The first two authors contributed equally to this work.
Akt inhibits MLK3/JNK3 signaling by inactivating Rac1: a protective mechanism against ischemic brain injury
Article first published online: 11 JUL 2006
Journal of Neurochemistry
Volume 98, Issue 6, pages 1886–1898, September 2006
How to Cite
Zhang, Q.-G., Wang, X.-T., Han, D., Yin, X.-H., Zhang, G.-Y. and Xu, T.-L. (2006), Akt inhibits MLK3/JNK3 signaling by inactivating Rac1: a protective mechanism against ischemic brain injury. Journal of Neurochemistry, 98: 1886–1898. doi: 10.1111/j.1471-4159.2006.04020.x
- Issue published online: 11 JUL 2006
- Article first published online: 11 JUL 2006
- Received February 13, 2006; revised manuscript received May 12, 2006; accepted May 16, 2006.
- mixed-lineage kinase 3;
- neuronal death;
The overall goal of this study was to determine the molecular basis by which mixed-lineage kinase 3 (MLK3) kinase and its signaling pathways are negatively regulated by the pro-survival Akt pathway in cerebral ischemia. We demonstrated that tyrosine phosphorylation of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) underlies the increased Akt-Ser473 phosphorylation by orthovanadate. Co-immunoprecipitation analysis revealed that endogenous Akt physically interacts with Rac1 in the hippocampal CA1 region, and this interaction is promoted on tyrosine phosphatase inhibition. The elevated Akt activation can deactivate MLK3 by phosphorylation at the Ser71 residue of Rac1, a small Rho family of guanidine triphosphatases required for MLK3 autophosphorylation. Subsequently, inhibition of c-Jun N-terminal kinase 3 (JNK3) results in decreased serine phosphorylation of 14-3-3, a cytoplasmic anchor of Bax, and prevents ischemia-induced mitochondrial translocation of Bax, release of cytochrome c and activation of caspase 3. At the same time, the expression of Fas-ligand decreases in the CA1 region after inhibition of c-Jun activation. The neuroprotective effect of Akt activation is significant in the CA1 region after global cerebral ischemia. Our results suggest that the activation of the pro-apoptotic MLK3/JNK3 cascade induced by ischemic stress can be suppressed through activation of the anti-apoptotic phosphatidylinositol 3-kinase/Akt pathway, which provides a direct link between Akt and the family of stress-activated kinases.
apoptosis signal-regulated kinase 1
bovine serum albumin
IV, cytochrome c oxidase subunit IV
Cdc42/Rac interactive binding
glycogen synthase kinase 3
c-Jun N-terminal kinase 3
mitogen-activated protein kinase
MAPK kinase kinase
mixed-lineage kinase 3
PBS with 0.1% Tween 20
PIP3-dependent protein kinase 1
pathway, phosphatidylinositol 3-kinase/Akt pathway
phosphatase and tensin homolog deleted on chromosome 10
protein tyrosine phosphatase
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
stress-activated protein kinase/extracellular signal-regulated kinase-1, or MAPK kinase 4
Transient, severe global ischemia in animals leads to selective and delayed neuronal death (also called programmed cell death or apoptosis), particularly of pyramidal neurons in the hippocampal CA1 region; neuronal death is not detected until 2–4 days after the induction of global ischemia in rats and gerbils (Pulsinelli et al. 1982; Tanaka et al. 2000). The cellular decision to undergo apoptosis is determined by the integration of multiple survival and death signals. The phosphatidylinositol 3-kinase (PI3K)/Akt (also known as PKB) pathway is a major cell survival pathway that has recently been studied extensively (Cantley 2002). The Akt family of serine/threonine-directed kinases is activated in a PI3K-dependent manner by a variety of stimuli, including growth factors, protein phosphatase inhibitors and ischemia (Alloatti et al. 2004). Phosphorylation of residues Thr308 and Ser473 is required for Akt activity. Recently, the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has been shown to negatively regulate the PI3K/Akt kinase-mediated pathway, which is implicated in the regulation of cell viability and apoptosis (Lian and Di Cristofano 2005). The PI3K/Akt pathway promotes cellular survival, in part, by phosphorylating and inhibiting death-inducing proteins, including glycogen synthase kinase 3 (GSK-3), Bcl-2/Bcl-xL-associated death protein (BAD), caspase 9, Forkhead transcription factors and apoptosis signal-regulated kinase 1 (ASK1) (Kim et al. 2001; Alloatti et al. 2004). However, the identification of additional target proteins of Akt should provide greater insight into the precise role of this kinase in the regulation of cell survival and apoptosis.
Considerable evidence has suggested that c-Jun N-terminal kinase (JNK) is an important kinase mediating neuronal cell death in response to cerebral ischemia. JNKs are members of the mitogen-activated protein kinase (MAPK) pathway that is activated in response to many extracellular stimuli and different forms of environmental stress. Recently, an intracellular serine/threonine kinase, mixed-lineage kinase 3 (MLK3), has been identified as a novel upstream activator of the JNK pathway (Gallo and Johnson 2002). MLK3 functions as a MAPK kinase kinase (MAPKKK) of the JNK stress pathway by directly phosphorylating and activating the JNK activators stress-activated protein kinase/extracellular signal-regulated kinase-1, or MAPK kinase 4 (SEK1/MKK4) and MKK7. MLK3 has received attention as an important mediator of JNK-mediated neuronal apoptosis and ischemic injury (Xu et al. 2001; Gallo and Johnson 2002; Zhang and Zhang 2005; Zhang et al. 2005). The molecular mechanisms that regulate MLK3 activity have been delineated extensively (Gallo and Johnson 2002). MLK3 contains several protein–protein interaction domains that may be important for its regulation and signaling specificity, including an N-terminal SH3 domain, a centrally located leucine zipper domain, a Cdc42/Rac interactive binding (CRIB) motif and a COOH-terminal region of 220 amino acids that is rich in proline, serine and threonine residues. Zhang and Gallo (2001) have recently reported that MLK3 is autoinhibited through an intramolecular interaction between its SH3 domain and a non-classical SH3 binding sequence located between its zipper and CRIB motifs. They have also found that activated forms of the small guanidine triphosphatases (GTPases) Cdc42 and Rac increase MLK3's autophosphorylation and substrate phosphorylation activity and change the subcellular localization of MLK3, both of which are correlated with changes in MLK3's in vivo phosphorylation status.
Several upstream members of the death-related MLK3/JNK pathway have also been defined. The most distal of these are the Rho small GTPase family members Rac1 and Cdc42. Small GTPases of the Rho subfamily have been traditionally linked to the regulation of cytoskeletal organization in many cell types. These proteins cycle between a GDP-bound state (inactive) and a GTP-bound state (active), which is able to interact and activate downstream signaling effectors. Previous evidence has revealed that Rho-related GTPases, including Rho, Rac and Cdc42, play crucial roles in the potent activation of the JNK family of MAPKs. For example, Rac1 and Cdc42 have been shown to activate the JNKs through interaction with p21-activated kinase (Coso et al. 1995; Tang et al. 1999). The over-expression of constitutively active forms of Rac1 and Cdc42 leads to activation of the JNK pathway and to the death of PC12 cells and sympathetic neurons (Chuang et al. 1997; Bazenet et al. 1998). In addition, inactivation of the small GTPase Rac1 protects the liver from ischemia/reperfusion injury in the rat (Harada et al. 2003). The ability to stimulate stress-activated protein kinases suggests that these Rho GTPases can promote the initiation of apoptotic programs mediated by JNK, thus resulting in cell death.
In this study, we found that global cerebral ischemia elevates the interaction between Rac1 and MLK3 following Rac1 activation, which is correlated with increased activation of the MLK3/JNK3 pathway. We demonstrated that orthovanadate (OV) induces inactivation and activation of PTEN and Akt, respectively, which, in turn, negatively regulate the interaction between Rac1 and Akt and Rac1 activation, suggesting that Rac1 is a target protein of Akt in vivo. Furthermore, inhibition of Rac1 activation by Akt down-regulates the MLK3/JNK3 pathway, which, in turn, regulates the activation of c-Jun and the expression of Fas-ligand (Fas-L), as well as the delayed neuronal cell death induced by cerebral ischemia. Recent studies have shown that JNK can promote Bax translocation to mitochondria through the phosphorylation of 14-3-3 protein, a cytoplasmic sequestration protein of Bax (Tsuruta et al. 2004). Because Bax and JNK are both implicated in stress-induced cell death, we also explored the regulation of the interaction between Bax and 14-3-3 for JNK-mediated apoptotic signals.
Materials and methods
Anti-phospho-Akt (Ser473, #4058), anti-Akt (#9272), anti-phospho-MLK3 (Thr277/Ser281, #2811), rabbit polyclonal anti-cytochrome c (#4280) and anti-cleaved caspase 3 (#9661) antibodies were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). Antibodies to PTEN (sc-9145), Rac1 (sc-217), p-Rac1 (Ser71, sc-12924-R), MLK3 (sc-13072), active JNKs (recognizes JNK1, JNK2 and JNK3, sc-6254), Fas-L (sc-6237), Fas (sc1023), Bax (sc-493), 14-3-3 (sc-1019) and phospho-c-Jun (sc-822) were all purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The monoclonal antibody of the cytochrome c oxidase subunit IV (Cox IV) was obtained from Molecular Probes (Eugene, OR, USA). Rabbit polyclonal anti-JNK3 antibody (#06-749), the Rac1 assay reagent kit (#14-325) and LY294002 were obtained from Upstate Biotechnology Inc. (Lake Placid, NY, USA). The corresponding secondary antibodies were purchased from Sigma (St. Louis, MO, USA). The nitrocellulose filter was obtained from Amersham (Pharmacia Biotech, Buckinghamshire, UK). 5-Bromo-4-chloro-3-indolyl-phosphate (BCIP) and nitroblue tetrazolium (NBT) were purchased from Promega (Madison, WI, USA). All the other chemicals were from Sigma unless indicated otherwise.
Animal model of ischemia and drug administration
Adult male SD rats (Shanghai Experimental Animal Center, Chinese Academy of Sciences, Shanghai, China), weighing 250–300 g, were used. Cerebral ischemia was induced by four-vessel occlusion (4-VO) as described previously (Zhang et al. 2005). Briefly, under anesthesia with chloral hydrate (350 mg/kg, intraperitoneally), vertebral arteries were electrocauterized and common carotid arteries were exposed. Rats were allowed to recover for 24 h and fasted overnight. Ischemia was induced by occluding the common arteries with aneurysm clips. Rats that lost their righting reflex within 30 s, and whose pupils were dilated and unresponsive to light during ischemia, were selected for the experiments. Rats with seizures were discarded. An EEG was monitored to ensure isoelectricity within 30 s after carotid artery occlusion. The carotid artery blood flow was restored by releasing the clips. The rectal temperature was controlled at 36.5–37.5°C before and after ischemia–reperfusion and after treatment with drugs via a temperature-regulated heating pad. Sham control animals received the same surgical procedures except that the carotid arteries were not occluded. When necessary, animals were given OV (15 mg/kg; Sigma) by intraperitoneal injections 20 min before ischemia. The final pH of the OV solution was adjusted to pH 7.4. Control rats received intraperitoneal injections of vehicle (0.9% saline). LY294002 (25 µg in 5 µL of 1% DMSO diluted in saline) was administered to the rats 20 min before ischemia through cerebral ventricular injection (anteroposterior, 0.8 mm; lateral, 1.5 mm; depth, 3.5 mm from the bregma).
For brain tissue preparation, rats were killed under anesthesia at several time points of reperfusion after 15 min of global cerebral ischemia. Whole brains were removed for dissections and the hippocampal CA1 regions were microdissected from both sides of the hippocampal fissure and immediately frozen in liquid nitrogen. When necessary, cytosol fractions and nuclear fractions were extracted as described previously (Ogita and Yoneda 1994). Briefly, tissue samples were homogenized in 1.5 mL of 10 mm HEPES, pH 7.9, 0.5 mm MgCl2, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 50 mm NaF, 5 mm dithiothreitol (DTT), 10 mmβ-phosphoglycerol, 1 mm Na3VO4, 1% Nonidet P40 (NP-40), 1 mm benzamidine and enzyme inhibitors [5 mg/mL phenylmethylsulfonyl fluoride (PMSF) and 5 mg/mL each of pepstatin A, leupeptin and aprotinin] and were then centrifuged for 10 min at 800 g. Supernatants were recentrifuged at 60 000 g for 30 min at 4°C. The supernatant, which corresponded to the cytosolic fraction, was collected, immediately frozen and stored at − 80°C until assay. The nuclear pellets were extracted with 20 mm HEPES, pH 7.9, 20% glycerol, 420 mm NaCl, 0.5 mm MgCl2, 1 mm EDTA, 1 mm EGTA, 1 mm DTT and enzyme inhibitors for 30 min at 4°C with constant agitation. After centrifugation for 15 min at 15 000 g, the supernatant was removed and stored at − 80°C until use. The protein concentrations were determined by the method of Lowry et al. (1951) with bovine serum albumin (BSA) as standard. To prepare mitochondrial fractions, the hippocampal CA1 region was immediately isolated. All procedures were conducted in a cold room. Non-frozen brain tissue was used to prepare mitochondrial fractions because freezing tissue causes the release of cytochrome c from the mitochondria. The hippocampal CA1 tissues were homogenized in 1 : 10 (w/v) ice-cold homogenization buffer. The homogenates were centrifuged at 800 g for 10 min at 4°C. The pellets were discarded, and the supernatants were centrifuged at 17 000 g for 20 min at 4°C to obtain the cytosolic fraction in the supernatants and the crude mitochondrial fractions in the pellets.
Immunoprecipitation and western blotting
For immunoprecipitation, the cytosolic fractions (each containing 400 µg of protein) were diluted four-fold with HEPES buffer containing 50 mm HEPES (pH 7.4), 150 mm NaCl, 10% glycerol, 1% Triton X-100 and 1 mm each of EGTA, EDTA, PMSF and Na3VO4. Samples were pre-incubated for 1 h with 20 µL Protein A/G, and then centrifuged to remove any protein adhered non-specifically to the Protein A/G. The supernatant was incubated with 2–5 µg of appropriate antibodies for 4 h at 4°C. After the addition of Protein A/G-sepharose, the mixture was incubated at 4°C for an additional 2 h. Samples were triple washed with HEPES buffer and eluted by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer, followed by boiling at 100°C for 5 min.
Western blot analysis was carried out by 10–15% SDS-PAGE according to the method described previously (Zhang et al. 2005). Proteins were electrotransferred onto nitrocellulose filter (pore size, 0.45 µm). After blocking for 2 h in PBS with 0.1% Tween 20 (PBST) and 3% BSA, the membranes were incubated overnight with primary antibody in PBST containing 3% BSA. Detection was carried out by the use of appropriate alkaline phosphatase-conjugated IgG (1 : 20 000) and developed with an NBT/BCIP assay kit (Promega).
Rac activity assays were performed according to the manufacturer's protocol (Rac/CDC42 assay kit; Upstate Biotechnology). Precipitated complexes were washed three times with magnesium-containing lysis buffer and boiled in sample buffer. Proteins were fractionated by SDS-PAGE and subjected to western blot analysis using anti-Rac1-specific antibody.
Rats were anesthetized with chloral hydrate and underwent transcardial perfusion with 0.9% saline followed by 4% paraformaldehyde in 0.1 m phosphate buffer. Brains were removed, post-fixed overnight in paraformaldehyde, processed and embedded in paraffin. Coronal brain sections (6 µm thick) were cut on a microtome (Leica RM2155, Nussloch, Germany). Sections were de-paraffinized in xylene and rehydrated in a gradient of ethanol and distilled water. The sections were stained with cresyl violet and examined with a light microscope. The neuronal density of the hippocampal CA1 pyramidal cells was expressed as the number of cells per 1 mm linear length of hippocampal CA1 pyramidal layer counted under a light microscope (× 400).
Four or five independent animals were sampled at each time point for western blotting study and histological examination. Semi-quantitative analysis of the bands was performed with Image J analysis software (Version 1.30v; Wayne Rasband, National Institutes of Health, Bethesda, MD, USA). All values are expressed as the means ± SD. Statistical analysis of the results was carried out by one-way anova, followed by the least-significant difference (LSD) test or Newman–Keuls test. Differences of p < 0.05 were considered to be significant.
Tyrosine phosphatase inhibition promotes tyrosine phosphorylation of PTEN and Ser473 phosphorylation of Akt in the hippocampal CA1 region following global ischemia–reperfusion
To study the activation of Akt protein kinase, we analyzed both total Akt and phosphorylated-Akt (p-Akt) in the post-ischemic hippocampal CA1 tissues by western blot analyses. Total Akt was unchanged in hippocampal CA1 regions after ischemia, but p-Akt was increased strongly over control levels at 30 min and 6 h of reperfusion (Figs 1a and b). Phosphorylation of Akt returned to control levels at 24 h, with no secondary increase at 48 and 72 h of reperfusion (data not shown). However, in groups pre-treated with the tyrosine phosphatase inhibitor OV, Akt-Ser473 phosphorylation was significantly elevated in the early reperfusion period (10 min), but not at 30 min or 6 h, or at later times of reperfusion, relative to the vehicle-treated group. Previous reports have shown that PTEN plays an important role in the PI3K pathway by catalyzing the degradation of phosphatidylinositol-3,4,5-trisphosphate (PIP3) generated by PI3K (Hlobilkova et al. 2003). Several growth factors induce PTEN phosphorylation, probably at tyrosine residues 240, 315 and 336, which is followed by decreased protein stability and enzymatic activity (Mills et al. 2001). To examine whether PTEN was involved in Akt activation by OV, homogenate samples of the hippocampal CA1 region were immunoprecipitated with anti-PTEN antibody and blotted with anti-phospho-tyrosine antibody. The result showed that tyrosine phosphorylation of PTEN was significantly increased at 10 min of reperfusion in OV treatment group (Figs 1c and d), whereas PTEN expression remained unchanged. This suggests that tyrosine phosphorylation of PTEN underlies the increased Akt-Ser473 phosphorylation in the early reperfusion period of global ischemia.
Tyrosine phosphatase inhibition significantly increases/decreases Rac1–Akt/Rac1–MLK3 interactions, Ser71 phosphorylation of Rac1 is increased, and Rac1 activation and the autophosphorylation of MLK3 are inhibited
We initially detected the altered Ser71 phosphorylation of Rac1, which is a consensus phosphorylation site by Akt. The association between Akt and Rac1 was further detected in samples from the hippocampal CA1 region by co-immunoprecipitation. As shown in Figs 2(a) and (b), both the association between Rac1 and Akt and the Ser71 phosphorylation of Rac1 were significantly elevated; however, pull-down assays for Rac1, as detected by western blot, showed that active GTP-bound Rac1 was significantly decreased. As protein phosphorylation is a dynamic and reversible event, it can alter the catalytic activity, conformation, subcellular localization and protein–protein interactions. To understand the function of a particular phosphorylation event after Rac1 phosphorylation, co-immunoprecipitation between Rac1 and MLK3 was identified, which is a critical first step towards understanding the regulatory role of this post-translational modification on MLK3 function. As shown in Figs. 2(c) and (d), the association between Rac1 and MLK3 and the autophosphorylation of MLK3 at 30 min and 6 h of ischemia–reperfusion were simultaneously inhibited. Our observations strongly suggest a novel functional interaction between Akt and MLK3, a pro-apoptotic protein kinase kinase kinase, in cerebral ischemia–reperfusion. Our study also suggests that the inhibition of MLK3 by Akt may be an integral component of the mechanism by which Akt functions as a survival factor and as a negative regulator of stress-activated signals.
Tyrosine phosphatase inhibition strongly decreases the phosphorylation of JNK3 in the hippocampal CA1 region following global ischemia–reperfusion
Because the MAPK JNKs lie downstream of MLK3 MAPKKK, we next examined the activation of JNK isoforms after MLK3 inhibition. Homogenate samples of the hippocampal CA1 region were initially analyzed for the phosphorylation of JNK1/2 by immunoblotting using an anti-phospho-JNK1/2 antibody. What was puzzling was that JNK1/2 activation was not significantly affected after tyrosine phosphatase inhibition (data not shown). Past studies have indicated that JNK1/2 are mainly responsible for the high level of basal JNK activity in the brain, whereas JNK3 is a critical component of stress-induced JNK signaling and neuronal apoptosis. We then examined the phosphorylation of the neural-specific isoform of JNK3, which is predominantly expressed in rodent brain. Interestingly, JNK3 activation at 30 min and 1 day of ischemia–reperfusion was strongly down-regulated on MLK3 inhibition (Figs 3a and b), whereas the protein expression of JNK3 showed no significant changes.
Tyrosine phosphatase inhibition decreases the phosphorylation of c-Jun and subsequent Fas-L expression in the hippocampal CA1 region following global ischemia–reperfusion
The time course of c-Jun phosphorylation was first investigated by western blotting in the hippocampal CA1 region after transient ischemia–reperfusion. As shown in Figs 4(a)and (b), c-Jun phosphorylation was significantly increased at several time points of reperfusion, peaked at 3 h of reperfusion, and was dramatically inhibited by OV (Figs 4c and e). Fas-L, a well-known downstream target protein of activator protein-1 (AP1)/c-Jun, was further investigated. As shown in Figs 4(a) and (b), Fas-L expression was strongly increased in the hippocampal CA1 region from 3 h of reperfusion and reached a peak level at 6 h of reperfusion. The application of OV diminished the increased Fas-L expression (Fig. 4d and f). However, the expression of c-Jun or Fas was not significantly affected.
Tyrosine phosphatase inhibition attenuates the serine phosphorylation of 14-3-3 by JNK, decreases the interaction between 14-3-3 and Bax, and reduces Bax translocation to the mitochondria in the hippocampal CA1 region induced by cerebral ischemia–reperfusion
Strong evidence shows that JNK is a key mediator for the transmission of apoptotic signals to mitochondrial apoptosis-related proteins. Recent studies have indicated that JNK phosphorylates the 14-3-3 protein and promotes Bax dissociation from 14-3-3 and translocation to the mitochondria. To elucidate whether the mitochondria-mediated pathway was involved in MLK3/JNK3 signaling, serine phosphorylation of 14-3-3, the interaction of 14-3-3 with Bax and the expression of Bax in the mitochondria and cytosol after brain ischemia were examined by co-immunoprecipitation and western blotting analyses. As indicated in Figs 5(a) and (b), the interaction between phospho-JNK and 14-3-3 and the serine phosphorylation of 14-3-3 were increased significantly and peaked at 6 h of reperfusion. However, the association of 14-3-3 with Bax decreased significantly from 3 h of reperfusion compared with the sham control. Meanwhile, as shown in Figs 5(c)–(e), tyrosine phosphatase inhibition attenuated the serine phosphorylation of 14-3-3, decreased the interaction of 14-3-3 with Bax in the cytosol, and subsequently prevented Bax translocation to the mitochondria at 6 h of reperfusion.
Tyrosine phosphatase inhibition attenuates cytochrome c release to the cytosol and caspase 3 activation in the hippocampal CA1 region induced by cerebral ischemia–reperfusion
The initiator caspase 8 by Fas-L and the release of cytochrome c to the cytosol, induced by cerebral ischemia–reperfusion, may cooperate to activate caspase 3, which is responsible for the cleavage of important cellular substrates, resulting in the classical biochemical and morphological changes associated with the apoptotic phenotype. The activation of caspase 3 was confirmed at 6 h of reperfusion by immunoblotting with an antibody recognizing the activated fragments for caspase 3 (Figs 6a and c). The administration of tyrosine phosphatase inhibitor can significantly attenuate the release of cytochrome c from the mitochondria to the cytosol and suppress the activation of caspase 3 induced by ischemia–reperfusion (Figs 6a–c). The level of Cox IV, which is a mitochondrial marker protein, was examined in the cytosolic and mitochondrial fractions using anti-Cox IV antibody. Cox IV was not detected in the cytosolic fraction, but was constantly expressed in the mitochondrial fractions, indicating that the detected cytosolic cytochrome c was not the result of mitochondrial damage during preparation of the cytosolic fraction. Collectively, tyrosine phosphatase inhibition not only attenuates c-Jun phosphorylation and down-regulates Fas-L expression, but can also provide the retention of cytochrome c inside the mitochondria and decrease the level of cleaved caspase 3 in the cytoplasm.
Tyrosine phosphatase inhibition rescues neurons from delayed neuronal death in the rat hippocampal CA1 region induced by global cerebral ischemia
To further investigate the potential protective effects against ischemic injury after the inhibition of MLK3/JNK3 signaling, histological analyses were performed on sections from sham, vehicle-treated control animals and animals treated with OV. Cresyl violet staining was used to examine the survived cells of CA1 pyramidal neurons. Normal cells showed round and pale-stained nuclei. Shrunken cells with pyknotic nuclei after reperfusion were counted as dead cells. As shown in Fig. 7, transient cerebral ischemia followed by 5 days of reperfusion induced severe cell death (Figs 7c and d). The neuronal density was not improved in the vehicle-treated groups (Figs 7e and f). However, tyrosine phosphatase inhibition limited neuronal degeneration (Figs 7g and h), which was abolished by treatment combined with the specific blockade LY294002 of PI3K (Figs 7i and j). The results indicate that the inhibition of MLK3/JNK3 signaling by Akt is capable of protecting against delayed neuronal injury induced by reperfusion after ischemia.
The JNK signaling pathway mediates many cellular events, including neuronal cell death. Recent studies have suggested that Akt negatively regulates the JNK signaling pathway and JNK-mediated apoptosis (Cross et al. 2000; Yang et al. 2004). However, the molecular mechanism by which Akt suppresses the JNK signaling pathway remains to be elucidated. Activated Akt has recently been shown to inhibit stress-activated signals through the phosphorylation of both SEK1/MKK4 and ASK1 in the JNK signaling cascade (Kim et al. 2001; Park et al. 2002; Yuan et al. 2003; Song and Lee 2005). The link between Akt and JNK led us to explore the role of Rac1, which is the target for Akt phosphorylation on Ser71, resulting in Rac1 suppression. Our data suggest that MLK3 is another target of Akt in brain ischemia in vivo, because Akt phosphorylation of Rac1 can alter the binding affinity of Rac1 for MLK3 and subsequent dimerization. Thus, cross-talk between the Akt and JNK pathways may regulate the level of cytoprotection versus ischemic neuronal cell death, and provides a new mechanism to explain the cytoprotective actions of Akt.
As mentioned above, MLK3 contains several potential protein–protein interaction domains that probably contribute to its regulation and signaling specificity; MLK3 is autoinhibited through an interaction between its SH3 domain and a non-canonical SH3 binding motif that is situated between the zipper and CRIB motifs. CRIB triggers the dimerization of MLK3 via its tandem leucine zippers, followed by the intramolecular phosphorylation and subsequent activation of MLK3 (Gallo and Johnson 2002). Autophosphorylation of Thr277 and Ser281 is essential for MLK3 kinase activity. Activated forms of the GTPases Cdc42 and Rac1 can bind MLK3 and remove the negative regulation of the SH3 domain on the MLK catalytic domain; co-expression of MLK3 and activated GTPase in cells increases the catalytic activity of MLK3 and alters its in vivo phosphorylation pattern (Teramoto et al. 1996; Bock et al. 2000). Recent evidence has suggested that the survival-promoting PI3K-targeted Akt kinase may phosphorylate Rac1 at Ser71 and abolish its GTPase activity (Kwon et al. 2000). In this study, we have demonstrated that Akt-mediated Rac1 phosphorylation on Ser71 results in a decrease in the binding of Rac1 with MLK3 and negative regulation of JNK signaling in the rat hippocampus. Thus, inhibition of the interaction between Rac1 and MLK3 may be a possible mechanism of Akt-mediated MLK3 inhibition after Rac1 phosphorylation. Other possibilities can also be proposed. For instance, Akt may interact directly and interfere with the activation of MLK3 (Barthwal et al. 2003). Further in vivo studies are needed to clarify this possibility.
Akt has long been studied with regard to its role in promoting cell survival through targets such as BAD and Forkhead, whereas Rac1 has been studied with regard to its role in cell motility and the actin cytoskeleton. Recently, the inhibition of Rac1 has been shown to promote cell survival through a decrease in JNK activation (Bazenet et al. 1998; Harrington et al. 2002; Ito et al. 2004). However, the signaling events downstream of Akt that regulate cell survival in cerebral ischemia are poorly defined. Our results show that the activation of MLK3 and consequent downstream signaling molecules can be negatively regulated by Akt stimulation in the early ischemia–reperfusion period. An increased association between Akt and Rac1, and decreased association between Rac1 and MLK3, in the hippocampal CA1 region after OV treatment further suggest a functional link between these kinases. Akt can therefore interact constitutively with Rac1, but MLK3 phosphorylation is influenced by increased Akt activation. Collectively, the phosphorylation and co-immunoprecipitation results present a potential mechanism for the neuroprotection of the activation of the Akt pathway after drug treatment.
A large and growing body of evidence suggests that the JNK/c-Jun pathway can function in a pro-apoptotic manner. JNK3 is predominantly expressed in the brain and is most consistently associated with neuronal death (Keramaris et al. 2005). Previous studies have shown that targeted disruption of the neural-specific Jnk3 gene, but not Jnk1 or Jnk2, renders mice highly resistant to glutamate excitotoxicity (Yang et al. 1997). In contrast, the Jnk1/Jnk2 compound mutation leads to neural tube defects and embryonic lethality (Kuan et al. 1999; Sabapathy et al. 1999). These studies indicate the functional diversity of JNK isoforms and suggest that JNK3 may have a preferential role in stress-induced neuronal apoptosis. Moreover, a recent study has shown that neural-specific JNK3 plays a critical role in c-Jun phosphorylation and brain ischemic apoptosis in vivo, whereas JNK1 and JNK2 deficiency do not appear to have an effect (Kuan et al. 2003), suggesting that JNK3 is a potential target for neuroprotection therapies in stroke. In this context, Akt has previously been reported to elicit the down-regulation of the JNK pathway. Indeed, inhibition of Rac1 activity is efficient in decreasing MLK3 autophosphorylation and JNK3 activation. It may be possible that Akt binds to a specific complex of scaffold–MLK3–MKK–JNK3 and negatively regulates JNK3 activation, because the formation of distinct signaling complexes is known to regulate the specificity of signal transduction pathways (Pawson and Scott 1997). For example, Akt2 preferentially binds the third SH3 domain of the plenty of SH3s (POSH) scaffold in the POSH–MLK–MKK–JNK complex, and this binding promotes the disassembly of the complex through Akt-mediated MLK3 phosphorylation, leading to decreased activation of the JNK signaling pathway (Figueroa et al. 2003). β-Arrestin 2 is another scaffold protein which preferentially enhances JNK3 phosphorylation through interactions with upstream activators ASK1 and MKK4 (McDonald et al. 2000). However, future studies in animal models will be required to mechanistically identify and assess other binding partners of MLK3 in post-ischemic cell death.
Activated JNK can relay extracellular signals by the alteration of gene transcription via phosphorylation, and can thereby modify the activity of the AP1 group of transcription factors. AP1 stimulation is mainly mediated by the phosphorylation of c-Jun by JNKs, which are the only kinases that phosphorylate at the Ser63 and Ser73 residues of c-Jun in vivo (Karin 1995). c-Jun promotes neuronal cell death by regulating the expression of proteins that directly or indirectly control cytochrome c release (Ham et al. 2000). One possible candidate is the Fas-L gene, a well-known downstream target of c-Jun, which has been shown to be a potential mediator of neuronal apoptosis after Nerve Growth Factor (NGF) withdrawal of PC12 and in cerebral ischemia (Zhang and Zhang 2005). Fas-L belongs to the cytokine family of death-inducing ligands. On interaction with Fas-L, Fas-R forms a complex with the Fas-associated death domain protein, which directly binds and activates caspase, resulting in the induction of apoptosis (DosReis and Borges 2003). Increased expression of c-Jun protein is associated with neuronal damage following global ischemia (Neumann-Haefelin et al. 1994). We have demonstrated herein that MLK3 inhibition is also capable of altering the activity of the transcription factor c-Jun that plays an important role in regulating Fas-L expression. We found that global ischemia induced a marked increase in Fas-L in CA1, evident at 6 h after ischemia; this observation reinforces the concept about the transcriptional control of Fas-L by c-Jun; it is also expected that elevated Fas-L will result in a concomitant sensitization of CA1 cells to Fas-L-mediated death. The ability of Rac1 to elicit the activation of c-Jun via MKK4 has been demonstrated (Coso et al. 1995; Minden et al. 1995; Teramoto et al. 1996; Hall 1999; Schuringa et al. 2001), suggesting that suppression of Rac1 activity by Akt is expected to reduce c-Jun transcriptional activity. In summary, our observations have established the important role of c-Jun phosphorylation by upstream kinases in the regulation of Fas-L expression in cerebral ischemia.
In addition to gene expression regulation, post-translational regulation of BH3-containing proteins is an important mechanism by which JNK promotes apoptotic cell death via the mitochondrial pathway. Recent studies have demonstrated that Bax plays an essential role in inducing apoptosis in response to stress stimuli (Lindsten et al. 2000; Wei et al. 2001; Zong et al. 2001), and Bax translocation appears to be important for neuronal cell death after ischemia (Snider et al. 1999; Ferrer and Planas 2003; Love 2003). A substantial proportion of Bax is bound to 14-3-3 proteins in the cytosol of resting cells. In response to stress stimuli, Bax dissociates from 14-3-3 and redistributes to the mitochondria (Samuel et al. 2001; Nomura et al. 2003). Moreover, recent studies have shown that phosphorylation of 14-3-3 by JNK promotes the dissociation of Bax from 14-3-3, leading to Bax translocation to the mitochondria and to apoptosis (Tsuruta et al. 2004). Here, we have shown that serine phosphorylation of 14-3-3 by JNK releases the pro-apoptotic protein Bax from 14-3-3. As a result of dissociation, Bax translocates to the mitochondria, where it associates with Bcl-2/Bcl-xL and induces cytochrome c release, either by forming a pore by oligomerization in the outer mitochondrial membrane or by opening other channels (Shimizu et al. 1999; Saito et al. 2000; Kuwana et al. 2002). Given that OV can inhibit the activation of JNK3, we suppose that it may have the ability to inhibit the phosphorylation of 14-3-3 protein and prevent Bax translocation to the mitochondria, and thus attenuate the release of cytochrome c and caspase 3 activation. This was indeed found to be the case from western blot results. At the same time, results from cresyl violet staining provided strong evidence that tyrosine phosphatase inhibition can protect the hippocampal CA1 neurons from delayed neuronal cell death. These results suggest that Bax translocation is a novel molecular link between JNK3 activation and the mitochondrial apoptosis signaling pathway in ischemic neurons.
In this study, we have demonstrated in vivo that Rac1 is a binding substrate for phosphorylation by Akt, and that this phosphorylation is associated with a decrease in the autophosphorylation of MLK3. This regulatory event has measurable consequences for MLK3 downstream signaling, including Fas-L expression and caspase activation induced by cerebral ischemia. These results suggest that Rac1 may be a physiological target of Akt, and raises the possibility that the ability of Akt to inhibit stress-activated kinases in specific cell contexts is a consequence of this interaction. In conclusion, as depicted in Fig. 8, the activation of the pro-apoptotic MLK3/JNK3 cascade induced by ischemic stress could be suppressed through the activation of the anti-apoptotic PI3K/Akt pathway, which is also a potential mechanism for the neuroprotection of OV.
This work was supported by a grant from the Key Project of the National Natural Science Foundation of China (No. 30330190).
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