 min, which involved neither B1R nor B2R. Therefore, we tried using the concentration on primary neurons and SH-SY5Y cells for further
research. And then we observed that exogenous active TK was able to increase the phosphorylation of EGFR and ERK1/2, as well as the number of processes and mean process length of neurons, which were block by EGFR inhibitor or RNA interference. However, we did not coprecipitate TK with EGFR (data not shown). Correlative experiments showed that members of the TK gene family could directly active G
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Fig. 5. TK-induced neurite outgrowth is dependent on EGFR phosphorylation. (A) Cultured primary neurons from 5 DIV were pre-incubated for 1 h in the presence or absence of the EGFR antagonist, AG1478 (300 nM), prior to treatment with 1 μM TK for 48 h, and then neurons were stained with MAP2 in 7 DIV. Images captured at 20×. Scale bar = 10 μm. (B) Neurons exposed to TK for 48 h showed increased number of processes and mean process length compared to control neurons, which were reduced by AG1478. N = 25–30 neurons each. **, P b 0.01 compared to control group. ##, P b 0.01 compared to TK stimulated group. (C, D) SH-SY5Y cells were pre-incubated for 1 h with or without the EGFR antagonist, AG1478 (300 nM), prior to treatment with 1 μM TK for 5 min, and EGFR or ERK1/2 phosphorylation was detected by Western blot. The p-EGFR or p-ERK1/2 was normalized to total amount of EGFR or ERK respectively. At least three individual experiments were performed. Values are represented as mean ± S.D. **, P b 0.01 compared to control group. ##, P b 0.01 compared to TK stimulated group.
protein-coupled PAR1, PAR2 and PAR4 [26,27]. TK might stimulate the PAR1-mediated protein kinase C and Src kinase pathway, leading to EGFR transactivation resulting from metalloproteinase-induced EGFR ligand shedding [5]. These combined results revealed a novel BK
receptors-independent mechanism of TK action which might contribute to the process of neurite growth via EGFR. The flotillin proteins were reported to function as key regulators of the cell-intrinsic program which was required for axon growth and
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Fig. 6. TK-induced neurite outgrowth is dependent on EGFR activation. (A) Cultured primary neurons from 3 DIV were performed RNA interference experiments. After transfection 48 h neurons were treated with or without 1 μM TK for 48 h, and were stained with MAP2 in 7 DIV. Images captured at 20×. Scale bar = 10 μm. (B) NC transfected neurons exposed to TK for 48 h showed increased number of processes and mean process length compared to NC group, which were blocked by knockdown of EGFR. N = 25–30 neurons each. **, P b 0.01 compared to NC group. ##, P b 0.01 compared to NC/TK group. (C, D) SH-SY5Y cells were transiently transfected with EGFR siRNA. After transfection 48 h cells were treated with or without 1 μM TK for 5 min, the level of EGFR or ERK1/2 was detected by Western blot. The p-EGFR or p-ERK1/2 was normalized to GAPDH or total ERK respectively. At least three individual experiments were performed. Values are represented as mean ± S.D. **, P b 0.01 compared to NC group. ##, P b 0.01 compared to NC/TK group.
regeneration in neurons [21,22]. In support of this theory, we proposed that Flot2 played a fundamental role in the TK-induced neurite outgrowth. Consistent with the published information, we found that Flot2 down-regulation by siRNAs would impair the process of neurite growth in primary neurons. In the current study, the formation and development of neurite processes induced by TK were inhibited in Flot2 knockdown neurons. Our evidence also suggested that there was a reduced phosphorylation of EGFR and ERK1/2 after TK stimulation in
the absence of Flot2, indicating that Flot2 was necessary for TK downstream signaling pathways. These research findings raised the question whether Flot2 was directly involved in the EGFR signaling of TK. Recent published data indicated that one significant function of flotillins might be to regulate the formation of macromolecular complexes/clusters at the plasma membrane which was necessary for initiating cellular processes such as signal transduction, endocytosis and proteolytic processing [28,29].
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Fig. 7. SiFlot2 blocks TK-induced neurite outgrowth through EGFR and ERK1/2 phosphorylation. (A) Cultured primary neurons from 3 DIV were performed RNA interference experiments. After transfection 48 h neurons were treated with or without 1 μM TK for 48 h, and were stained with MAP2 in 7 DIV. Images captured at 20×. Scale bar = 10 μm. (B) NC transfected neurons exposed to TK for 48 h showed increased number of processes and mean process length compared to NC group, which were blocked by knockdown of Flot2. N = 25–30 neurons each. **, P b 0.01 compared to NC group. ##, P b 0.01 compared to NC/TK group. (C, D) SH-SY5Y cells were transiently transfected with Flot2 siRNAs. After transfection 48 h cells were treated with or without 1 μM TK for 5 min, the level of EGFR or ERK1/2 was detected as described. The p-EGFR or p-ERK1/2 was normalized to total cell EGFR or ERK respectively. At least three individual experiments were performed. Values are represented as mean ± S.D. **, P b 0.01 compared to NC group. ##, P b 0.01 compared to NC/TK group.
In line with this, we were able to coimmunoprecipitate EGFR together with Flot2 and vice versa. We detected p-EGFR as well in the TK-stimulated samples which were precipitated with Flot2. Flotillins and EGFR were reported to colocalize after endocytosis [30], therefore it could be reasonable to hypothesize that Flot2 was involved in the TK-mediated activation of EGFR. By means of immunofluorescence
staining, we observed that TK stimulation resulted in translocation of EGFR and Flot2 from plasma and cytoplasm membrane to around the nuclei. Taken together, our experiments indicated that EGFR and Flot2 formed constitutive complex in the TK stimulation, which might act as a molecular mediator to activate the downstream signaling toward ERK1/2.
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Fig. 8. TK-induced neurite outgrowth is dependent on ERK1/2 phosphorylation. (A) Cultured primary neurons from 5 DIV were pre-incubated for 1 h with or without the ERK1/2 antagonist, PD98059 (10 μM), prior to treatment with 1 μM TK for 48 h, and then neurons were stained with MAP2 in 7 DIV. Images captured at 20×. Scale bar = 10 μm. (B) Neurons exposed to TK for 48 h showed increased number of processes and mean process length compared to control neurons, which were reduced by PD98059. N = 25–30 neurons each. *, P b 0.05 and **, P b 0.01 compared to control group. ##, P b 0.01 compared to TK stimulated group. (C, D) SH-SY5Y cells were pre-incubated for 1 h with or without the ERK1/2 antagonist, PD98059 (10 μM), prior to treatment with 1 μM TK for 5 min, and EGFR or ERK1/2 phosphorylation was detected as described. The p-EGFR or p-ERK1/2 was normalized to total cell EGFR or ERK respectively. At least three individual experiments were performed. Values are represented as mean ± S.D. **, P b 0.01 compared to control group. ##, P b 0.01 compared to TK stimulated group.
It has been noted that ERKs, members of the classical MAPK family, were crucial in regulating cellular survival, cell growth and proliferation [31]. Previous information suggested that TK promoted the activation of ERK1/2, which was needed for axon growth and regeneration [21,22], with the involvement of EGFR phosphatase activity [5,32]. Consistent
with these studies, we verified that the effect of TK in neurite outgrowth was blocked by ERK inhibitor, which was also reduced by EGFR antagonist or RNA interference and Flot2 down-regulation. Focus on ERK1/2 activity, we perform our tests by means of Western blot and immunocytochemistry (data not shown), and found that TK could promote the
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Fig. 9. EGFR and Flot2 form constitutive complex in the TK stimulation. (A) SH-SY5Y cells (unstimulated or 1 μM TK stimulated for 5 min) were lysed and immunoprecipitated with antibody against EGFR. Co-immunoprecipitation of EGFR with Flot2 was determined in all samples. (B) SH-SY5Y cells (unstimulated or 1 μM TK stimulated for 5 min) were lysed and immunoprecipitated with antibody against Flot2. Co-immunoprecipitation of Flot2 with EGFR was determined in all samples. In the TK-stimulated samples, two bands against EGFR or p-EGFR antibody were detected by immunoprecipitate that were precipitated with Flot2. Equal loading was verified with GAPDH.
phosphorylation of ERK1/2 which significantly decreased after the intervention with AG1478 or EGFR down-regulation, Flot2 siRNAs and PD98059 respectively. All experimental evidence that we have collected agreed with our hypothesis that TK promoted neurite outgrowth through ERK1/2 activation which occurred downstream of EGFR and Flot2 complex. In conclusion, we demonstrated that TK provided beneficial effects of neurite outgrowth through EGFR, Flot2 and ERK1/2 pathway, which was a novel transmembrane signaling mechanism independent of BK receptors. Combining our previous work [24,33,34] with updated findings here, we indicated the potential of TK as a therapeutic target for
patients with neural injury especially in stroke. However, future studies should explore the neurogenesis role of TK in stroke models. And it will be valuable to exhibit the exact molecular chain of events which takes place during the course of neurological functional reconstruction in vivo.
Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (Nos. 81271295, 81000487).
Fig. 10. TK facilitates the translocation of EGFR and Flot2 to around the nuclei. Starved SH-SY5Y cells were treated with 1 μM TK for 5 min, and then were stained with EGFR (in green), Flot2 (in red) or nuclei (in blue). The addition of TK remarkably promoted the green and the red fluorescence to accumulate around the nuclei, which colocalized with yellow fluorescence in overlay. Images captured at 20×.
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