Increased inflammation and brain injury after transient focal cerebral ischemia in activating transcription factor 3 knockout mice

Neuroscience 220 (2012) 100–108

INCREASED INFLAMMATION AND BRAIN INJURY AFTER TRANSIENT FOCAL CEREBRAL ISCHEMIA IN ACTIVATING TRANSCRIPTION FACTOR 3 KNOCKOUT MICE L. WANG, a,b1 S. DENG, c1 Y. LU, a,b Y. ZHANG, a,b L. YANG, a,b Y. GUAN, d H. JIANG a,b* AND H. LI a,b*2

Conclusions: Our study demonstrated that ATF3 was markedly induced by brain ischemia. ATF3 deficiency exacerbated the inflammatory response and brain injury after cerebral ischemia, potentially through further activation of the NF-jB signaling pathway. ATF3 is likely an important protective regulator in cerebral ischemic injury. Ó 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

a

Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan 430060, PR China

b

Cardiovascular Research Institute of Wuhan University, Wuhan 430060, PR China

c Department of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, PR China

Key words: ATF3, cerebral ischemia, inflammation, NF-jB, CREB.

d

Department of Thoracic and Cardiovascular Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, PR China

INTRODUCTION

Abstract—Background and Purpose: Activating transcription factor 3 (ATF3) is a stress-induced transcription factor that has been shown to repress inflammatory gene expression in multiple cell types and diseases. This study was conducted to investigate the role of ATF3 in the pathological processes of cerebral ischemia and its influence on post-ischemic inflammation. Methods: Wild-type (WT) and ATF3 knockout (KO) mice were subjected to middle cerebral artery occlusion (45 min) followed by reperfusion. Infarct volume, brain edema, and neurological deficits were examined. Neural apoptosis, inflammatory gene expression, cellular inflammatory response and Matrix Metallo Proteinases 9 (MMP9) activity were assessed. Activity of the nuclear factor-kappa B (NF-jB) signaling pathway and cAMP-responsive element-binding protein (CREB) was studied. Results: Knockout of ATF3 significantly exacerbated the infarct volume and worsened neurological function after brain ischemia. Neural apoptosis, inflammatory gene expression and cellular inflammatory response were upregulated in ATF3 KO mice. The MMP9 mRNA expression and protein activity were increased in ATF3 KO mice. KO of ATF3 led to an elevation in the activity of the NF-jB signaling pathway and inhibition of CREB activity.

Stroke is the second leading cause of death and the most frequent cause of disability in adults worldwide (Mathers et al., 2009). Within a few minutes of ischemia onset, neurons are irreversibly injured in the ischemic core. In the penumbra, the tissue is damaged but not yet dead. Evidence indicates that inflammation is an important mechanism in the process of stroke pathogenesis and may determine the fate of brain tissue in the penumbra areas. After cerebral ischemic injury, the expression of various cytokines, chemokines, and cellular adhesion molecules is increased, and a mass of inflammatory cells are activated and recruited to the ischemic areas (Jin et al., 2010). Previous evidence revealed that several transcription factors participate in this inflammatory process, including activator protein 1 (AP-1), nuclear factor-kappa B (NF-jB), and signal transducer and activator of transcription 3 (STAT3) (Stephenson et al., 2000; Yi et al., 2007). These transcription factors are activated by ischemic injury to induce signaling cascades and regulate inflammatory gene expression. Activating transcription factor 3 (ATF3) is a member of the mammalian activation transcription factor/cAMPresponsive element-binding protein (CREB) family of transcription factors. It both homodimerizes and heterodimerizes with other members of the CREB/ATF family or with AP-1 and CCAAT/enhancer binding protein (C/ EBP) family proteins (Chen et al., 1994). ATF3 has different activities as a homodimer or heterodimer, and its transcriptional activity differs according to its counterpart. The expression of ATF3 is ubiquitous, but it is maintained at a very low level in the absence of cellular stressors; however, it is induced quickly under stress. Abundant evidence shows that the ATF3 gene is induced in many tissues by various stress signals. In neurons, ATF3 is induced by hypoxic stress, leukemia inhibitory factor

*Correspondence to: H. Jiang or H. Li, Department of Cardiology, Renmin Hospital of Wuhan University, Jiefang Road 238, Wuhan 430060, PR China. Tel: +86-27-88041911; fax: +86-27-88040334. E-mail addresses: [email protected] (H. Jiang), lihl@whu. edu.cn (H. Li). 1 These authors contributed equally to this work. 2 Tel/fax: +86-27-88076990. Abbreviations: AP-1, activator protein 1; ATF3, Activating transcription factor; CBF, cerebral blood flow; CREB, cAMP-responsive elementbinding protein; HDAC1, histone deacetylase 1; ICAM, intercellular adhesion molecule; IL, interleukin; KO, knockout; MCA, middle cerebral artery; MCP, monocyte chemoattractant protein; MMP9, Matrix Metallo Proteinases 9; NF-jB, nuclear factor-kappa B; TLR, Toll-like receptor; tMCAO, transient middle cerebral artery occlusion; TNF, tumor necrosis factor; TTC, 2,3,5-triphenyl-2H-tetrazolium chloride; VCAM, vascular cell adhesion molecule; WT, wild-type.

0306-4522/12 $36.00 Ó 2012 IBRO. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuroscience.2012.06.010 100

L. Wang et al. / Neuroscience 220 (2012) 100–108

stimulation, nerve growth factor withdrawal, and ischemic injury (Tsujino et al., 2000; Kiryu-Seo et al., 2008; Zhang et al., 2011). Previous studies indicate that ATF3 represses the expression of inflammatory genes in multiple cell types and diseases, such as lipopolysaccharide (LPS)-treated macrophages, murine asthma and islet transplantation (Gilchrist et al., 2006, 2008; Zmuda et al., 2010). However the functional role of ATF3 in the pathophysiological processes of stroke and its influence on post-ischemic inflammation have not yet been identified. In this study, ATF3 knockout (KO) mice on a C57BL/6 background were utilized to produce a model of transient focal cerebral ischemia. Infarct volumes were larger, neurological score outcomes were worse, and inflammatory gene expression and inflammatory cell recruitment were dramatically higher in ATF3 KO mice than in wild-type (WT) mice. These findings indicate that ATF3 plays a role in inflammation repression and neuron protection in ischemic stroke.

EXPERIMENTAL PROCEDURES Animals All animal procedures were approved by the Wuhan University Animal Ethics Committee. The animal experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised 1996. The ATF3 KO mice were kindly provided by Dr. Tsonwin Hai from the Department of Molecular and Cellular Biochemistry and the Center for Molecular Neurobiology, Ohio State University, Columbus, Ohio, USA. Genotyping was performed by polymerase chain reaction (PCR), as described previously (Hartman et al., 2004). Male, 10- to 12-week-old WT and ATF3 KO mice on a C57BL/6J background were used in this study.

Mouse transient focal cerebral ischemia model Procedures for transient middle cerebral artery occlusion (tMCAO) were described previously (Connolly et al., 1996). Briefly, the animals were anesthetized with 2.5–3% isoflurane in O2. Rectal temperature was maintained at 37 ± 0.5 °C with heating pad. A probe was fixed to the skull (2 mm posterior and 5 mm lateral to the bregma) and connected to a laser-Doppler flowmetry (Periflux System 5010; Perimed, Sweden) for continuous monitoring of cerebral blood flow (CBF). For tMCAO, a 6–0 silicon-coated monofilament surgical suture (Doccol, Redland, CA) was inserted into the left external carotid artery, advanced into the internal carotid artery, and wedged into the cerebral arterial circle to obstruct the origin of the middle cerebral artery (MCA). An interruption of the CBF in the MCA territory was confirmed by documenting a >80% decline in relative CBF. The filament was left in place for 45 min and then withdrawn. A return to >70% of basal CBF within 10 min of suture withdrawal confirmed a reperfusion of the MCA territory.

Neurological deficit scores Three days after tMCAO, the neurological deficits were tested with a 9-point scale (Xia et al., 2006). No neurological deficit scored as 0; left forelimb flexion when suspended by the tail or failure to extend right forepaw fully as 1; left shoulder adduction when suspended by the tail as 2; reduced resistance to lateral push toward the left side as 3; spontaneous movement in all directions with circling to the left exhibited only if pulled by tail

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as 4; circle or walk spontaneously only to the left as 5; walk only when stimulated as 6; no response to stimulation as 7; and stroke-related death as 8.

Measurement of infarct volume Infarct volume and swelling were measured at 3 days after tMCAO by 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) staining. Brains were cut into 1-mm-thick coronal sections using a mouse brain matrix, stained with 2% TTC in phosphate buffer (pH 7.4) for 15 min at 37 °C, then transferred to 10% formalin solution and fixed overnight. Fixed sections were photographed, and volume of infarct area was quantitated with Image-Pro Plus 6.0. To correct for the effect of edema, the area of infarction was measured by subtracting the area of the nonlesioned ipsilateral hemisphere from that of the contralateral hemisphere. The volume of infarction was calculated by integration of the lesion areas at the seven measured levels of the brain.

Immunofluorescence staining Mice were anesthetized 72 h after tMCAO with sodium pentobarbital and were perfused through the left ventricle with 0.1 mol/L sodium phosphate buffer under 100 mm Hg pressure for 5 min followed by a fixative solution that contained 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4) for 15 min. The brains were removed carefully, postfixed for 6–8 h in the same fixative solution at room temperature and then immersed in a 0.1 mol/L phosphate buffer that contained 30% sucrose overnight at 4 °C. Brains were embedded in OCT, and serial frontal sections were cut with a cryostat microtome. For immunofluorescence staining, the sections were washed in phosphate buffer solution (PBS) containing 10% goat serum. Next, sections were incubated in primary antibody solutions overnight at 4 °C. Primary antibodies included: anti F4/80 (Serotec, MCA497), anti 7/4 (abcam, ab53457), and anti glial fibrillary acidic protein (GFAP) (Epitomics, 146464). After sections were washed in PBS, they were incubated in each respective secondary antibody for 1 h: Anti-rat IgG Alexa Fluor 555 Conjugate (Cell Signaling Technology, 4417) for anti F4/80 and anti 7/4, goat anti rabbit IgG Alexa Fluor 488 Conjugate (Invitrogen, A11008) for anti GFAP. Finally, the nuclei were labeled with 4’,6-diamidino-2phenylindole (DAPI) Visualization was performed under fluorescence microscopy (OLYMPUS DX51) using the software DP2BSW (version 2.2). Image analysis was performed with ImagePro Plus (version 6.0).

TUNEL staining Twenty-four hours after the onset of ischemia, the brains were collected and sliced as described above. For NeuN immunofluoresence staining, the sections were washed in PBS that contained 10% goat serum and 0.1% Triton X-100. Next, sections were incubated in the anti-NeuN antibody for 2 h and incubated in secondary antibody for 1 h at 37 °C. After the NeuN immunofluorescence staining was completed, TUNEL staining was performed using the In Situ Cell Death Detection Kit (Roche Diagnostics) according to the manufacturer’s protocol. Finally, the nuclei were labeled with DAPI. DNA fragmentation was quantified under highpower magnification (200X). The percentages of TUNEL-positive cells relative to all DAPI-positive cells were counted by an investigator who was blinded to the studies.

Tissue preparation For quantitative real-time PCR and western blotting analysis, mice were anesthetized with sodium pentobarbital and perfused through the left ventricle with cold sodium phosphate; the brains were then quickly removed. To collect tissue in an unbiased

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manner that reflected the global extent of the infarcts, the olfactory bulbs and front and back 1 mm of the brain tissue were excised in all mice, and then, the left hemispheres of the remaining brains were collected (including the infarct area and peri-infarct area). Brain tissue was frozen in liquid nitrogen immediately and was then transferred to a 80 °C freezer for storage.

Quantitative real-time PCR Total RNA was prepared from snap-frozen tissue specimens using TRIzol (Invitrogen) and was reverse transcribed from 2 lg of RNA of each sample using the Transcriptor First Strand cDNA Synthesis Kit (Roche). The specific mRNA expression levels of ATF3, vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1, cluster of differentiation 36 (CD36) monocyte chemoattractant protein (MCP)-1, interleukin (IL)-1b, IL-6, IL-10, and tumor necrosis factor (TNF)-a were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to calculate relative mRNA expression levels. Quantitative RT-PCR analysis was performed using the LightCycler 480 SYBR Green 1 Master Mix (Roche) and the LightCycler 480 QPCR System (Roche). The following sequence-specific primers were used: ATF3 forward: 50 -TGTCAGTCACCAAGTCTGAGGCGG-30 ; ATF3 reverse: 50 -GCCGGTGCAGGTTGAGCATGTA-30 ; MCP-1 forward: 50 -TGGCTCAGCCAGATGCAGT-30 ; MCP-1 reverse: 50 -CCAGCCTACTCATTGGGATCA-30 ; TNF-a forward: 50 -CATCTTCTCAAAATTCGAGTGACAA-30 ; TNF-a reverse: 50 -TGGGAGTAGACAAGGTACAACCC-30 ; IL-1b forward: 50 -CCGTGGACCTTCCAGGATGA-30 ; IL-1b reverse: 50 -GGGAACGTCACACACCAGCA-30 ; IL-6 forward: 50 -AGTTGCCTTCTTGGGACTGA-30 ; IL-6 reverse: 50 -TCCACGATTTCCCAGAGAAC-30 ; IL-10 forward: 50 -TGAATTCCCTGGGTGAGAAG-30 ; IL-10 reverse: 50 -CTCTTCACCTGCTCCACTGC-30 ; VCAM-1 forward: 50 -ATTTTCTGGGGCAGGAAGTT-30 ; VCAM-1 reverse: 50 -ACGTCAGAACAACCGAATCC-30 ; ICAM-1 forward: 50 -GGAGCCTCCGGACTTTCGATCT-30 ; ICAM-1 reverse: 50 -AGCGGCAGGGTTCTGTCGAA-30 ; CD36 forward: 50 -TGGGTTTTGCACATCAAAGA-30 ; CD36 reverse: 50 -GATGGACCTGCAAATGTCAGA-30 ; GAPDH forward: 50 -ACTCCACTCACGGCAAATTC-30 ; GAPDH reverse: 50 -TCTCCATGGTGGTGAAGACA-30 .

Western blotting Western blotting was conducted to determine the protein level of phosphorylated IjB kinase (IKK) a/b (ser176/180) (p-IKK a/b, Cell Signaling Technology, 2697), IKK b (Cell Signaling Technology, 2370), phosphorylated IjBa (Ser32/36) (p-IjBa, Bioworld, BS4105), phosphorylated NF-jB p65 (ser536) (p-NF-jB, Bioworld, BS4138), NF-jB p65 (Cell Signaling Technology, 4764), phosphorylated-CREB (Ser 133) (Cell Signaling Technology, 9198), CREB (Santa Cruz, sc69367). Western blot analysis was performed with a 50-lg protein extract separated on 8– 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) gels that was subsequently transferred to PVDF membrane (Millipore). Blocking of membranes (5% skimmed milk powder), washes (PBS) and secondary anti-body (Goat-anti Rabbit IRDye 800CW or Goat-anti Mouse IRDye 800CW, Licor Biosciences) incubations were all performed at room temperature for 1 h, whereas primary antibodies were allowed to incubate overnight at 4 °C. Signals were detected using the Odyssey infrared imaging system (Li-Cor Biosciences). The specific protein expression levels were normalized to GAPDH (Cell Signaling Technology, 2118).

Gelatin zymography Matrix Metallo Proteinases-9 (MMP-9) activity in brain homogenates was determined by gelatin zymography as previously described (Wakisaka et al., 2010). Briefly, mice were perfused with ice-cold PBS, and brains were then quickly removed and dissected on ice. The ipsilateral and contralateral hemispheres (coronal levels 2–4 mm relative to the bregma) were homogenized with lysis buffer (50 mmol/L Tris–HCl pH 7.5, 150 mmol/L NaCl, 10 mmol/L CaCl2, 0.05% Brij-35, 0.02% Sodium azide, 2 mmol/L PMSF, and 2% DMSO). Supernatants obtained from homogenized brain tissue were then mixed with an equal volume of 2X SDS gel sample loading buffer (126 mmol/L Tris–HCl, 20% glycerol, 4% SDS, 0.005% bromophenol blue, pH 6.8). Equal amounts of protein (50 lg) were loaded into each lane and were separated on NovexÒ 10% Zymogram (Gelatin) gels (Invitrogen, EC6171BOX). Gels were run at 120 V at 4 °C until the dye reached the bottom of the gel. Next, the gels were incubated with 1X NovexÒ Zymogram Renaturing Buffer (Invitrogen, LC2670) for 30 min at room temperature with gentle agitation and then incubated in 1X NovexÒ Zymogram Developing Buffer (Invitrogen, LC2671) at room temperature for 30 min with gentle agitation. The gels were changed to fresh 1X NovexÒ Zymogram-Developing Buffer once and were incubated at 37 °C for 48 h. Gels were stained with the Colloidal Blue Staining Kit (Invitrogen, LC6025) for 3–12 h and were then destained with water for 3–7 h. Areas of protease activity appeared as clear bands against a dark background. Images of the gels were scanned using densitometry (Fluochem E, Cell Biosciences), and quantification was performed with Quantity One 4.6.2 software (Bio-Rad Laboratories).

Statistical analysis Data are expressed as the means ± SEM. Differences among the groups were determined by a two-way ANOVA followed by a post hoc Tukey test. Comparisons between the two groups were performed by the unpaired Student’s t-test. A P-value of <0.05 was accepted as the level of statistical significance.

RESULTS The expression of ATF3 was dramatically increased after cerebral ischemia Although minimal expression of ATF3 mRNA was observed in normal adult mouse brains, ATF3 mRNA was dramatically elevated in the ischemic brains of adult mice. Real-time PCR revealed that ATF3 mRNA expression increased 60-fold at 6 h after ischemic onset and subsequently fell to 20 times the baseline level after 24 h (Fig. 1A). To confirm ATF3 expression, double immunofluorescence for ATF3 and the neuronal marker NeuN was performed. ATF3 was mainly expressed in the nuclei of the injured neurons in ischemic brain at 6 and 24 h after ischemic onset (Fig. 1B). ATF3 deletion exacerbated cerebral injury after tMCAO According to laser Doppler flowmetry monitoring, CBF was similar between the WT and ATF3 KO mice during both ischemia and reperfusion (Fig. 2A). Infarct volumes increased and neurological deficit scores worsened in ATF3 KO mice. In the WT mice, MCA occlusion for 45 min followed by 23 h and 15 min of reperfusion led to ipsilateral cerebral infarcts with an average volume of

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Fig. 1. The expression levels of ATF3 mRNA and protein are significantly elevated in ischemic brains. (A) Real-time PCR revealed that ATF3 mRNA expression increased 60-fold at 6 h (#P < 0.05 vs. WT, ⁄P < 0.05 vs. sham, n = 6) after ischemic onset and fell to 20 times the baseline level at 24 h (#P < 0.05 vs. WT, ⁄P < 0.05 vs. sham, n = 6) after ischemic onset in WT mice. In ATF3 KO mice, the mRNA of ATF3 cannot be detected. (B) Double immunofluorescence for ATF3 and the neuronal marker NeuN showed the expression of ATF3 and NeuN were overlapped which indicated that ATF3 was mainly expressed in the nuclei of the neurons in the ischemic brain at 6 and 24 h after ischemic onset.

Fig. 2. ATF3 deficiency exacerbated brain injury after tMCAO. (A) There was no significant difference in the CBF between ATF3 KO mice and WT mice during ischemia or the reperfusion process. (B) ATF3 deficiency significantly increased the infarct volume (#P < 0.05 vs. WT, n = 7). (C) ATF3 deficiency significantly worsened neurological function after tMCAO (#P < 0.05 vs. WT, n = 17). (D) ATF3 KO mice lost more weight than WT mice (P = 0.093, n = 17).

21.22 ± 3.11 mm3, as determined by TTC staining. ATF3 KO mice had a significantly larger average infarct volume (47.61 ± 4.04 mm3), an approximate 40% increase. The increase in infarct size in ATF3 KO mice was mainly in the posterior part of the MCA territory (Fig. 2B), which indicates that ATF3 plays an important role in preventing the development of ischemic infarction in the penumbral

region. Neurological deficits were tested 24 h after the onset of ischemia with a 9-point scale according to previous research. Neurological deficit scores were worse in ATF3 KO mice compared to WT mice (Fig. 2C). The weight lost percentage of ATF3 KO mice was a little lower than WT mice, but there were no significant statistical differences between the two groups (Fig. 2D).

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Fig. 3. Neural apoptosis was upregulated in ATF3 KO mice. TUNEL and NeuN double staining showed that the apoptotic cells were mainly neurons. ATF3 deficiency significantly increased the rate of apoptosis in neurons in the peri-infarct area (#P < 0.05 vs. WT, n = 4).

Neuronal apoptosis increased in ATF3 KO mice Neuronal apoptosis was detected by TUNEL and NeuN staining. There were abundant TUNEL-positive cells in the peri-infarct area at 24 h after the onset of cerebral ischemia. NeuN immunofluorescence staining revealed that most TUNEL-positive cells also expressed NeuN, indicating the apoptotic cells at 24 h were mainly neurons. The number of TUNEL-positive nuclei and total nuclei (DAPI-labeled) were counted, and the apoptotic rate was calculated as follows: apoptotic rate (%) = TUNELpositive nuclei/total nuclei. The apoptotic rate in the periinfarct area of ATF3 KO mice was 49.33 ± 6.40%, which was considerably higher than that for WT mice (19.36 ± 0.89%, Fig. 3). Inflammatory gene expression and inflammatory cell recruitment are increased in ATF3 KO mice Quantitative real-time PCR was performed to detect inflammatory gene expression at 6 and 24 h after the onset of cerebral ischemia. The specific mRNA levels were normalized to GAPDH. The adhesion molecules VCAM-1 and ICAM-1, the chemokine MCP-1, the cytokines IL-1b, IL-6, and TNFa, and the scavenger receptor CD36 were elevated after transient cerebral ischemia, and the elevation was exacerbated in ATF3 KO mice (Fig. 4A). Inflammatory cell recruitment was detected by immunofluorescence staining. Anti-CD45 antibody was used to detect leukocytes, anti-7/4 antibody was used to detect neutrophils, anti-F4/80 antibody was used to detect macrophages/microglia, and anti-GFAP antibody was

used to detect astrocytes. Inflammatory cell recruitment was significantly increased in ATF3 KO mice (Fig. 4B). MMP9 mRNA levels and enzymatic activity were elevated in ATF3 KO mice MMP9 mRNA levels were detected by quantitative realtime PCR. MMP9 mRNA levels were increased at 6 and 24 h after transient focal cerebral ischemia, and this increase was markedly greater in ATF3 mice (Fig. 5A). Gelatin zymography was performed to evaluate the activity of MMP-9 (97 kDa) (Fig. 5B). The sham group expressed very low levels of the active form of MMP-9. After transient focal cerebral ischemia, the active form of MMP-9 in the ipsilateral hemisphere increased, and KO of ATF3 significantly facilitated the induction of the active form of MMP-9 (Fig. 5C). Higher activity of the NF-jB signaling pathway and the inhibition of phosphorylated CREB were detected in ATF3 KO mice NF-jB signaling pathway activity was detected by western blotting. P-IKKa/b, p-IjBb, and p-NF-jB p65 were distinctly elevated in the ischemic brains of ATF3 KO mice. There were no differences in total IKK, IjB, or NF-jB p65 protein levels between WT mice and ATF3 KO mice. These results indicate that NF-kB signaling pathway activation is exaggerated in ATF3 KO mice. Phosphorylation of CREB was also detected. The protein levels of phosphorylated CREB were significantly inhibited in the ischemic brains of ATF3 KO mice (Fig. 6).

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Fig. 4. The inflammatory response was aggravated in ATF3 KO mice after tMCAO. (A) Quantitative real-time PCR revealed significant increases in the mRNA levels of VCAM-1, ICAM-1, CD36, MCP-1, IL-1b, IL-6, IL-10, and TNFa in the ischemic brains of ATF3 KO mice (#P < 0.05 vs. WT,⁄P < 0.05 vs. sham, n = 6). (B) Immunofluorescence staining revealed that the recruitments of neutrophils (7/4 positive cells), macrophages/ microglia (F4/80 positive cells) and astrocytes (GFAP positive cells) were significantly increased in ATF3 KO mice (#P < 0.05 vs. WT, n = 4).

DISCUSSION We investigated the effects of the KO of ATF3 on infarct size, neurological deficit scores, neuronal apoptosis, inflammatory gene expression and cellular inflammatory response to transient cerebral ischemia. KO of ATF3 markedly exacerbated ischemic injury; the KO resulted in increased infarct volume and neurological deficit scores. ATF3 KO promoted inflammatory gene expression and cellular inflammatory response in the ischemic brain. In addition, KO of ATF3 led to an elevation in the activity of the NF-jB signaling pathway, which plays an important role in the inflammatory response after ischemic stroke.

ATF3 is a stress response gene that is induced quickly under various stressors, such as oxidative stress, cytokine treatment, allergies, and tumorigenesis (Gilchrist et al., 2006, 2008; Okamoto et al., 2006; Yin et al., 2010). The expression of ATF3 is ubiquitous; its expression can be induced in multiple cell types, including inflammatory cells, neurons, glial cells and endothelial cells. In this study, mRNA levels and protein expression were dramatically upregulated after tMCAO in the ischemic brains of WT mice. Immunofluorescence staining revealed that ATF3 expression increased mainly in neurons at 6 and 24 h after ischemic onset, which was in accordance with previous studies (Ohba et al., 2003;

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Fig. 5. The mRNA level and activity of MMP9 were upregulated in the ischemic brains of ATF3 KO mice. (A) Quantitative real-time PCR showed that MMP9 mRNA levels increased at 6 and 24 h after transient focal cerebral ischemia and increased more markedly in ATF3 KO mice (#P < 0.05 vs. WT,⁄P < 0.05 vs. sham, n = 6). (B) Gelatin zymography revealed that the activity of MMP9 in the left hemisphere (L) was upregulated 24 h after ischemic onset, and ATF3 KO significantly facilitated the induction of the active form of MMP-9, the activity of MMP9 in the right hemisphere was not changed after ischemic onset both in WT mice and ATF3 KO mice (⁄P < 0.05 vs. sham WT, #P < 0.05 vs. WT tMCAO 24 h, n = 6).

Fig. 6. ATF3 deficiency led to increased activity of the NF-jB signaling pathway and inhibited activity of CREB. Western blot analysis revealed that phosphorylation of IKKb, IjBa, and NF-jB p65 was upregulated in ATF3 KO mice and that the phosphorylation of CREB was significantly inhibited in ATF3 KO mice at 6 h after ischemia onset (#P < 0.05 vs. WT, n = 6). The total protein levels of IKKb, IjBa, NF-jB p65 and CREB were not changed in ATF3 KO or WT mice.

Song et al., 2011). This remarkable increased expression of ATF3 implied that ATF3 played an important role in cerebral ischemic pathophysiological process. In this study, mRNA levels of a number of inflammatory genes were markedly upregulated in ATF3 KO mice at 6 and 24 h after tMCAO. These genes included VCAM-1, ICAM-1, CD36, MCP-1, TNF-a, IL-1b and IL-6, which have been demonstrated to play important roles in stroke pathology. The adhesion molecules VCAM-1 and ICAM-1 play a critical role in blood inflammatory cell adhesion in the ischemic brain (Supanc

et al., 2011); the chemokine MCP-1 plays an important role in inflammatory cell mobilization and recruitment (Dimitrijevic et al., 2006); TNF-a, IL-1 and IL-6 are important cytokines which participate in glial cell activation and neuronal apoptosis (Vila et al., 2000; Clausen et al., 2008; Watters and O’Connor, 2011); scavenger receptor CD36 acting as a coreceptor for Toll-like receptor (TLR) 2 participates in the inflammatory response after cerebral ischemia (Abe et al., 2010). ATF3 is a transcription factor that both homodimerizes and heterodimerizes with other members of the CREB/ATF family, including C/EBPg,

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CHOP/DDIT3, ATF2, Jun, JunB. ATF3 could bind to CRE-like site and serve as a transcriptional repressor in TLR-signaling pathways. Previous studies also showed that ATF3 could indirectly interact with NF-jB and repress the transcriptional activity of NF-jB (Ashburner et al., 2001; Gilchrist et al., 2006). The promoter of VCAM-1, ICAM-1, CD36, MCP-1, TNF-a, IL-1b and IL-6 either contains a CREB binding site or contains a NFjB binding site or both. The up regulation of these inflammatory cytokines in the ischemic brain of ATF3 KO mice probably attributes to the absence of transcriptional repression of ATF3. Consistent with the up regulation of inflammatory cytokines, the cellular inflammatory response was also exacerbated in ATF3 KO mice after tMCAO. The infiltration of neutrophils, the recruitment of macrophages, and the activation of astrocytes were all increased in the ischemic brains of ATF3 KO mice. Increased inflammatory response is harmful to neurons in the penumbra, and this may lead to much more serious neuronal injury. Consistent with the aggravated inflammation, apoptosis of neurons in the peri-infarcted area increased, the infarct volume increased, and neurologic deficits worsened in the ATF3 KO mice. Multiple cell types involved in stroke pathology, including neurons, glial cells, neutrophils and macrophages, could generate MMP9 (Toft-Hansen et al., 2006; Rosell et al., 2008; del Zoppo, 2010). MMP9 plays important roles in the acute and sub-acute pathological processes after cerebral ischemia. For example, MMP9 degrades the basement membrane of the blood–brain barrier, leading to brain edema and facilitating infiltration of inflammatory cells. Additionally, MMP9 acts directly on neurons to induce neuronal damage. In this study, MMP9 expression and activity were upregulated in ATF3 KO mice. The upregulation of MMP9 transcription and activity may partly account for the exacerbated brain injury after tMCAO. In this study, we found that the NF-jB signaling pathway was excessively activated in the ischemic brains of ATF3 KO mice; this was evidenced by increases in the protein levels of phosphorylated IKK, phosphorylated IjB and phosphorylated NF-jB. The NF-jB signaling pathway plays a key role in inflammatory disease. Activated NF-jB promotes the expression of many inflammatory genes, including ICAM-1, MCP-1, TNF-a, IL-1b and IL-6. MMP9 expression is also regulated by NF-jB (Li et al., 2010a; Smale, 2011). The indirect interaction of ATF3 and NF-jB has been identified in lipopolysaccharide-activated macrophages and ischemia–reperfusion model of the kidney (Gilchrist et al., 2006; Li et al., 2010b): ATF3 interacted directly with histone deacetylase 1 (HDAC1) and recruited HDAC1 into the p65/RelA subunit of NF-jB to suppress NF-jB-dependent gene expression. Therefore, the deficiency of ATF3 could lead to an up-regulated transcriptional activity of NF-jB and increase the NF-jB-dependent gene expression. The inflammatory cytokines could strongly activate the NF-jB signaling pathway, so the overall excessive activation of NF-jB signaling pathway in ATF3 KO mice in this study was probably because the excessively expressed

inflammatory cytokines induced positive feedback of inflammatory response. Another finding in this study was that the phosphorylation of CREB was significantly inhibited in the ischemic brains of ATF3 KO mice, while the total protein level of CREB was not changed. The direct interaction of ATF3 with CREB has been demonstrated previously (Kim et al., 2010). CREB is an important protective transcription factor in stroke pathology; activated CREB protects the brain from ischemic injury by promoting neurotrophic factor expression (Dworkin and Mantamadiotis, 2010). In this study, the inhibited CREB activity might partially account for the worsened outcomes of ATF3 KO mice after ischemic injury.

CONCLUSIONS In conclusion, we have shown that ischemic brain injury markedly induced ATF3. ATF3 deficiency promoted the expression of a number of inflammatory genes and exacerbated the cellular inflammatory response, potentially through the activation of the NF-jB signaling pathway. In addition, the phosphorylation of CREB was inhibited in ATF3 KO mice, indicating that ATF3 is necessary for CREB activation after cerebral ischemia. Thus, ATF3 should be an important protective regulator in cerebral ischemic injury. Inducing ATF3 in advance or exogenously increasing ATF3 expression could provide a new therapeutic intervention for ischemia-mediated acute cerebral injury.

AUTHORS’ CONTRIBUTIONS L.W. participated in the conception of the study and designed the study and drafted the manuscript. S.D. L.Y. carried out the molecular studies. Y.L. Y.G. participated in the animal model preparation and performed the statistical analysis. Y.Z. carried out the immunofluorescent staining. H.J. H.L. conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. Acknowledgments—We thank Dr. Tsonwin Hai at the Department of Molecular and Cellular Biochemistry and Center for Molecular Neurobiology of Ohio State University for providing the ATF3 KO mice. This work was supported by National Natural Science Foundation of China (NO. 81100230), National Science and Technology Support Project (NO. 2011BAI15B02), Natural Science Foundation of Hubei Province of China (NO. 2010CDB06206).

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(Accepted 6 June 2012) (Available online 13 June 2012)