Annexin A1 translocates to nucleus and promotes the expression of pro-inflammatory cytokines in a PKC-dependent manner after OGD/R

Abstract

Annexin A1 (ANXA1) is a protein known to have multiple roles in the regulation of inflammatory responses. In this study, we find that after oxygen glucose deprivation/reoxygenation (ODG/R) injury, activated PKC phosphorylated ANXA1 at the serine 27 residue (p27S-ANXA1), and promoted the translocation of p27S-ANXA1 to the nucleus of BV-2 microglial cells. This in turn induced BV-2 microglial cells to produce large amounts of pro-inflammatory cytokines. The phenomenon could be mimicked by either transfecting a mutant form of ANXA1 with its serine 27 residue converted to aspartic acid, S27D, or by using the PKC agonist, phorbol 12-myristate 13-acetate (PMA) in these microglial cells. In contrast, transfecting cells with an ANXA1 S27A mutant (serine 27 converted to alanine) or treating the cells with the PKC antagonist, GF103209X (GF) reversed this effet. Our study demonstrates that ANXA1 can be phosphorylated by PKC and is subsequently translocated to the nucleus of BV-2 microglial cells after OGD/R, resulting in the induction of pro-inflammatory cytokines.

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Annexin A1 (ANXA1), a member of the vertebrate annexin class A family of proteins, also previously known as lipocortin 1, has received more and more attention in light of recent research ndings1,2. ANXA1 has been known to have multiple roles in important biological processes such as cell dierentiation3, proliferation4, plasma membrane repair5, epithelial repair6, and cell apoptosis7. For instance, ANXA1 can bind to negatively charged cellular phospholipids, vesicles and cytoskeletal proteins such as F-actin8, demonstrating a possible role in intracellular trafficking9. In addition, there is compelling evidence of a role of extracellular ANXA1 in multiple anti-inammatory processes10,11, including regulation of neutrophil migration, macrophage phagocytosis12,13, and induction of changes in cell polarity of microglial cells aer ischemia like injury in vitro14,15. Although the function of ANXA1 in the central nervous system is still elusive, enhanced ANXA1 expression has been found at demyelinating brain lesions in patients suering from multiple sclerosis16, Parkinsons disease17, and Alzheimers disease1. Our recent data showed that ANXA1 can be translocated into the nucleus following oxygen glucose deprivation/reoxygenation (OGD/R injury) to induce neuronal cell death7, a role that is currently being examined in detail. Together these data highlight multiple roles of ANXA1 under normal and pathological conditions.

Protein kinase C (PKC) can be activated during ischemic injury in multiple tissues, including the heart18,

liver19, and kidney20, which suggests a conserved role in the ischemic response pathway. However, whether PKC is a direct mediator of this pathway or is simply activated during ischemic-like injury remains controversial because of contradictory reports on the expression level, activity and distribution of PKC aer injury. This is, therefore the main focus of our studies. There are also conicting standpoints about the role of PKC in ischemic tissues, which

Department of Neurobiology, Tongji Medical College, Huazhong University of Science and Technology,

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are subject to debate. Some studies have shown that PKC levels and PKC activity increased aer ischemia damage based on measurements taken at relatively early time points in some in vivo models21, as well as aer OGD/R treatments, and excitotoxic injury in vitro22. Moreover, treating cells with PKC nonspecic inhibitors protected cells against excitotoxic cell death in vitro23,24 and against ischemic-like injury in vivo25. Another nding suggests that PKC acts as a switch to amplify pro-inammatory pathways, which would be sufficient to induce neuronal cell death26. Taken together these ndings indicate that PKC is activated during ischemia-like injury and may play a detrimental role in the pathophysiology of ischemic injury. Albeit, the precise molecular mechanisms underlying these biological processes are still not fully understood.

It was recently demonstrated that microglial cells were not only immunocentric, but in addition exerted neurobiological functions in both healthy and pathological contexts. In the disease context, the widespread consensus is that microglial cells are in a dynamic state with a potential to contribute to both central nervous system damage and repair27. The emerging roles of microglial cells are currently being investigated in the healthy and diseased brain with a growing interest in their diverse functions27. Interestingly, microglial cells are now being considered to be the CNS counterparts of peripheral macrophages, given the fact that they respond rapidly (within minutes) to immunological stimuli together with a burst of pro-inammatory mediators28,29. Indeed, a number of studies have found that microenvironmental conditions can selectively modify unique microglia phenotypes and functions. The location of microglia in the ischemic brain changes their activation and cell fate. In the ischemic core, where blood ow is reduced to near zero, cell death is nearly universal by 24hours30. Moreover constitutive expression of ANXA1 has been reported in glial cells scattered throughout normal adult human brains and in rodent, and specically in microglial cells9,31. Thus, ANXA1, appears to be constitutively expressed in cells of the innate immune system of the normal brain, but whether it plays a role in regulating microglia function or not remains elusive.

In this study, we demonstrate an OGD/R related mechanism mediating the production of pro-inammatory cytokines by BV-2 microglial cells. Following OGD/R injury, we found that increased PKC expression and activity leads to phosphorylation of ANXA1, which promotes its translocation to the nucleus, and induces the production of pro-inammatory mediators by BV-2 microglial cells.

Thus far, the pivotal pathological progression of brain ischemia-reperfusion is post-ischemic inammation, and the release of inammatory cytokines from immune cells like microglial cells. In order to detect the levels of inammatory cytokines released from BV-2 microglial cells under ischemia conditions, whole cell lysates and cell culture supernatants were collected from microglial cell cultures following OGD/R and subjected to SDS-PAGE analysis or, ELISA detection respectively. Our results show that the expression levels of the pro-inammatory cytokines, IL-1, IL-6 and TNF- were higher when compared to those of non-hypoxic conditions (Fig.1A,B, Fig. S2). In contrast, the levels of the anti-inammatory cytokines, IL-4, IL-10, and TGF- were found to be lower than the levels of in the non-hypoxic controls (Fig.1C,D). Importantly, the levels of pro- and anti-cytokine secretion correlate with the cellular expression levels of the same cytokine in both BV-2 and primary microglial cells (Fig.1E,F).

ANXA1 has been considered a pivotal player in the pathological progression of the inammatory response2. However, most previous studies have focused on the anti-inammatory and pro-repair action of ANXA1, based on the expression and translocation of ANXA1 to the membrane and/or its secretion into the extracellular matrix (ECM)1,32. In this study, we explored the protein expression levels of ANXA1 in BV-2 microglial cells and found that ANXA1 protein levels were higher following OGD/R injury as detected by SDS-PAGE analysis (Fig.2A,B), as well as by ANXA1 antibody staining of BV-2 microglial cells (Fig.2C, compare top panels with bottom panels).

Furthermore, it has been demonstrated that PKC activation can have multiple eects on ischemia/reperfusion-mediated nervous system damage23,33. For instance, our previous data showed that in neurons, phosphorylation of ANXA1 at serine 5, mediated by transient receptor potential melastatin 7 (TRPM7) allows ANXA1 to be trans-located to the nucleus to induce cell death aer OGD/R injury7. In addition, Varticovski and colleagues reported that ANXA1 can be identied as a PKC substrate and in turn can be phosphorylated by PKC on the ANXA1 serine residue at position 2734. Consequently, we want to examine further whether PKC was activated by oxygen deprivation and whether the residue 27 of ANXA1 could indeed be phosphorylated by PKC. To this end, we explored the expression of PKC in BV-2 microglial cells using immunocytochemistry and immunouorescence analysis. Our data showed that PKC protein levels were upregulated in whole cell lysates (Fig.3A,B) and in immunouorescence labeling of BV-2 cells following OGD/R injury (Fig.3C, bottom panels). We also found that PKC activity was upregulated aer OGD/R as detected by a pan phospho-PKC antibody (Fig.4A,B).

Next, we performed co-immunoprecipitation (co-IP) studies to further investigate the relationship between PKC and ANXA1 aer OGD/R injury. In our co-IP studies we found upregulated levels of total and active PKC proteins associated with ANXA1 under OGD/R conditions when compared to controls (Fig.4C,D, compare lane 3 to lanes 12). Lastly, we used a serine phospho-specic poly-clonal antibody to detect the phosphorylation level of ANXA1 by PKC. In our SDS-PAGE analysis of BV-2 microglial cell lysates aer OGD/R treatments, we found an increase in the phosphorylation levels of ANXA1 serine residue aer OGD/R injury when compared to controls (Fig.4E,F, compare lane 3 to lanes 12, Fig. S1).

Together the data shown above demonstrate the presence of high protein levels of ANXA1 (Fig.2C), upregulated levels of total PKC (Fig.3AC), active PKC (Fig.4A,B), and PKC associated with ANXA1 (Fig.4C,D), in BV-2 microglial cells

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Figure 1. Expression and secretion of inammatory cytokines in BV-2 and primary microglia cells under normal conditions and aer OGD/R injury. (A) Western blot analysis of BV-2 microglial cell lysates showing the expression of IL-1, IL-6, and TNF- before and aer OGD/R treatments. (B) Western blot quanticationsof IL-1, IL-6, and TNF- intensities normalized to their respective controls (dened as 1.0). Data are expressed as meanSEM; n=3; *P< 0.05 versus controls. (C) Western blots showing expression levels of IL-4, IL-10, and TGF- in BV-2 microglia whole cell lysates. (D) Western blot quantications of IL-4, IL-10, and TGF- intensities normalized to their respective controls (dened as 1.0). Data are expressed as meanSEM; n=3; *P<0.05 versus controls. (E) ELISA detection of secreted lymphokines in supernatants of BV-2 microglial as well as (F) of isolated primary microglial cells. Data are expressed as meanSEM; n=3; *P< 0.05 versus controls.

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Figure 2. Expression and translocation of ANXA1 aer OGD/R treatments. (A) Western blot analysis showing ANXA1 protein levels in BV-2 microglial cells before and aer OGD/R. (B) Western blot intensities were quantied and normalized to their respective controls (dened as 1.0). Data are expressed as mean SEM; n=3; #P< 0.01 versus controls. (C) Immunouorescence analysis of ANXA1 (red, le panels) in BV-2 microglial cells and in nuclei (blue, middle panels). Merged images are shown in the overlay pictures with partially enlarged details (right panels) in both control (top panels) and OGD/R conditions (bottom panels). Data are representative of three independent experiments. Bar=27m.

aer OGD/R treatments. We next sought to investigate whether PKC activation aected the translocation of ANXA1 to the nucleus. For this, we used EGFP-tagged constructs of ANXA1 that were subjected to site-directed mutagenesis at the serine 27 residue, the site shown to be phosphorylated by PKC34. In addition, we used an agonist and an antagonist of PKC to examine the eect of active PKC signaling on the distribution of the ANXA1 protein. Interestingly, the mutant protein ANXA1 S27A (with serine 27 mutated to alanine) was strongly trans-located to the cell membrane aer OGD/R treatments (Fig.5A,B, compare lane 3 to lanes 12; and e, top panels), a phenomenon that could also be observed using the PKC antagonist, GF109203X, which resulted in decreased ANXA1 protein levels in the nucleus (Fig.5A,B, compare lane 4 to lanes 12).

On the contrary, the mutant protein ANXA1-S27D (with serine 27 mutated to aspartic acid) into BV-2 micro-glial cells resulted in decreased ANXA1 protein levels in the plasma membrane and cytoplasm, and increased levels of translocated ANXA1 in the nucleus (Fig.5C,D, compare lane 3 to lanes 12; and e, bottom panels), a similar eect on ANXA1 protein levels was seen aer treatment of BV-2 microglial cells with phorbol ester (PMA) (Fig.5C,D; compare lane 4 to lanes 12). We then wondered whether nuclear translocation of ANXA1 aected the pro-inammatory action of BV-2 microglial cells.

Given that ANXA1 was previously thought to be a secreted protein and exert an anti-inammatory and pro-repare action in ischemic tissue1, we asked whether ANXA1 could aect the expression of inammatory cytokines in OGD/R induced inammation. To do this, we set out to examine the relationship between the dierent subcellular distributions of ANXA1 and the upregulation of inammatory cytokines. When BV-2 microglial cells were transfected with ANXA1-S27A constructs following by OGD/R treatment, the pro-inammatory cytokines, IL-1, IL-6, and TNF-, were found to be expressed at lower levels than those of control groups (Fig.6AD, compare lane 3 to lanes 12). The same phenomenon could be seen using the PKC antagonist, GF109203X (Fig.6AD, compare lane 4 to lanes 12). In contrast, BV-2 microglial cells transfected with the

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Figure 3. Expression of PKC in BV-2 microglial cells aer OGD/R treatments. (A) Western blots showing protein levels of PKC in BV-2 microglial cell lysates under normal conditions and aer OGD/R treatments.(B) Western blot intensities of PKC expression levels were quantied and normalized to their respective controls (dened as 1.0). Data are expressed as meanSEM; n=3; *P< 0.01 versus control. (C) Immunocytochemistry analysis of PKC (red, le panels) in BV-2 microglial cells and their nuclei (blue, middle panels). Merged images are shown in the overlay pictures with partially enlarged details (right panels) in both control (top panels) and OGD/R conditions (bottom panels). Data are representative of three independent experiments. Bar=27m.

ANXA1-S27D mutant (Fig.6EH, lane 3) or treated with PMA (Fig.6EH, lane 4) exhibited a reversed outcome with higher levels of pro-inammatory cytokines expressed (Fig.6EH, compare lanes 34 with lanes 12, Fig. S3).

Next, we investigated the expression levels of the anti-inammatory cytokines, IL-4, IL-10, and TGF-, using the conditions described above and found that ANXA1-S27A and GF109203X treatments resulted in the increased expression of these anti-inammatory cytokines (Fig.7AD). In contrast, when BV-2 microglial cells were transfected with ANXA1-S27D or treated with PMA, the anti-inammatory cytokines examined here were expressed at much lower levels than in control groups (Fig.7EH).

In this study, we report an increase in PKC levels and activity aer OGD/R treatment of BV-2 microglial cells. In addition, active PKC was found in association with ANXA1, which led to the phosphorylation of ANXA1 on serine 27. Phosphorylated ANXA1 was in turn translocated to the nucleus of BV-2 microglial cells, where it promoted the production of pro-inflammatory cytokines, while actively suppressing the production of anti-inammatory cytokines aer OGD/R treatment. These eects could be prevented using the PKC antagonist, GF109203X or by transfecting BV-2 microglial cells with an unphosphorylatable ANXA1 construct containing a serine to alanine mutation at position 27 (S27A). In contrast, using phorbol ester (PMA) to activate the PKC pathway, or a serine to aspartic acid mutant (S27D) of ANXA1 that renders it constitutively active, enhanced the production of pro-inammatory cytokines in BV-2 microglial cells.

ANXA1 is an endogenous protein known to have potential anti-inammatory functions in the peripheral nervous system9. However, ANXA1s role in regulating inammatory activities of the central nervous system (CNS) remains poorly understood. A few studies have proposed a protective role of ANXA1 in the CNS ischemic response35,36 as well as in the progression of neurodegenerative diseases37,38.

Previous studies have reported an anti-inammatory and pro-resolving function of ANXA1 in the nervous system. In a recent publication, our group showed that enhanced ANXA1 binding to the formyl peptide receptor

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Figure 4. Phosphorylation levels of PKC and ANXA1 and their protein-protein interaction aer OGD/R treatments. (A) PKC phosphorylation levels in BV-2 microglial cells aer OGD/R treatment as detected with an anti-phospho-PKC polyclonal antibody. (B) Quantication of western blots intensities normalized to their respective controls (dened as 1.0). Data are expressed as meanSEM; n=3; #P< 0.01 versus controls.(C) Co-immunoprecipitation of PKC with ANXA1 in BV-2 microglial cells under negative (IgG), control (Con) and OGD/R conditions as indicated. (D) Quantication of western blot intensities normalized to their respective controls (dened as 1.0). Data are expressed as meanSEM; n=3; #P< 0.01 versus appropriate controls. (E) Serine phosphorylation levels of ANXA1 as detected with a specic phospho-serine polyclonal antibody aer immunoprecipitation. (F) Quantication of western blot intensities normalized to their respective controls (dened as 1.0). Data are expressed as meanSEM; n=3; #P< 0.01 versus controls.

(FPR) induces morphological changes in microglial cells to an alternative phenotype of M2 polarized cells, which protects neurons against ischemia-like injury.

Using rodent microglia cultures, it was shown that the N-terminal fragment of ANXA1, Ac226, prevents lipopolysaccharide (LPS) mediated stimulation of cyclo-oxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS), as well as the release of nitric oxide (NO)39,40. In addition, ANXA1 is known to inhibit phospholipase A2 activity41, thereby preventing the release of arachidonic acid (AA), an essential fatty acid for prostanoid synthesis. Within the hypothalamic regulatory center, ANXA1 is also thought to mediate the antipyretic actions of glucocorticoid (GCs)42. These functions, in addition to the blocking of prostaglandin E2 (PGE2) synthesis, include the inhibition of the pro-inammatory cytokines, IL-1, IL-6 and IL-8, all of which are notably elevated in the striatum and cerebrospinal uid (CSF) in subjects with idiopathic Parkinsons disease43,44. Intra-cerebral administration of ANXA1 fragments also inhibits neuroendocrine and febrile responses to peripheral or centrally administered cytokines45,46. Furthermore, ANXA1 can bind to cell surface receptors to exert paracrine or autocrine eects on multiple biological events as described above. Although this so far eluded full conrmation, a growing body of evidence indicates that the eects of ANXA1 in the immune47 and neuroendocrine systems9,48 might be mediated by the FPR family of receptor proteins. The functional implications of ANXA1 gene expression changes in microglia and astrocytes are still unknown, but they can be used as potential targets for limiting neuro-inammation and

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Figure 5. Translocation of ANXA1 aer OGD/R injury. BV-2 microglial cells were transfected with wild-type (WT) ANXA1 or mutated ANXA1-S27A, or WT ANXA1 with addition of the PKC antagonist, GF109203X, (1M); or the ANXA1-S27D mutant or WT ANXA1 together with the PKC activator, phorbol ester (PMA, 1M). (A) Western blot analysis showing the translocation of ANXA1 in BV-2 microglial cells treated as indicated. The top western blot panel indicates the levels of translocated ANXA1 at the plasma membrane; in the cytoplasm (second panel from top), and in the nucleus (fourth panel from top). (B) Western blots intensities normalized to their respective controls (dened as 1.0). Data are expressed as mean SEM; n=3; #P<0.05

versus ANXA1. #*P< 0.05 versus ANXA1+OGD/R. (C) Western blots showing the translocation of ANXA1 in BV-2 microglia cells treated as indicated. Top western blot panel indicates the levels of translocated ANXA1 at the plasma membrane; in the cytoplasm (second panel from top), and in nucleus (fourth panel from top). (D) Western blot intensities normalized to their respective controls (dened as 1.0). Data are expressed as meanSEM; n=3; #P< 0.01 versus ANXA1. #*P< 0.05 versus ANXA1+OGD/R. (E) Transfected EGFP-tagged ANXA1 constructs into BV-2 microglial cells (in green, le panels), and their nuclei (in blue, middle panels). Merged images are shown in the overlay pictures with partially enlarged details (right panels) for ANXA-S27A (top panels) and ANXA1-S27D (bottom panels). Bar=20m. Data are representative of three independent experiments.

combatting neurodegeneration. Of note, these described actions of ANXA1 are mostly based on the translocation of ANXA1 to the plasma membrane and its subsequent secretion into the extracellular matrix.

Importantly, ANXA1 has been shown to have multiple opposing physiological roles. In our studies, we show that translocation of ANXA1 into neuronal nuclei aer OGD/R injury, induces neuronal death. We have also found that ANXA1 is translocated to the nucleus of BV-2 microglial cells (Fig.2C) and induces cells to produce pro-inammatory cytokines (Fig.6EH, Fig. S3C,D), while it also suppresses the production of anti-inammatory cytokines (Fig.7EH).

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Figure 6. Translocation of ANXA1 aects the expression of pro-inammatory cytokines in BV-2 microglial cells. Western blot analysis showing the expression of IL-1, IL-6, and TNF- in BV-2 microglia cells. (A) BV-2 microglial cells were transfected with either vector control, WT ANXA1, or ANXA1-S27A mutant, or WT ANXA1 together with GF109203X (1M) aer OGD/R treatments. (BD) Western blot intensities of IL-1, IL-6, and TNF- normalized to their respective controls (dened as 1.0). Data are presented as mean SEM for three independent experiments. Asterisks indicate statistically signicant dierence (*P<0.05, **P<0.01). (E) BV-2 microglial cells transfected with either vector, or WT ANXA1, or with ANXA1-S27D, or WT ANXA1 with addition of PMA (1M) aer OGD/R treatments. (FH) Western blot intensities of IL-1, IL-6, and TNF- normalized to their respective controls (dened as 1.0). Data are presented as mean SEM for three independent experiments. Asterisks indicate statistically signicant dierence (*P<0.05, **P<0.01).

PKC has been implicated in mediating ischemia/reperfusion lesions in multiple organs20,49. Recent reports have linked PKC activity to regulatory events in several signaling pathways, including the mediation of excitatory or inhibitory amino acid release50, and cytokine induced superoxide production51. Here we found that

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Figure 7. Translocation of ANXA1 to the nucleus aected the expression of anti-inammatory cytokines in BV-2 microglial cells. Western blot showing the expression of IL-4, IL-10, and TGF- in BV-2 microglia cells. (A) BV-2 microglial cells were transfected with either vector control alone, WT ANXA1, mutated ANXA1-S27A, or WT ANXA1 with addition of GF109203X (1 M) and subjected to analysis aer OGD/R treatments. (BD) Western blot intensities of IL-4, IL-10, and TGF- normalized to their respective controls (dened as 1.0). Data are presented as mean SEM for three independent experiments. Asterisks indicate statistically signicant dierence (*P<0.05, **P<0.01). (E) BV-2 microglial cells were transfected with either vector control alone, WT ANXA1, mutated ANXA1-S27D, or WT ANXA1 with addition of PMA (1M) and analyzed aer OGD/R treatments. (FH) Western blot intensities of IL-4, IL-10, and TGF- were quantied and normalized to their respective controls (dened as 1.0). Data are presented as mean SEM for three independent experiments. Asterisks indicate statistically signicant dierence (*P<0.05, **P<0.01).

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the expression and activation of PKC was upregulated in BV-2 microglia cells aer OGD/R treatments, which resulted in increased levels of ANXA1 that were co-immunoprecipitated with PKC. In vivo experiments of

32P-labeled mesangial cells, phosphorylation was increased by treating the cells with PKC activators, such as angiotensin II or by using common phorbol esters (i.e. PMA, TPA). Moreover, a phosphoamino acid analysis, revealed that phosphorylation of ANXA1 occurs only on serine residues52. Consistent with this we found that phosphorylation of ANXA1 at serine 27 residue in BV-2 microglial cells was upregulated aer OGD/R treatment (Fig.4E), implying a role for PKC in ANXA1 phosphorylation in microglial cells aer OGD/R injury.

Importantly, most studies elucidating the role of PKC pathways, report a rapid loss of total PKC levels and activity aer ischemic injury, suggesting that PKC is degraded under these conditions33,53. The loss of total PKC activity, also seen in in vitro culture models of ischemic and excitotoxic cell death54,55, correlates with neurode-generative processes56, implying that maintaining PKC activity may confer protection against excitotoxic damage. These apparently conicting reports may stem from examination of varying animal models, brain regions, duration and intensities of the ischemia/reperfusion insult, and maybe compounded by the dierent, possibly opposing roles of individual PKC isozymes.

In the injured brain, activated microglia cells participate in the course of inflammation, a process that includes the actions of various kinds of cytokines. Some of these cytokines are necessary to protect neurons, others can be particularly harmful. Nonetheless, these actions depend on dierences in polarization of microglia cells. Microglial cells, as the main immune cells of the CNS, are responsible for monitoring the brain microenvironment. Microglial activation results in the synthesis and secretion of a host of mediators, including prostaglandins (PGs), nitric oxide (NO) arising from upregulation of cyclo-oxygenase 2 (COX-2) and the inducible form of nitric oxide synthase (iNOS), respectively, as well as pro-inammatory cytokines, such as interleukin-1 (IL-1), interleukin-6 (IL-6), and tumour necrosis factor alpha (TNF-). This process is called persistent neuro-inammation, or reactive gliosis, which develops in many acute and chronic neurological conditions, such as stroke, Parkinsons and, Alzheimers disease, as well as motorneuron and prion diseases5759. Excessive production of pro-inammatory mediators such as cytokines, prostanoids, and free radicals, are thought to contribute to the neuropathological process and neuronal loss during ischemia. Inammatory responses in brain ischemia/reperfusion lead to pivotal injuries in neurons that would eventually result in neuronal death. Mediators of inammation released from microglial cells in the CNS are thus key mediators of ischemic brain injury.

IL-1, IL-6 and TNF are key pro-inammatory cytokines that when induced can excessively activate micro-glial cells, forming a vicious cycle of pro-inammatory responses that continuously damage neurons and other important nervous system structures. On the contrary, IL-4, IL-10, and TGF- are important anti-inammatory and pro-repare cytokines in the ischemic brain. Notably, these cytokines can preclude and reduce the imminent harm mediated by microglial cells aer ischemic injury.

In this study, we investigated the immunity property of BV-2 microglial cells, and they match the immunity property of innate immune cells (Fig. S4) and found that pro-inammatory cytokines are upregulated while anti-inammatory cytokines are suppressed aer phosphorylated ANXA1 is translocated to the nucleus of BV-2 microglial cells (Figs6 and 7). This work highlights the importance of regulating pro-inammatory cytokines and suggests a way in which we could preclude the translocation of ANXA1 into nucleus to protect neurons from death aer ischemic injury. We are now evaluating possible therapeutic targets based on our results that can prevent neuronal cell loss in ischemic brain injury.

All animal experiments were approved by the Huazhong University of Science and Technology Institutional Animal Care and Use Committee, and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Culture of primary microglial cells were performed as previously described60. Two freshly perfused adult mouse brains were used per experiment. Primary microglial cells were cultured in DMEM/F12 media with 10% FBS and 1% Pen/Step. The immortalized BV-2 microglial cell line was grown in Dulbeccos Modied Eagle Medium (DMEM) with high glucose supplemented with 10% Fetal Bovine Serum (FBS), 100U/ml penicillin and 100g/ml streptomycin at 37C in a 100% humidied atmosphere of 95% air and 5% CO2. Before all experimental procedures, BV-2 microglial cells were serum starved overnight (or for 12h). Cells were then incubated with phorbol 12-myristate 13-acetate (PMA, 1M) (Beyotime, Shanghai, China) or GF103209X (GF, 1M) (PeproTech, Hamburg, Germany) for 30min before cell protein was extracted.

Culture medium was changed into glucose-free DMEM and washed with 1 X PBS three times, cultures were then transferred to an incubator containing 5% CO2 and 95% N2 at 37C for 1h. Following Oxygen Glucose Deprivation (OGD) treatment, the cultures were re-oxygenated under normoxic conditions in high glucose-containing DMEM at37C in a humidied 5% CO2 incubator for 24h before they were collected for analysis.

Culture supernatants were collected aer OGD/R treatments. The production of IL-1, IL-6, IL-12, IL-17, TNF-, IL-4, IL-10 and TGF- were measured with a commercial ELISA kit (Biolegend, San Diego, CA) following manufacturers instructions and expressed as pg/mL.

BV-2 microglia cells cultured on sterile glass cover slips were washed with 1 X PBS and xed with 4% paraformaldehyde (PFA) for 15min. The cells were then permeabilized with 0.2% Triton X-100 in 1 X PBS for 5min. Cells were blocked in 1 X PBS containing 1% bovine serum albumin (BSA) for 30min. The cells were incubated with primary antibodies overnight at 4C. The primary antibodies used were as follows: rabbit anti-ANXA1 (1:100, Santa Cruz Biotechnology, Dallas, TX), PKC (1:100, Santa Cruz Biotechnology, Dallas, TX).

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Aer washing, Alexa Fluor 594-conjugated anti- rabbit Ig G were applied at a dilution of 1:2000 for 1 h, and

4,6-diamidino-2-phenylindole (DAPI, Roche, Shanghai, China) was used for the identication of nuclei. Cover slips were mounted with glycerinum, and cells were imaged with an Olympus immunouorescence microscope (Olympus, Tokyo, Japan).

Protein immunoprecipitation was performed according to manufacturers instructions of a commercially available immunoprecipitation kit, Protein A/G PLUS-Agarose, (Santa Cruz, Biotechnology, Dallas, TX) with only minor modications. For this, cellular lysates were divided into two parts, one used for the immunoprecipitation assays and, the other for total protein analysis. For immunoprecipitation analysis, proteins were incubated with a polyclonal antibody against ANXA1 (1:200 (ug), Santa Cruz Biotechnology, Dallas, TX) at 4 C on a vertical rotator overnight, followed by protein A/G plus agarose added (Santa Cruz, Biotechnology, Dallas, TX) for 4h. Aer washing 5 times with lysis buer, samples were eluted by boiling in 1 X SDS/PAGE buer for 7 min. Proteins were then separated by SDS-PAGE and examined by immunoblotting using antibodies against PKC (1:1000, Santa Cruz Biotechnology, Dallas, TX), phosphor serine (1:250, Abcam, Cambridge, MA).

An ANXA1 cDNA construct containing a point mutation in a key phosphorylation site, 27 serine (S27) was generated. In addition, an ANXA1 cDNA construct tagged with a C-terminal enhanced green uorescent protein (EGFP) was generated by attaching pEGFP-N1 [GenBank: U55762] to the wild-type (WT) construct between the restriction sites XhoI at the N-terminal coding region and the BamHI sites, replacing the stop codon. Replacement of the S27 amino acid with alanine (A) was introduced by sequential site-directed mutagenesis reactions employed the QuikChange site-directed mutagenesis kit (TransGen Bioteck, Beijing, China). Synthetic oligonucleotide primers containing the desired mutation site were extended in polymerase chain reaction (PCR) followed the kit protocol described. The products were DMT-treated to digest the parental template and transformed in DMT chemically competent E. coli cells. Plasmids were amplied in E. coli Trans5 cells (TransGen Bioteck, Beijing, China) and puried with a Plasmid Purication Kit (QIAGEN, Shanghai, China).

BV-2 microglia cells were plated in six-well plates in DMEM (Invitrogen, Carlsbad, CA) containing 10% FBS (Gibco) at 3 105 cells per well for 24h before transfection in a humidied atmosphere of 5% CO2 and 95% air at 37C. Cells were then transfected with

ANXA1 cDNA using the expression plasmid, pEGFP-N1 (Invitrogen, Carlsbad, CA) according to the manufacturers introduction and, cells were harvested aer a 48 h incubation period. All the cDNA plasmids described above were prepared using Endo-Free Plasmid Kits (QIAGEN, Shanghai, China) to avoid contamination of

endotoxins.

For experimental assays in which cell surface ANXA1 was examined, we used previously described procedures48, which were modied briey as follows. BV-2 microglial cells were rst washed for 15min on ice in HEPES buer (25mM) containing protease and phosphatase inhibitors (1mM PMSF, 1 mg/ml leupeptin, 1 mg/ml pepstatin, 1 mg/ml aprotinin, 1 mM Na3VO4, 1 mM NaF; all from Sigma-Aldrich, St. Louis, MO), and a Ca2 chelating agent (1 mM EDTA-EGTA, Sigma-Aldrich, St. Louis, MO), which removes proteins attached to the cell surface. Subsequent washes were condensed with tubular ultraltration modules provided by Millipore (Billerica, MA), and retained for ANXA1 protein measurement.

Cells were lysed in radioimmunoprecipitation assay (RIPA) buer supplemented with a protease inhibitor cocktail (Roche, Shanghai, China) for whole cell protein preparations. Nuclear and cytoplasmic fractionations were performed with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientic, Rockford, IL) according to the manufacturers protocol. Total protein concentration was measured and equal proteins were loaded and separated by 10% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Subsequently, proteins were transferred to PVDF membranes and then blocked with 5% milk or 5% BSA in Tris-buered saline with Tween 20 (TBST) before immunodetection with the following antibodies: PKC (1:1000, Santa Cruz Biotechnology, Dallas TX), p-PKC (1:300, Cell Signaling Technology, Danvers, MA), ANXA1 (1:1000, Santa Cruz Biotechnology, Dallas TX), IL-1 (1:500, Abcam, Cambridge, MA), IL-6 (1:500, Abcam, Cambridge, MA), TNF-(1:1000, Abcam, Cambridge, MA), IL-4 (1:500, Abcam, Cambridge, MA), IL-10 (1:500, Abcam, Cambridge, MA), TGF-(1:1000, Abcam, Cambridge, MA), -actin (1:4000, Santa Cruz Biotechnology, Dallas TX). Aer primary antibody incubation for 12h, the PVDF membranes were washed with TBST (1530min at room temperature) before incubated with secondary antibody for 1 h. Specic binding was visualized by ECL reaction. The western blot bands were quantied using Image J Soware (version 1.41).

Data are expressed as means (SEM) of the indicated number of independent experiments. The statistical signicance between multiple groups was analyzed by one-way ANOVA, the least signi-cant dierence (LSD) post hoc test was used for multiple comparisons, and the students t test was used to detect the signicance of dierences between two means. P< 0.05 was considered statistically signicant.

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This study was supported by grants 31171029 and 31471015 from the National Natural Science Foundation of China.

Z.B.M. designed and performed the experiments, analyzed the data, and wrote the manuscript. J.W. and L.L. provided useful advice, X.L., S.X.L. and Q.X. performed cell culture. J.S. supervised the experimental work, conceived the study, and participated in its design and coordination. All authors of this paper have read and approved the nal version of the manuscript.

Supplementary information accompanies this paper at http://www.nature.com/srep

Competing nancial interests: The authors declare no competing nancial interests.

How to cite this article: Baoming, Z. et al. Annexin A1 translocates to nucleus and promotes the expression of pro-inammatory cytokines in a PKC-dependent manner aer OGD/R. Sci. Rep. 6, 27028; doi: 10.1038/ srep27028 (2016).

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