Skip to main content
NCBI home page
Immunology logoLink to Immunology
. 2010 Aug;130(4):556–563. doi: 10.1111/j.1365-2567.2010.03256.x

β-Arrestin 2 regulates toll-like receptor 4-mediated apoptotic signalling through glycogen synthase kinase-3β

Hui Li 1,2, Xiuli Sun 3,2, Gene LeSage 2, Yi Zhang 2, Zhihou Liang 1, Jixiang Chen 1, Gregory Hanley 4, Lei He 2, Shenggang Sun 1, Deling Yin 2
PMCID: PMC2913266  PMID: 20497256

Abstract

Toll-like receptor 4 (TLR4), a key member of the TLR family, has been well characterized by its function in the induction of inflammatory products of innate immunity. However, the involvement of TLR4 in a variety of apoptotic events by an unknown mechanism has been the focus of great interest. Our investigation found that TLR4 promoted apoptotic signalling by affecting the glycogen synthase kinase-3β (GSK-3β) pathway in a serum-deprivation-induced apoptotic paradigm. Serum deprivation induces GSK-3β activation in a pathway that leads to subsequent cell apoptosis. Intriguingly, this apoptotic cascade is amplified in presence of TLR4 but greatly attenuated by β-arrestin 2, another critical molecule implicated in TLR4-mediated immune responses. Our data suggest that the association of β-arrestin 2 with GSK-3β contributes to the stabilization of phospho-GSK-3β, an inactive form of GSK-3β. It becomes a critical determinant for the attenuation of TLR4-initiated apoptosis by β-arrestin 2. Taken together, we demonstrate that the TLR4 possesses the capability of accelerating GSK-3β activation thereby deteriorating serum-deprivation-induced apoptosis; β-arrestin 2 represents an inhibitory effect on the TLR4-mediated apoptotic cascade, through controlling the homeostasis of activation and inactivation of GSK-3β.

Keywords: apoptosis, β-arrestin 2, glycogen synthase kinase-3β, serum deprivation, Toll-like receptor 4

Introduction

Toll-like receptor 4 (TLR4), an extensively investigated member of the TLR family, represents the first line of defence against invading pathogens in the innate immune system.1 For conventional TLR4 signalling, it specifically recognizes lipopolysaccharide (LPS) from the outer membrane of Gram-negative bacteria and activates two major signalling pathways, nuclear factor-κB (NF-κB) pathway and mitogen-activated protein kinase pathway, both of which control the expression of key immunoregulatory genes.1 In addition to the widely accepted inflammatory response induced by exogenous infection, activation of TLR4 occurs as a result of non-infectious insults such as hypoxia, ischaemia,2,3 concomitantly with cell damage and apoptosis. It is increasingly clear that TLR4 is physiologically relevant in cell death under stressful conditions through poorly defined mechanisms.2,4–6

Although the preponderance of literature ties glycogen synthase kinase-3β (GSK-3β) to cytokine production by activation of TLR4,7,8 actually, as a critical element downstream element of the phosphoinositide 3 kinase (PI3K)/Akt pathway, GSK-3β promotes mitochondria-mediated apoptotic signalling by a broad range of insults.9–13 The GSK-3β is constitutively active whereas phosphorylation of GSK-3β at the regulatory serine residue of position 9 causes its inactivation and turns off downstream effectors.14 Homeostasis of phosphorylation and dephosphorylation of GSK-3β is temporally and spatially controlled in mammalian cells to avoid detrimental responses.15,16 Numerous negative regulators leading to loss of GSK-3β activity, function to inhibit GSK-3β-dependent apoptosis. However, there is still little work focusing on the roles of GSK-3β in the TLR-mediated apoptotic signalling pathway.

β-Arrestin 2, as a scaffold protein, has been traditionally associated with termination of G protein coupled receptor signalling.17 As a result of the identification of new β-arrestin-interacting partners, more novel roles of β-arrestin 2 have been exploited. The interaction of β-arrestin 2 with its signalling partners usually modulates phosphorylation, ubiquitination and/or subcellular distribution of the binding molecules.18 Recruitment of β-arrestin 2 to multiple downstream effectors of the TLR4 signalling pathway negatively regulates the activation of NF-κB and activator protein 1.18–21 Accumulating evidence suggests that β-arrestins function in the anti-apoptotic pathway by impacting the activity of interacted kinases.22–24 In the case of neurokinin-1 receptor, β-arrestin forms a complex with the internalized receptor, src, and extracellular signal-regulated kinase 1/2, thereby facilitating proliferative and anti-apoptotic effects following substance p stimulation.24

In the current study we sought to investigate a possible role of GSK-3β in TLR4-mediated apoptotic signalling and attempted to clarify the underlying mechanism by which TLR4 impairs the cell survival pathway. We established the non-infectious injury cell model through serum deprivation (SD) to determine if and how TLR4 participates in the apoptotic signalling and provided insight into the detrimental effects of TLR4 on SD-induced apoptosis. Our studies reveal that GSK-3β-dependent apoptosis is aggravated in the existence of TLR4. Furthermore, β-arrestin 2 acts as a defender against apoptotic signalling through alteration of GSK-3β phosphorylation.

Materials and methods

Reagents

Total/phospho-GSK-3β (serine 9), total/phospho-Akt (serine 473), pro-/cleaved-caspase-3 antibodies were purchased from Cell Signal Technology (Beverly, MA). Anti-β-arrestin 2 was obtained from Santa Cruz (Santa Cruz, CA) and the GSK-3β inhibitor SB216763 and the PI3K inhibitor LY294003 were obtained from Tocris Bioscience (Bristol, UK).

Cell culture and transfection

All cells were maintained in a basal medium (Dulbecco’s modified Eagle’s medium; Invitrogen Corporation, Carsbad, CA) supplemented with 10% fetal bovine serum in a humidified incubator at 37° under 5% CO2/95% air. Mouse embryonic fibroblasts (MEFs) with or without β-arrestin 2 were generous gifts from Dr Robert Lefkowitz, Duke University Medical Center. The human embryonic kidney 293 (HEK293) cells stably transfected with hTLR4 (HEK293/TLR4) or hTLR2 (HEK293/TLR2) were kindly provided by Dr Evelyn A. Kurt-Jones of the University of Massachusetts Medical School. β-Arrestin 2 full-length vector, short hairpin RNA (shRNA) vector and corresponding cloning vectors were generous gifts from Dr Gang Pei, Shanghai Institutes for Biological Sciences, China. The plasmids pcDNA3-GSK3β (S9A) and pcDNA3-GSK3β (K85A) were kindly provided by Dr Michael Martin (University of Louisville School of Dentistry, Louisville, KY). HEK293 and HEK293/TLR4 cells (3 × 105/dish) were seeded on 35-mm dishes 24 hr before transfection. Transfection was performed with 1 μg vector using LipofectAMINE 2000 reagent (Invitrogen Corporation, Carlsland, CA) according to the manufacturer’s instructions. Forty-eight hours later, the full medium was replaced with the basal medium for later SD experiments.10,11

Quantification of apoptosis by TUNEL assay

Apoptotic cells were determined by terminal deoxynucleotidyl transferase biotin dUTP nick end labelling (TUNEL) assay using an in situ cell death detection kit (Roche Diagnostic, Indianpolis, IN) as described in our previous publications.25,26 The 3′-OH ends of fragmented nucleosomal DNA were specifically labelled in situ in the presence of exogenously added terminal transferase biotin-labelled dUTP, and were detected with alkaline-peroxidase-conjugated anti-fluorescein antibody. Cells were fixed on coverslips with ice cold 4% paraformaldehyde for 30 min and exposed for the appropriate time to a permeabilization solution (0·1% Triton X-100, 0·1% sodium citrate). Coverslips were coated with poly-d-lysine. After washing, 50 μl of TUNEL reaction mixture was placed on the cells and then incubated in a humidified atmosphere for 60 min at 37°. Fifty microlitres of substrate solution was added onto coverslips following convert-AP incubation. Finally coverslips were washed with phosphate-buffered saline and mounted with citiflor. Apoptosis was quantified by scoring the percentage of cells with positive staining at the single cell level. Apoptotic versus total cells were counted in at least five randomly chosen microscopic fields (magnification 20 ×) and 1000 total cells.

Western blot analysis

Western blot was performed as described previously.27 Briefly, protein was extracted by Lysis buffer containing 1% nonidet P-40, 50 mm HEPES, 150 mm NaCl, 1 mm ethylenediaminetetraacetic acid, 1 mm phenylmethylsulphonyl fluoride, 0·1% sodium dodecyl sulphate (SDS), 0·1% deoxycholate and 500 μm orthovanadate. Bradford method was used to determine protein concentration. Cell lysates were resolved by 12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA) and immunoblotted with antibodies following the protocols provided by the manufacturers. The immunocomplex was visualized by an enhanced chemiluminescence reagent (Pierce Biotechnology, Rockford, IL) using appropriate horseradish-peroxidase-conjugated antibodies (Bio-Rad, Hercules, CA). Band intensity was quantified by densitometric analyses using a densitometer.

Statistical analysis

Data were selected using a minimum of three experiments and expressed as means ± SD. The statistical significance of differences in groups was assessed using one-way analysis of variance and Student’s t-test. Differences were considered significant at P<0.05.

Results

Over-expression of TLR4 exerts an additive effect on SD-induced apoptosis

An over-expression of TLR4 has been reported to potentiate basal NF-κB activation and cytokine production.28 In an attempt to investigate the effects of TLRs on apoptosis, HEK293/TLR4, HEK293/TLR2 and HEK293 cells were treated with SD. Numbers of apoptotic cells were quantified after TUNEL assay. Increased apoptosis occurred in both HEK293 and HEK293/TLRs cells, suggesting that SD culture for the times indicated did indeed cause cell apoptosis (Fig. 1a). Interestingly, HEK293/TLR4 exhibited approximately 5% spontaneous cell death without stimulation and apparently ∼ 40% apoptotic cells after 48 hr of SD (Fig. 1a). Furthermore, a higher degree of apoptotic cells was observed in HEK293/TLR4 than that observed in HEK293/TLR2 or HEK293 cells (Fig. 1a).The TLR4 mediated apoptosis is executed by the caspase family based on previous reports.29 Cleavage of caspase-3 was readily detected in HEK293/TLR4 for a period of 48 hr of starvation (Fig. 1b). This indicates over-expressing TLR4 other than TLR2 develops intensified apoptotic events in presence of SD.

Figure 1.

Figure 1

Open in a new tab

Toll-like receptor 4 (TLR4) induces excessive apoptosis following serum deprivation (SD). (a) HEK293/TLR4, HEK293/TLR2 and HEK293 cells were cultured without serum for the indicated times and then apoptosis was quantified as described in the Materials and methods. TLR4 significantly potentiated apoptotic cell death after SD. Error bar ± SD from the mean. (b) Cleavage of caspase-3 was analysed by Western blot after SD for 12 and 24 hr. Similar results were obtained from three independent experiments.

TLR4 accelerates GSK-3β dephosphorylation in response to SD

The above findings prompt an examination of mechanistic links between TLR4 and subsequent apoptotic events. We try to determine whether the abnormal death of HEK293/TLR4 cells is the result of the perturbation of cell intrinsic survival pathways. Inactivation of GSK-3β by upstream PI3K/Akt is the dominant mechanism for the serum-dependent survival pathway.11 Deregulated GSK-3β activity becomes a crucial contributor to SD-mediated apoptosis.9 Hence, GSK-3β phosphorylation was further analysed by Western blot. Without treatment, HEK293/TLR4 cells exhibited a higher level of pAkt/pGSK-3β signalling than that seen in HEK293 cells as shown in Fig. 2(a). Following starvation synchronously in both cells, GSK-3β was progressively dephosphorylated in a time-dependent manner. Interestingly, mild dephosphorylation of GSK-3β occurred in HEK293 cells whereas significant dephosphorylation of GSK-3β occurred in HEK293/TLR4 cells, with an identical alteration of dephosphorylation of Akt, indicating that TLR4 contributes to more GSK-3β activation by SD even with an elevated basal level of pGSK-3β. These results suggest that SD culture activates GSK-3β through promoting GSK-3β dephosphorylation, nevertheless, TLR4 appears to exhibit a dual effect: it inhibits GSK-3β activation under resting conditions but substantially induces activation of GSK-3β upon stimulation of SD.

Figure 2.

Figure 2

Open in a new tab

Glycogen synthase kinase-3β (GSK-3β) dephosphorylation is enhanced by Toll-like receptor 4 (TLR4). Phosphorylation of GSK-3β and Akt was determined by Western blot analysis. The GSK-3β phosphorylation was quantified by densitometry and presented as mean ± standard deviation from three independent experiments (*P<0·05, **P<0·01).

TLR4-mediated apoptosis is associated with GSK-3β

Over-expression of active GSK-3β is sufficient to induce apoptosis in multiple cells.10,12 To confirm whether the impaired survival of TLR4 coincides with enhanced activation of GSK-3β, HEK293/TLR4 cells were pre-treated with the GSK-3β pharmacological inhibitor SB216763 for 24 hr or transfected constitutively with the inactivated mutant GSK-3β (K85A) before SD experiments.8 The percentage of SD-induced apoptotis was decreased by SB216763 in a dose-dependent manner in HEK293/TLR4 (Fig. 3a), but the inhibitory effect on HEK293 cells was not as evident, implying that TLR4-mediated apoptosis involves GSK-3β. Additionally, the inactive GSK-3β (K85A) mutant seems to be effective in rescuing cells from the SD-induced damage in HEK293/TLR4 but not in HEK293 cells (Fig. 3b). Together, these data support the idea that TLR4 activation of GSK-3β is responsible for the enhancement of SD-induced apoptotic signalling.

Figure 3.

Figure 3

Open in a new tab

Pharmacological inhibition or expression of an inactive mutant form of glycogen synthase kinase-3β (GSK-3β) protects trophic-deprived HEK293/TLR4 from apoptosis. (a) HEK293/TLR4 and HEK293 cells were exposed to increasing concentrations (5, 10, 25 μm) of GSK-3β inhibitor SB216763 for 24 hr and then proceeded to starvation for 24 hr. Apoptosis was determined by TUNEL assay. (b) GSK-3β inactive mutant K85A was transfected into HEK293/TLR4 and HEK293 cells, the empty cloning vector pcDNA3.0 was used as a control. Two days after transfection, cells were subjected to non-serum culture for a period of 24 hr. The TUNEL assay was performed to evaluate the percentage of apoptotic cells. Data represent mean values ± standard deviation from three independent experiments (*P<0·05, **P<0·01).

β-Arrestin 2 stabilizes GSK-3β phosphorylation

Arrestins have been shown to be central players in the regulation of multiple kinase pathways,22 many of which are known to regulate cellular growth and proliferation. We found that endogenous β-arrestin 2 was rapidly degraded in HEK293/TLR4 cells in response to SD but not in HEK293 cells (data not shown). β-Arrestin-2-specific interaction with GSK-3β was well described in vivo in the presence of dopamine receptor agonists.30,31 To address whether β-arrestin 2 is involved in the regulation of GSK-3β activity, β-arrestin 2+/+ and β-arrestin 2−/− MEFs underwent SD individually to identify the different responsiveness of the phenotypes to GSK-3β phosphorylation. Our data showed that in the absence of β-arrestin 2, MEFs displayed marked GSK-3β activation, indicated by decreased pGSK-3β even during a short period of starvation, whereas a marginal change of pGSK-3β occurred in β-arrestin 2+/+ MEFs (Fig. 4a). In β-arrestin 2−/− MEFs, pGSK-3β failed was not detected by Western blot after 6 hr of SD. β-Arrestin 2 appears to possess the capability of stabilizing phosphorylated GSK-3β in response to extracellular stimulation. We then asked whether the degradation of β-arrestin 2 was attributable to the exaggeration of SD-induced apoptotic death in HEK293/TLR4 cells. The β-arrestin 2 expression vector was therefore transfected into HEK293/TLR4 before starvation. As anticipated, transduced β-arrestin 2 restored the pGSK-3β level in HEK293/TLR4 cells (Fig. 4b), similar to MEFs in the presence of β-arrestin 2. The converse experiment, knocking down β-arrestin 2 using β-arrestin 2 shRNA vector, was performed as shown in Fig. 4(a,c) and decreased pGSK-3β was noted by β-arrestin 2 RNAi transfection. These data suggest that β-arrestin 2 stabilized pGSK-3β, very close to the scaffold role in activation of Jun N-terminal kinase and extracellular signal-regulated kinase.17

Figure 4.

Figure 4

Open in a new tab

β-Arrestin 2 facilitates glycogen synthase kinase-3β (GSK-3β) phosphorylation. (a) GSK-3β phosphorylation was analysed by Western blot in β-arrestin 2+/+ and β-arrestin 2−/− mouse embryonic fibroblasts (MEFs) starved for the indicated times. (b) β-Arrestin 2 expression vector was transfected into HEK293/TLR4, the corresponding cloning vector pcDNA3.0 as a control. Forty-eight hours after transfection, cells were cultured without serum for the indicated times. β-Arrestin 2 and GSK-3β phosphorylation were detected by Western blot. (c) β-Arrestin 2 short hairpin RNA vector or the control vector pU6 was transfected into HEK293/TLR4 cells. Expression of β-arrestin 2 was examined 48 hr later to verify the effective knock-down. GSK-3β phosphorylation was monitored in the absence of serum for 12 and 24 hr. The results are representative of three independent experiments.

β-Arrestin 2 suppresses SD-induced apoptosis in requirement of GSK-3β inactivation

The association of β-arrestin 2 with GSK-3β potentially correlates with the anti-apoptotic property of β-arrestins.22–24 We compared the SD-induced apoptotic percentage of β-arrestin 2+/+ with β-arrestin 2−/− MEFs. As shown in Fig. 5(a), β-arrestin 2−/− MEFs showed TUNEL-positive cells at higher rate for a period of 24 hr, whereas β-arrestin 2+/+ MEFs seemed relatively resistant to SD-induced apoptosis, which is consistent with the previous observation in N-formyl-peptide-receptor-induced apoptotic events.22 Apoptosis of HEK293/TLR4 was also assessed in the absence or presence of β-arrestin 2. Results also showed that β-arrestin 2 caused reduced apoptosis upon stimulation of SD (Fig. 5b), in agreement with the observation from MEFs. Nevertheless, β-arrestin 2 failed to inhibit apoptosis with statistical significance when co-transfected with GSK-3β active mutant S9A, or pre-treatment with the PI3K inhibitor LY294002, both of which are known to produce active GSK-3β, directly or indirectly,8,11 indicating that highly active GSK-3β is able to mask the anti-apoptotic effect of β-arrestin 2. Therefore, we conclude that GSK-3β inactivation is required for the inhibition of SD-induced apoptosis by β-arrestin 2.

Figure 5.

Figure 5

Open in a new tab

β-Arrestin 2 protects trophic-deprived mouse embryonic fibroblasts (MEFs) and HEK293/TLR4 from apoptosis in the requirement of glycogen synthase kinase-3β (GSK-3β) inactivation. (a) Serum deprivation (SD) –induced apoptosis in β-arrestin 2+/+ MEFs (open circles) and β-arrestin 2−/− MEFs (black circles) was determined by TUNEL assay. Error bar, ± standard deviation from the mean. (b) β-Arrestin 2 expression vector was transfected into HEK293/TLR4 cells in the presence of GSK-3β active mutant S9A or phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (20 μm), TUNEL-positive cells were scored 24 hr after SD. Data represent mean values ± standard deviation from three independent experiments (*P<0·05).

Discussion

Although TLRs are well-defined receptors in the innate immune response against invading pathogens, an additional role of cell surface TLR4 is to sense danger signals from tissue damage, necrotic cells or stressful survival conditions where the infection is not necessary.3 The TLR4 appears to be functionally activated when exposed to such danger signals.1,3 Activation of apoptosis through TLR4 signalling is an alternative regulatory mechanism for deciding cell fate.29,32,33 The current study was designed to identify the potential mechanism accounting for the increased susceptibility to cell damage resulting from trophic withdrawal in the presence of TLR4.

Apoptotic signalling induced by TLR4 shares a number of components from its immune signalling pathway, MyD88, IRF3 for instance.34–36 The GSK-3β previously has been identified as a vital regulator in pro-inflammatory and anti-inflammatory cytokine production through transcription factor cAMP response element binding protein and c-Jun, following LPS treatment.7,8 Also, it has been well characterized as having roles in inhibition of cell proliferation and induction of cell death.9,10,37 The mechanism of how TLR4 induction of apoptosis occurs via GSK-3β is to be addressed in our study.

The GSK-3β is activated in serum deprivation culture because starvation inhibits the upstream PI3K/Akt pathway.10–12 Intriguingly, TLR4 causes dramatic GSK-3β activation relative to the same condition without TLR4. It raises the possibility that the regulation of GSK-3β activity may account for the excessive apoptotic event induced by TLR4. This study demonstrates that excessive apoptosis is attenuated by GSK-3β inhibition. Notably, a reduced apoptotic signal can be achieved by the GSK-3β inhibitor SB216763 or the inactive mutant GSK-3β (K85A). Nevertheless, spontaneous apoptosis has been recorded in HEK293/TLR4 here and is difficult to explain as simple GSK-3β-dependent apoptosis because GSK-3β is in a pre-inhibited state in the resting HEK293/TLR4. This might reflect a complicated and paradoxical GSK-3β regulation system toward apoptosis in different cell states. Alternative apoptotic signalling other than GSK-3β-dependent apoptosis presumably occurs in quiescent conditions whereas GSK-3β-dependent apoptosis emerges upon the extracellular stimulation.

Translocation of β-catenin, resulting from GSK-3β activation, was believed to be involved in the impaired cell proliferation by activation of TLR4.38 Here we provide an alternative explanation for the impaired cell survival by TLR4. β-Arrestin 2 not only terminates G-protein couple receptor signalling but also regulates other signalling pathways.18β-Arrestin 2 signalling complex with Akt/GSK-3β has been well established by Beaulieu et al.,30,31 which illustrates the activation of GSK-3β by β-arrestin 2 through scaffolding PP2A to Akt.30 Conversely, β-arrestin 2 suppresses GSK-3β activity through stabilization of pGSK-3β in the SD-induced apoptotic paradigm in the present study. The different regulation in specific physiological conditions may account for such discrepancy. Moreover, β-arrestin 2 is required for serum-dependent cell survival, just like the PI3K pathway, both of which converge on the inactivation of GSK-3β. It is currently uncertain how β-arrestin 2 stabilizes pGSK-3β, despite the confocal images supporting the effective co-localization of GSK-3β with β-arrestin 2 (data not shown) and our unpublished data suggest that β-arrestin 2 advances GSK-3β phosphorylation in the presence of LPS. However, our data strongly indicate that β-arrestin-2-mediated inactivation of GSK-3β prevents SD-induced apoptosis. Apparently, over-activation of GSK-3β leads to the failure of inhibited apoptosis by β-arrestin 2. The egradation of β-arrestin 2 in HEK293/TLR4 is possibly responsible for the amplification of the GSK-3β-dependent apoptotic cascade. Hence, apart from the well-defined effects on NF-κB1, IκBα, TRAF6 and GRK5 in the TLR4 cascade,18,19,39 GSK-3β is expected to be the additional potent effecter of β-arrestin 2 in the TLR4-primed apoptotic cascade.

Generally, β-arrestin 2 mediates signalling regulation through directly binding to the respective signalling molecules. It gives rise to the question of whether β-arrestin 2 scaffolds with GSK-3β, and subsequently a complex is formed to serve as a molecular switch for activation of proliferative or apoptotic pathways. We have tried but failed to resolve the problem by searching for the putative interaction between β-arrestin 2 and GSK-3β by co-immunoprecipitation, but the correlated study is well underway. However, β-arrestin 2 association of GSK-3β is strongly considered in a growing list of signal patterns that modulate the induction of apoptosis by TLR4.

The mechanism by which SD influences the TLR4 signalling pathway is unclear. Considering most effects of SD on the disruption of cell intrinsic signalling pathways, the mechanism might be indirect, through the release of endogenous ligands.2,3 In any case, inactivation of GSK-3β is a key step that couples TLR4 to the downstream effects. The data presented here are the first to implicate GSK-3β in TLR4-mediated apoptosis. This signalling mechanism has several novel aspects as well as significant implications for TLR studies. We demonstrate that under the stimulation of SD, TLR4 activates the intensive cell death pathway. This pathway includes mechanisms dependent, as well as independent, of GSK-3β signalling. β-Arrestin 2, perhaps serving a scaffolding function with GSK-3β, facilitates and stabilizes pGSK-3β, thereby exerting its anti-apoptotic effect, which may represent a novel mechanism of β-arrestin 2 prevention from apoptosis. In all, our findings provide the evidence that TLR4 promotes apoptotic signalling via regulation of GSK-3β, and β-arrestin 2 bridges GSK-3β inactivation with apoptotic signalling. β-Arrestin 2–GSK-3β functional association, as a therapeutic target, could potentially be designed to regulate TLR4-mediated apoptotic signalling.

Acknowledgments

This work was supported by the National Institutes of Health (NIH) grant DA020120 and the East Tennessee State University Research Development Committee (ETSU RDC) grant 2-25491 to D. Yin. The authors wish to express their appreciation to Dr Gang Pei, Shanghai Institutes for Biological Sciences for β-arrestin 2 full-length vector and shRNA vector; to Dr Robert Lefkowitz, Duke University Medical School, for providing β-arrestin 2+/+ and β-arrestin 2−/− MEFs; to Dr Evelyn A. Kurt-Jones, University of Massachusetts Medical School, for HEK293/TLR4 cells; and to Dr Michael Martin, University of Louisville School of Dentistry, for the plasmid pcDNA3-GSK3β (S9A) and pcDNA3-GSK3β (K85A).

Glossary

Abbreviations:

GSK-3β

glycogen synthase kinase-3β

LPS

lipopolysaccharide

MEF

mouse embryonic fibroblast

NF-κB

nuclear factor-κB

PI3K

phosphoinositide 3-kinase

SD

serum deprivation

shRNA

short hairpin RNA

TLR4

toll like receptor 4

TUNEL

terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling

Disclosures

The authors have no financial conflict of interest.

References

  • 1.Li X, Jiang S, Tapping RI. Toll-like receptor signaling in cell proliferation and survival. Cytokine. 2010;49:1–9. doi: 10.1016/j.cyto.2009.08.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Blanco AM, Vallés SL, Pascual M, Guerri C. Involvement of TLR4/type I IL-1 receptor signaling in the induction of inflammatory mediators and cell death induced by ethanol in cultured astrocytes. J Immunol. 2005;175:6893–9. doi: 10.4049/jimmunol.175.10.6893. [DOI] [PubMed] [Google Scholar]
  • 3.Miyake K. Innate immune sensing of pathogens and danger signals by cell surface Toll-like receptors. Semin Immunol. 2007;19:3–10. doi: 10.1016/j.smim.2006.12.002. [DOI] [PubMed] [Google Scholar]
  • 4.Barton GM, Medzhitov R. Toll like receptor signaling pathways. Science. 2003;300:1524–5. doi: 10.1126/science.1085536. [DOI] [PubMed] [Google Scholar]
  • 5.Marsh B, Stevens SL, Packard AE, Gopalan B, Hunter B, Leung PY, Harrington CA, Stenzel-Poore MP. Systemic lipopolysaccharide protects the brain from ischemic injury by reprogramming the response of the brain to stroke: a critical role for IRF3. J Neurosci. 2009;29:9839–49. doi: 10.1523/JNEUROSCI.2496-09.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Szczepanski MJ, Czystowska M, Szajnik M, et al. Triggering of Toll-like receptor 4 expressed on human head and neck squamous cell carcinoma promotes tumor development and protects the tumor from immune attack. Cancer Res. 2009;69:3105–13. doi: 10.1158/0008-5472.CAN-08-3838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Martin M, Rehani K, Jope RS, Michalek SM. Toll-like receptor-mediated cytokine production is differentially regulated by glycogen synthase kinase 3. Nat Immunol. 2005;6:777–84. doi: 10.1038/ni1221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Wang H, Garcia CA, Rehani K, Cekic C, Alard P, Kinane DF, Mitchell T, Martin M. IFN-β production by TLR4-stimulated innate immune cells is negatively regulated by GSK3-β. J Immunol. 2008;181:6797–802. doi: 10.4049/jimmunol.181.10.6797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Venè R, Larghero P, Arena G, Sporn MB, Albini A, Tosetti F. Glycogen synthase kinase 3β regulates cell death induced by synthetic triterpenoids. Cancer Res. 2008;68:6987–96. doi: 10.1158/0008-5472.CAN-07-6362. [DOI] [PubMed] [Google Scholar]
  • 10.Eom TY, Roth KA, Jope RS. Neural precursor cells are protected from apoptosis induced by trophic factor withdrawal or genotoxic stress by inhibitors of glycogen synthase kinase 3. J Biol Chem. 2007;282:22856–64. doi: 10.1074/jbc.M702973200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Hetman M, Cavanaugh JE, Kimelman D, Xia Z. Role of glycogen synthase kinase-3β in neuronal apoptosis induced by trophic withdrawal. J Neurosci. 2000;20:2567–74. doi: 10.1523/JNEUROSCI.20-07-02567.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Pap M, Cooper GM. Role of glycogen synthase kinase-3 in the phosphatidylinositol 3-kinase/Akt cell survival pathway. J Biol Chem. 1998;273:19929–32. doi: 10.1074/jbc.273.32.19929. [DOI] [PubMed] [Google Scholar]
  • 13.Liu S, Yu S, Hasegawa Y, Lapushin R, Xu HJ, Woodgett JR, Mills GB, Fang X. Glycogen synthase kinase 3beta is a negative regulator of growth factor-induced activation of the c-Jun N-terminal kinase. J Biol Chem. 2004;279:51075–81. doi: 10.1074/jbc.M408607200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Doble BW, Woodgett JR. GSK-3: tricks of the trade for a multi-tasking kinase. J Cell Sci. 2003;116(Pt 7):1175–86. doi: 10.1242/jcs.00384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Wakefield JG, Stephens DJ, Tavaré JM. A role for glycogen synthase kinase-3 in mitotic spindle dynamics and chromosome alignment. J Cell Sci. 2003;116(Pt 4):637–46. doi: 10.1242/jcs.00273. [DOI] [PubMed] [Google Scholar]
  • 16.Delcommenne M, Tan C, Gray V, Rue L, Woodgett J, Dedhar S. Phosphoinositide-3-OH kinase-dependent regulation of glycogen synthase kinase 3 and protein kinase B/AKT by the integrin-linked kinase. Proc Natl Acad Sci U S A. 1998;95:11211–6. doi: 10.1073/pnas.95.19.11211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Luttrell LM, Lefkowitz RJ. The role of beta-arrestins in the termination and transduction of G-protein-coupled receptor signals. J Cell Sci. 2002;115(Pt 3):455–65. doi: 10.1242/jcs.115.3.455. [DOI] [PubMed] [Google Scholar]
  • 18.Wang Y, Tang Y, Teng L, Wu Y, Zhao X, Pei G. Association of beta-arrestin and TRAF6 negatively regulates Toll-like receptor-interleukin 1 receptor signaling. Nat Immunol. 2006;7:139–47. doi: 10.1038/ni1294. [DOI] [PubMed] [Google Scholar]
  • 19.Witherow DS, Garrison TR, Miller WE, Lefkowitz RJ. Beta-arrestin inhibits NF-kappaB activity by means of its interaction with the NF-kappaB inhibitor IkappaBalpha. Proc Natl Acad Sci U S A. 2004;101:8603–7. doi: 10.1073/pnas.0402851101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Parameswaran N, Pao CS, Leonhard KS, Kang DS, Kratz M, Ley SC, Benovic JL. Arrestin-2 and G protein-coupled receptor kinase 5 interact with NFkappaB1 p105 and negatively regulate lipopolysaccharide-stimulated ERK1/2 activation in macrophages. J Biol Chem. 2006;281:34159–70. doi: 10.1074/jbc.M605376200. [DOI] [PubMed] [Google Scholar]
  • 21.Fan H, Luttrell LM, Tempel GE, Senn JJ, Halushka PV, Cook JA. Beta-arrestins 1 and 2 differentially regulate LPS-induced signaling and pro-inflammatory gene expression. Mol Immunol. 2007;44:3092–9. doi: 10.1016/j.molimm.2007.02.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Revankar CM, Vines CM, Cimino DF, Prossnitz ER. Arrestins block G protein-coupled receptor-mediated apoptosis. J Biol Chem. 2004;279:24578–84. doi: 10.1074/jbc.M402121200. [DOI] [PubMed] [Google Scholar]
  • 23.Povsic TJ, Kohout TA, Lefkowitz RJ. β-arrestin1 mediates insulin-like growth factor 1(IGF-1) activation of phosphatidylinositol 3-kinase (PI3K) and anti-apoptosis. J Biol Chem. 2003;278:51334–9. doi: 10.1074/jbc.M309968200. [DOI] [PubMed] [Google Scholar]
  • 24.DeFea KA, Vaughn ZD, O’Bryan EM, Nishijima D, Déry O, Bunnett NW. The proliferative and antiapoptotic effects of substance P are facilitated by formation of a β-arrestin-dependent scaffolding complex. Proc Natl Acad Sci U S A. 2000;97:11086–91. doi: 10.1073/pnas.190276697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Yin D, Tuthill D, Mufson RA, Shi Y. Chronic restraint stress promotes lymphocyte apoptosis by modulating CD95 expression. J Exp Med. 2000;191:1423–8. doi: 10.1084/jem.191.8.1423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Chen L, Zhang Y, Sun X, et al. Synthetic resveratrol aliphatic acid inhibits TLR2-mediated apoptosis and an involvement of Akt/GSK3beta pathway. Bioorg Med Chem. 2009;17:4378–82. doi: 10.1016/j.bmc.2009.05.029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Zhang Y, Zhang Y, Miao J, Hanley G, Stuart C, Sun X, Chen T, Yin D. Chronic restraint stress promotes immune suppression through Toll-like receptor 4-mediated phosphoinositide 3-kinase signaling. J Neuroimmunol. 2008;204:13–9. doi: 10.1016/j.jneuroim.2008.08.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Bihl F, Salez L, Beaubier M, et al. Overexpression of Toll-like receptor 4 amplifies the host response to lipopolysaccharide and provides a survival advantage in transgenic mice. J Immunol. 2003;170:6141–50. doi: 10.4049/jimmunol.170.12.6141. [DOI] [PubMed] [Google Scholar]
  • 29.Banerjee A, Grumont R, Gugasyan R, White C, Strasser A, Gerondakis S. NF-κB1 and c-Rel cooperate to promote the survival of TLR4-activated B cells by neutralizing Bim via distinct mechanisms. Blood. 2008;112:5063–73. doi: 10.1182/blood-2007-10-120832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Beaulieu JM, Marion S, Rodriguiz RM, et al. A beta-arrestin 2 signaling complex mediates lithium action on behavior. Cell. 2008;132:125–36. doi: 10.1016/j.cell.2007.11.041. [DOI] [PubMed] [Google Scholar]
  • 31.Beaulieu JM, Sotnikova TD, Marion S, Lefkowitz RJ, Gainetdinov RR, Caron MG. An Akt/beta-arrestin 2/PP2A signaling complex mediates dopaminergic neurotransmission and behavior. Cell. 2005;122:261–73. doi: 10.1016/j.cell.2005.05.012. [DOI] [PubMed] [Google Scholar]
  • 32.Hsu LC, Park JM, Zhang K, et al. The protein kinase PKR is required for macrophage apoptosis after activation of Toll-like receptor 4. Nature. 2004;428:341–5. doi: 10.1038/nature02405. [DOI] [PubMed] [Google Scholar]
  • 33.Chao W, Shen Y, Zhu X, Zhao H, Novikov M, Schmidt U, Rosenzweig A. Lipopolysaccharide improves cardiomyocyte survival and function after serum deprivation. J Biol Chem. 2005;280:21997–2005. doi: 10.1074/jbc.M413676200. [DOI] [PubMed] [Google Scholar]
  • 34.Chao W. Toll-like receptor signaling: a critical modulator of cell survival and ischemic injury in the heart. Am J Physiol Heart Circ Physiol. 2009;296:H1–12. doi: 10.1152/ajpheart.00995.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Cohen-Sfady M, Pevsner-Fischer M, Margalit R, Cohen IR. Heat shock protein 60, via MyD88 innate signaling, protects B cells from apoptosis, spontaneous and induced. J Immunol. 2009;183:890–6. doi: 10.4049/jimmunol.0804238. [DOI] [PubMed] [Google Scholar]
  • 36.Zhu X, Zhao H, Graveline AR, Buys ES, Schmidt U, Bloch KD, Rosenzweig A, Chao W. MyD88 and NOS2 are essential for toll-like receptor 4-mediated survival effect in cardiomyocytes. Am J Physiol Heart Circ Physiol. 2006;291:H1900–9. doi: 10.1152/ajpheart.00112.2006. [DOI] [PubMed] [Google Scholar]
  • 37.Ougolkov AV, Fernandez-Zapico ME, Savoy DN, Urrutia RA, Billadeau DD. Glycogen synthase kinase-3beta participates in nuclear factor kappaB-mediated gene transcription and cell survival in pancreatic cancer cells. Cancer Res. 2005;65:2076–81. doi: 10.1158/0008-5472.CAN-04-3642. [DOI] [PubMed] [Google Scholar]
  • 38.Sodhi CP, Shi XH, Richardson WM, et al. Toll-like-receptor-4 inhibits enterocyte proliferation via impaired β-catenin signaling in necrotizing enterocolitis. Gastroenterology. 2010;138:185–96. doi: 10.1053/j.gastro.2009.09.045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Sun J, Lin X. Beta-arrestin 2 is required for lysophosphatidic acid-induced NF-κB activation. Proc Natl Acad Sci U S A. 2008;105:17085–90. doi: 10.1073/pnas.0802701105. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Immunology are provided here courtesy of British Society for Immunology

RESOURCES