Novel Insights into MSK1 Phosphorylation by MRKβ in Intracerebral Hemorrhage-Induced Neuronal Apoptosis

Intracerebral Hemorrhagic Stroke Model in Rats

All surgical experiments and postoperative animal care were carried out in accordance with the guidelines prescribed by the Chinese National Experimental Animals for Medical Purposes Committee, Jiangsu Branch. Adult male Sprague-Dawley rats (weighing 220–250 g) were obtained from Nantong University Laboratory Animal Center and housed in a temperature-controlled (21°C) and 60–80% humidity environment. Surgeries were performed in mice according to a previous published paper with minor modifications ²⁴ . The rats were anesthetized intraperitoneally with chloral hydrate (10% solution) and positioned in a stereotactic frame. Then, autologous freely dripping blood (100 μL) was collected by cutting the tail tip. Autologous whole blood was injected stereotaxically into the right basal ganglia (coordinates 0.2 mm anterior, 5.5 mm ventral, 3.5 mm lateral to the bregma) at a rate of 10 μL/min by a microinfusion pump. The sham-operated group had only a needle insertion. The sterile syringe was left in place for over 10 min, removed slowly, and the scalp was closed with sutures afterward. Surgical animals (n = 4 per time point) were sacrificed to extract the protein for western blot analysis at 3, 6, and 12 h at 1, 3, 5, and 7 days following ICH. The sham-operated rats (n = 3) were sacrificed on the second day. Additional experimental animals (n = 6 per time point) for sections were sacrificed at each time point for histopathological analysis.

Behavioral Testing Procedures

Cell Cultures and Treatment

Primary cortical neurons were obtained from dissociated cortex of embryonic day-18 (E18) rats. In brief, the cerebral cortices were mechanically dissected and digested with trypsin. After digestion, the precipitate was re-suspended, and the isolated cells were seeded in the culture medium, supplemented with neurobasal medium containing 5% fetal bovine serum, 5% horse serum. Cells were maintained at 37°C in a humidified 5% CO₂ incubator. Half of the culture medium was changed every 2–3 days. Culture neuronal cells were used at day 8. To mimic hemorrhagic conditions in vitro, cells were exposed to hemin (Sigma) in 50 μmol/L for the indicated times.

Western Blotting Analysis

The protein was separated by SDS-PAGE, transferred onto PVDF membranes (Bio-Rad, Hercules, CA, USA), and immunoblotted with antibodies. The primary antibodies used were as follows: rabbit anti-MRKβ (1:1000; Abcam, Cambridge, UK), mouse anti-MSK1 (1:1000; Abcam), mouse anti-p-MSK1 (1:500; Abcam), rabbit anti-Bax (1:1000; Abcam), and mouse anti-caspase-3 (1:500; Abcam). Internal controls included mouse anti- GAPDH (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or mouse anti-β-actin (1:1000; Sigma-Aldrich, St. Louis, MO, USA). After incubation with HRP-conjugated secondary antibodies (anti-rabbit or anti-mouse; 1:2,000; Promega, Madison, WI, USA) for 2 h at 37°C, proteins were visualized using an ECL detection system (ECL, Pierce Company, Waltham, MA, USA). The optical density of the band was assessed using Image J.

Sections and Immunohistochemistry

At the defined survival times, rats were anesthetized and perfused with 0.9% saline and 4% paraformaldehyde separately through the ascending aorta. The brains were removed and fixed in the same fixative overnight, and then the fixative was replaced with 20% sucrose for 2–3 days, and then 30% sucrose for 3–4 days. The tissues were embedded in OCT compound. Then, 5-μm coronary frozen sections were prepared using a freezing microtome. All sections were stored at –20°C until use. For immunohistochemistry (IHC), sections were first blocked with confining liquid consisting of 10% donkey serum, 0.3% Triton X-100, and 1% bovine serum albumin (BSA) for 2 h at RT. Then, the sections were incubated overnight at 4°C with anti-MRKβ antibody (rabbit, 1:100; Abcam) or anti-p-MSK1 antibody (1:100; Abcam) followed by incubation with a biotinylated secondary antibody. Staining was visualized using DAB chromogen. The stained sections were mounted with a Leica light microscope (Leica, Wetzlar, Germany), coupled to a Leica DFC300FX microscope camera.

Immunofluorescent Staining

Rat brain sections were prepared as described above. Cultured neurons were fixed with 4% paraformaldehyde and permeabilized for 3 min with 1% Triton X-100 at room temperature (RT). Brain sections or neuronal cultures were first incubated in blocking solution containing 3% BSA and 0.1% TritonX-100 for 2 h at RT. Then, the sections were labeled with the primary antibodies against the protein of interest overnight at 4°C, and lastly incubated with the specific FITC- and TRITC-conjugated secondary antibodies (1:200; Abcam) for 2 h at RT. Nuclei were stained with DAPI (1:2000, Sigma). The immunoreactive complexes were visualized and analyzed with a Leica TCSSP8 confocal system. Quantitative analysis of the extent of colocalization was performed with ImageJ software.

Co-immunoprecipitation

Tissues surrounding hematomas (2 mm) from the ICH or neurons treated with hemin were lysed in 500 μl RIPA buffer (50 mM Tris-HCl, 1% NP-40, 1% PMSF, 1 mM EDTA-Na, 0.02% sodium azide, 0.1% SDS, 0.5% sodium deoxycholate, 150 mM NaCl). The lysates were centrifuged at 13,000 rpm for 20 min at 4°C to collect the supernatants. The lysate supernatant was incubated with protein G (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C for 2 h followed by incubated with either anti-MRKβ or anti-p-MSK1 antibodies at 4°C overnight. The precipitates were isolated by centrifugation, washed three times with lysis buffer, and immune complexes were eluted by heating at 100°C in loading buffer and used for immunoblotting analysis.

Nuclear and Plasma Separation

The nucleus and plasma separation of neurons was performed according to the nucleus and plasma separation kit (Sangon Biotech Cooperation, Shanghai, China) instructions. After separation, the samples were kept at –80°C until analysis.

Small Interfering RNA Transfection

Three small interfering RNAs (siRNAs) targeting the MRKβ and MSK1 genes were designed and synthesized, and the most effective siRNA (as identified by Western blotting) was applied for further experiments. The sequences of three siMRKβ were made to target the sequence: 5’-GATCCGCCAGTGGTTAGATACTCTG-3’, 5’-TTGAGCTCATGTCGTCTCTCGGTGC-3’, and 5’-AGCTTAAGCCTCTCTTCCATAACCA-3’. The sequences of three siMSK1 were made to target the sequence: 5’-AATCGCATAGCGTATGCCGTT-3’, 5’-AGCCACATGCACGATGTAGGA-3’, and 5’-CATCAGCCACGAGACATCAAC-3’. Scrambled RNA oligonucleotides were used as controls. All plasmids were verified by DNA sequencing analyses. Cells were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), as suggested by the manufacturer. The transfection efficiency was measured by Western blotting. The concentration of siRNA used was 20 μM.

Analysis of Apoptosis by Flow Cytometry

The apoptosis analysis was conducted following the protocol of Annexin V/PI apoptosis detection kit (Calibur, BD Biosciences, Franklin Lakes, NJ, USA). The neurons were treated with hemin for 12 h before they were analyzed by FACS. After treatment, up to 2–9 × 10⁵ cells for each sample were analyzed using CellQuest software.

Statistical Analysis

Stata 7.0 software and GraphPad Prism were used for statistical analysis and data plotting. Quantitative values were presented as means ± SD. Differences of the mean value between groups were calculated using the student’s t test. One-way ANOVA followed by Tukey’s post hoc multiple-comparison tests were used. The experiments were repeated at least three times. P < 0.05 was considered statistically significant.

Spatiotemporal Changes of MRKβ and p-MSK1 Expression after ICH

To assess acute and chronic changes in sensorimotor function and plasticity in the ICH model, two behavioral tests were used: a forelimb placing test and a corner turn test ²⁵ . Rats that received an intracerebral infusion of autologous whole blood had significant forelimb placing deficits and significant increases in the percentage of right (ipsiversive) turns compared with sham controls from days 1 through 14 (Fig. 1A, B). This result showed that the ICH group exhibited apparent neurological deficits in the two tests over the 1st day (*P < 0.05). Western blotting was performed to investigate the temporal levels of MRKβ and p-MSK1 in brain tissues around the hematoma at various time points after ICH. Compared with the sham group, MRKβ protein level increased gradually at 3 h following the ICH operation, and reached maximal levels at day 3. In addition, p-MSK1 (S376) expression was also upregulated in a time-dependent manner. However, total MSK1 expression did not change among the different time groups (Fig. 1C, D). IHC staining with anti-MRKβ and anti-p-MSK1 antibodies on transverse cryosections of brain tissues was performed to examine brain histological changes 3 days after ICH. As shown in Fig. 2, the sham-operated group showed relatively low MRKβ-positive and p-MSK1-positive staining, similar to the level of the contralateral group. On the contrary, the number of MRKβ- and p-MSK1-positive cells in the ipsilateral group was significantly upregulated close to the hematoma (Fig. 2E, F, M, N), which was consistent with the Western blotting data.

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Fig. 1.

Time-dependent variation in MRKβ/MSK1/p-MSK1 protein expression following ICH in rats. Forelimb placing scores (A) and Corner turn testing (B) were displayed at various time points following ICH. (C) Western blot was employed to detect the protein levels of MRKβ, MSK1, and p-MSK1 adjacent to the hematoma at different survival times. (D) The relative protein contents of MRKβ, MSK1, and p-MSK1 were calculated by densitometry analysis, and the data were normalized to corresponding GAPDH levels. The behavioral testing experiments were repeated at least three times.

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Fig. 2.

Expression and distribution of MRKβ and p-MSK1 immunohistochemistry surrounding the hematoma. (A, B) MRKβ was relatively low in the sham group. At day 3 after ICH, the number and intensity of MRKβ positive cells had significantly increased in the ipsilateral group (E, F) compared with the sham control group (A, B) or the contralateral group (C, D). (G) Quantitative analysis of MRKβ-positive cells per square millimeter. (H, I) p-MSK1 staining was detected in sham group. (M, N) Significant increase of p-MSK1 staining was detected 3 days post-ICH in the ipsilateral group (M, N) compared with the sham control group (H, I) or the contralateral group (J, K). (O) Quantitative analysis of p-MSK1-positive cells per square millimeter. *Significant difference at P < 0.05 compared with sham group. Error bars represent SEM. Scale bars 50 μm (A, C, E, H, J, M), 20 μm (B, D, F, I, K, N). The experiments were repeated at least three times.

Subcellular Localization of MRKβ and p-MSK1

To further confirm the subcellular localization of MRKβ and p-MSK1 after ICH, immunofluorescence staining was performed with cell-specific markers. As shown in Fig. 3, double-labeling immunofluorescence staining showed there was no co-localization of MRKβ and GFAP in the brain tissues 3 days after ICH (Fig. 3A–C). In contrast, obvious MRKβ immunoreactivity was observed in NeuN+ cells with neuronal morphology (Fig. 3 G, H). Meanwhile, it was found that p-MSK1 was expressed widely in neurons, with a relatively low level of expression observed in astrocytes in the ICH groups (Fig. 3 D–J). Furthermore, triple-immunofluorescent labeling showed MRKβ+/p-MSK1+ staining was expressed in neurons, which indicated that MRKβ and MSK1 might interact and participate in neuronal apoptosis after ICH.

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Fig. 3.

Immunofluorescence staining for MRKβ and p-MSK1 in brain. (A–C) In the rat after ICH, horizontal sections labeled with MRKβ and astrocyte marker (GFAP). (D–F) Double immunofluorescence staining for p-MSK1 and GFAP. (G–J) Triple immunofluorescence staining for MRKβ, p-MSK1 and neuronal marker (NeuN).The yellow color visualized in the merged images represents the colocalizations of p-MSK1 with GFAP (F). The purple visualized in the merged images represents the colocalizations of p-MSK1 with MRKβ and NeuN (J). Total p-MSK1+ and MRKβ+ cells were counted in the same area after ICH. Scale bars 20 μm (A–J). (L, M) Immunoprecipitation showed that the interaction of MRKβ and p-MSK1 in the experimental group 3 days after ICH.

Interaction of MRKβ and p-MSK1 in Vivo and in Vitro

To verify this possible interaction between MRKβ and p-MSK1 following ICH, an immunoprecipitation assay was performed in the tissues surrounding the hematoma at day 3 following ICH (Fig. 3 L, M). Given that MRKβ and p-MSK1 are closely involved in neuronal apoptosis, we set out to determine the possible functions of the MRKβ-p-MSK1 complex during this process. Rat primary neurons were stimulated with hemin (100 μmol/L) to establish the apoptosis model. Then, we detected the protein levels of MRKβ, p-MSK1, and t-MSK1. The levels of MRKβ and p-MSK1 increased significantly after treatment, with both peaking at 12 h. However, total MSK1 did not change (Fig. 4A, B). To further evaluate the intracellular interaction between MRKβ and p-MSK1, we exposed the neurons to hemin to mimic ICH in vitro for further investigation. Immunoprecipitation assays demonstrated that hemin enhanced the interaction of MRKβ and p-MSK1 in neurons (Fig. 4C, D). In addition, immunofluorescence staining showed the co-localization of MRKβ and p-MSK1 in neurons (Fig. 4E). Interestingly, an obvious alteration in MRKβ subcellular localization was detected by immunofluorescent staining. As shown in Fig. 4E, MRKβ expression was mainly in the cytoplasm in normal neurons, but accumulated in the nucleus 6 h after hemin treatment. Then, we further confirmed the nuclear transportation of MRKβ by Western blotting. Consistent with the immunofluorescence results, the expression of MRKβ in the nucleus increased to a maximum at 6 h post hemin treatment, and decreased gradually thereafter (Fig. 4F). Based on this result, we hypothesized that MRKβ could transfer to the nucleus, where it phosphorylates p-MSK1 and participates in neuronal apoptosis.

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Fig. 4.

MRKβ nuclear transportation and interaction with p-MSK1 in hemin-treated neurons. (A, B) Western blot showing expression patterns of MRKβ, p-MSK1 and MSK1 in neurons after hemin treatment at different times. (C, D) Immunoprecipitation showing that the interaction of MRKβ and p-MSK1 improved significantly in neurons suffered from hemin compared with the sham group. (E) Immunofluorescence assay examining the subcellular distribution and colocalization of MRKβ (red) and p-MSK1 (green) in rat primary neurons following hemin stimulation. Scale bar, 10 μm. (F) Nuclear and cytoplasmic extracts of neurons were prepared as described in ‘Materials and Methods’ and examined by Western blot to determine the nuclear translocation of MRKβ stimulated by hemin. Each experiment consisted of at least three replicates per condition.

MRKβ-mediated MSK1 Phosphorylation Induced Activation of the NF-κB Pathway

Uninjured neurons were collected and the knockdown efficiency of MRKβ siRNA and MSK1 siRNA was examined by Western blotting (Fig. 5A, B). MRKβ protein expression was markedly reduced in MRKβ siRNA-transfected neurons treated with 100 μmol/L hemin for 6 h, compared with the control group. MRKβ downregulation with siRNAs also decreased the protein level of p-MSK1(Ser376) induced by hemin. However, there was no obvious decrease in total MSK1 expression among the various group (Fig. 5C), suggesting that MSK1 may possibly play biological roles after being phosphorylated by the new activator, MRKβ. However, following MSK1 knockdown, there was no significant change in MRKβ expression (Fig. 5D). These results indicate that MRKβ may regulate MSK1 expression in rat primary neurons stimulated with hemin via an upstream phosphorylation cascade. It was reported that phosphorylation of the NF-κB p65 subunit at Ser276 induced by oxidative stress is likely mediated by MSK1. Thus, to further investigate the activation of NF-κB and possible interactions with the MRKβ-p-MSK1 complex in the process of neuronal apoptosis, the main phosphorylation site of p65 (Ser276) was examined. Western blotting confirmed that hemin induced significant phosphorylation of p65 at the second amino acid residue (Ser276). Furthermore, Ser276 phosphorylation induced by hemin was significantly downregulated in neurons transfected with siRNA targeting MRKβ or MSK1 (Fig. 5C, D). Relative protein expression levels were measured and compared with β-actin to create the quantification graphs (Fig. 5E–J).

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Fig. 5.

MRKβ-mediated MSK1 phosphorylation induced the activation of NF-κB pathway. (A) Western blot showed the effects of siRNAs for MRKβ on expression of MRKβ in neurons. (B) Western blot showed the effects of siRNAs for MSK1 on expression of MSK1 in neurons. (C) Effects of siRNA for MRKβ on hemin-induced expression of MRKβ, p-MSK1 (Ser376), MSK1, and p-p65 (Ser276) were detected by Western blotting. (D) Effects of siRNA for MSK1 on hemin-induced expression of MRKβ, p-MSK1 (Ser376), MSK1, and p-p65 (Ser276) were detected by Western blotting. The bar chart indicated the density of MRKβ/ MSK1/p-MSK1/p-p65 versus β-actin (E–J). The data are mean ± SEM. (n = 3, # means P < 0.05).

Regulation of MRKβ-p-MSK1 on Cell Apoptosis in Vitro

To further examine the precise role of MRKβ-p-MSK1 in the process of neuronal apoptosis following ICH, a series of experiments were performed. Western blotting analysis reflected a time-dependent increased synthesis of the activated forms of caspase-3 and Bax, both in brain tissues surrounding the hematoma and in neurons, after stimulation with hemin (Fig. 6A–D). Immunofluorescent staining was also employed to examine the distribution and co-localization of MRKβ, p-MSK1, and activated caspase-3 in the ipsilateral brain cortex. Double immunofluorescent staining showed co-localization of activated caspase-3 and NeuN in the brain cortex 1 day after traumatic brain injury, confirming neuronal apoptosis following ICH (Fig. 6K–M). Additionally, caspase-3 fluorescence appeared in many MRKβ- or p-MSK1-expressing cells at day 1 after ICH (Fig. 6E–J). Flow cytometry using an Annexin V/PI apoptosis detection kit was performed to determine the presence of apoptotic cells. Hemin stimulation caused a notable increase in neuronal apoptosis, whereas both MRKβ-siRNA and p-MSK1-siRNA remarkably induced a higher level of apoptotic cells compared with the control group (Fig. 6 N, O). These results indicated that activated MRKβ-p-MSK1 inhibited neuronal cell death and induced the development of a neuronal protective mechanism against ICH-induced apoptosis.

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Fig. 6.

Modulations of MRKβ-p-MSK1 on neuronal apoptosis after ICH. (A, C) Western blot analysis showed the protein level of active caspase-3 and Bax increased adjacent to the hematoma after ICH. (B, D) Western blot showed active caspase-3 and Bax increased in neurons after hemin exposure. (E–M) Horizontal sections of brain labeled with active caspase-3 (Green F, I, L), NeuN (Red K), MRKβ (Red H) and p-MSK1 (Red E). The yellow color visualized in the merged images represented colocalization of active caspase-3 with NeuN, MRKβ and p-MSK1. (as the white arrows show in G, J, M). Scale bars 20 μm. (N) The apoptotic cells were determined using an Annexin V/PI apoptosis detection Kit (Sigma, St. Louis, MO, USA) for flow cytometry (Calibur, BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer’s instructions. (O) The apoptotic index of different groups according to annexin V/PI staining. * P < 0.05, n = 3.