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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-5850fjev1 20/13/2369    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Weiss, T. W. Articles by Medcalf, R. L. Search for Related Content PubMed PubMed Citation Articles by Weiss, T. W. Articles by Medcalf, R. L.

Oncostatin M is a neuroprotective cytokine that inhibits excitotoxic injury in vitro and in vivo

Australian Centre for Blood Diseases, Monash University, Alfred Medical Research and Education Precinct, Prahran, Victoria, Australia

¹Correspondence: Australian Centre for Blood Diseases, Monash University, 89 Commercial Rd., Prahran 3181, Victoria, Australia. E-mail: robert.medcalf{at}med.monash.edu.au

SPECIFIC AIMS

Excitotoxic pathways initiated after excessive glutamate release have been implicated in acute and chronic disorders of the central nervous system (CNS). Members of the interleukin-6 (IL-6) cytokine family have been shown to have beneficial or detrimental effects in some neuronal conditions. Oncostatin M (OsM) has unique actions among this family, but its role during excitotoxic injury has not been addressed. Our aim was to investigate the effect of OsM using in vitro and in vivo models of excitotoxic injury.

PRINCIPAL FINDINGS
1. OsM attenuates NMDA-induced neuronal cell death in vitro
Cultures of mouse cortical neurons were treated with 50 µM NMDA in the presence or absence of murine OsM. As shown in Fig. 1 A, 24 h treatment with OsM alone had no effect on neuronal viability whereas treatment with NMDA caused at least 40% cell death. Cotreatment with NMDA, together with 50 ng/ml OsM, reduced the degree of NMDA-induced cell death to 20% (i.e., a 50% reduction).

We next determined whether pretreatment of cells with OsM prior to challenge with NMDA would confer more effective protection. Cortical neurons were treated with OsM (50 ng/ml) for 24 h. Culture media were replaced with media containing fresh OsM (50 ng/ml) or NMDA (50 µM) (added alone or in combination) and the degree of cell death was assessed 24 h later. As shown in Fig. 1B , pretreatment of cells with 50 ng/ml OsM fully reversed the toxic effect of NMDA, indicating a profound neuroprotective property of this cytokine. To determine the dose dependency of the effect, neurons were pretreated with 10 ng/ml OsM. As shown, this lower concentration of OsM attenuated the degree of NMDA-induced injury by 25% (P<0.01) (Fig. 1B ). Caspase-3/-7 activation assays also showed that OsM inhibited NMDA-induced apoptosis.

2. The effect of OsM on excitotoxic injury induced by other glutamate analogues
To determine whether OsM could protect cells from excitotoxic injury initiated by other glutamate analogues, the effect of OsM was determined on neurons treated with 100 µM kainic acid (KA) or alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA). Treatment of neurons with either KA or AMPA alone caused significant cell death after 24 h. Cotreatment with OsM also conferred a protective effect, albeit smaller (20 to 30%), against both KA- and AMPA-induced cell death. However, in contrast to the results observed with NMDA, OsM pretreatment produced no additional protective effect against either AMPA- or KA-induced cell death. These data indicate that the protective effect of OsM on excitotoxic cell death is more specific to NMDA-induced injury.

3. OsM selectively down-regulates NMDA receptor subunit gene expression
The NMDA receptor is a multisubunit complex, and changes in the expression of individual subunits can affect NMDA receptor function. To investigate whether OsM affects the expression of NMDA receptor subunits, neuronal cultures were incubated in the presence or absence of OsM for 24 h. RNA was collected and changes in mRNA expression levels for NMDA receptor subunits were determined by RT-polymerase chain reaction (RT-PCR). mRNA levels for NR1, NR2A, and NR2B were unchanged after OsM treatment. However, expression of NR2C receptor subunit was reduced by OsM. This suppressive effect of OsM on NR2C expression was also evident at the NR2C protein level as assessed by Western blot analysis of OsM-treated primary neurons in vitro and in vivo in the brains of mice stereotactically injected with OsM. These data indicate that OsM specifically down-regulates expression of the NR2C subunit of the NMDA receptor.

4. OsM inhibits NMDA-induced increase in free intracellular calcium levels
NMDA-induced cell death is preceded by an increase in the level of free intracellular calcium ([Ca²⁺]_i). To determine whether OsM could influence the effect of NMDA at this level, we used real-time confocal imaging to visualize changes in [Ca²⁺]_i in individual neurons after NMDA treatment. Treatment of primary neurons with OsM for 5 min prior to NMDA addition attenuated an NMDA-induced increase in [Ca²⁺]_i by 30% (P<0.05).

5. OsM is neuroprotective in vivo
The striatal region of mice was stereotaxically injected with NMDA (1 µl of 50 mM NMDA) in the presence or absence of OsM (5 µg/ml) or vehicle and the lesion volume was determined 24 h later. As shown in Fig. 2 , codelivery of OsM reduced the NMDA-induced lesion volume by 40%. Hence, OsM confers neuroprotection against NMDA-induced toxicity in vivo.

CONCLUSIONS AND SIGNIFICANCE

OsM is known for its pro- and anti-inflammatory capabilities, its effect on hematopoiesis, and as a modulator of the proliferative process. OsM is also expressed by microglia, astrocytes, and neurons, and expression levels are elevated in the brain during some acute and chronic conditions. In this study, we used in vitro and in vivo models of excitotoxic injury and identified a novel and potentially important neuroprotective role for OsM.

With the exception of specific NMDA receptor antagonists (e.g., MK801), the complete reversal of NMDA-induced excitotoxic injury is a rare occurrence, and to the best of our knowledge, the profound neuroprotective effect of OsM is unprecedented for any other cytokine. Our study also revealed that the protective effect of OsM was more selective to NMDA-induced toxicity than to either KA or AMPA toxicity.

Although we cannot exclude the possibility that OsM directly antagonizes the NMDA receptor, a more likely scenario is that OsM was influencing expression of protective genes or regulating NMDA receptor expression. For the latter, OsM was shown to specifically down-regulate expression of the NR2C receptor subunit. Down-regulation of NR2A and NR2C subunit expression by cytokines (e.g., FGF) and by brain-derived neurotrophic factor (BDNF) has been reported before, but this is the first report showing that OsM selectively down-regulates expression of the NR2C gene. Although our study does not allow us to formally conclude that the decrease in NR2C is part of the mechanism underlying the protective effect of OsM, this is a reasonable possibility. Changes in NMDA receptor expression can influence the degree of neuronal damage after stroke and traumatic brain injury. Indeed, targeted disruption of the gene for the NR2C subunit confers protection from ischemic damage subsequent to permanent middle cerebral artery occlusion.

OsM is a pleiotropic cytokine that influences the expression pattern of many genes via the janus-activated kinase (JAK)-STAT or MAPK pathways. Thus, apart from its effect on NR2C expression, OsM treatment was also shown to increase mRNA levels of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), neuroserpin (NS-1), and the OsM receptor (OsM-Rßbeta;) (data not shown). TIMP-1 has been described to produce a neuroprotective effect by influencing the activity of matrix metalloproteases. Neuroserpin, on the other hand, inhibits the activity of tissue-type plasminogen activator (t-PA), a serine protease that is a detrimental in excitotoxic injury. Our observation that OsM increases the expression of its own receptor could facilitate an amplification loop.

NMDA toxicity is invariably coupled with acute increases in the level of free intracellular calcium. One could speculate that the OsM receptor (gp130/OsMRßbeta;) is coupled to the NMDA receptor and/or that the JAK-STAT/MAPK pathways initiated by OsMRßbeta; engagement have a profound and rapid negative impact on the downstream effector arm of NMDA signaling. With this in mind, it could be envisaged that the OsMRßbeta; and the NMDA receptor are colocalized on neurons.

Results obtained from our in vivo model of excitotoxic injury were consistent with our in vitro findings whereby OsM was shown to substantially reduce NMDA-mediated lesion volume. This finding strengthens the basis for our conclusion that OsM is a neuroprotective cytokine. This in vivo finding is relevant, as there has been much interest in the possible neuroprotective effects of gp130 ligands. Most studies have addressed the role of IL-6 in models of excitotoxic injury, but these in vitro investigations have yielded conflicting results, perhaps because of the different neuronal cell types used. A schematic representation outlining the possible mechanisms behind the neuroprotective role for OsM is shown in Fig. 3 .

In conclusion, we report a profound and unprecedented neuroprotective role for OsM. There has been a long-standing desire to protect neurons against glutamate-mediated excitotoxicity, as glutamate is generally considered to play an important role in neuronal cell death in a variety of acute and chronic neurodegenerative diseases. Our observation that OsM is neuroprotective in vivo raises the possibility of therapeutic options for this cytokine and also provides impetus to explore in further detail the effects of other members of the gp130 ligand family in the CNS.

View larger version (19K): [in this window] [in a new window]   Figure 1. Oncostatin M inhibits NMDA-mediated excitotoxic injury in vitro. A) Cortical neurons were incubated in the absence or presence of 50 µM NMDA and/or 50 ng/ml oncostatin M (OsM) and the degree of cell death determined 24 h later using the MTS cell viability assay. Percent viability was determined relative to untreated (control) cells. *Significant difference (P<0.01; n=3) compared to cells treated with NMDA alone. B) Cortical neurons were left untreated or pretreated with either 50 or 10 ng/ml OsM for 24 h. Culture media were then replaced with either fresh media or OsM (50 or 10 ng/ml) together with NMDA (50 µM) as indicated, and the degree of cell death was determined 24 h later using the MTS cell viability assay. Percent viability was determined relative to untreated (control) cells and results presented are mean values ±SD of quadruplicate determinations from 3 independent experiments. *P < 0.001 and **P < 0.01, significant difference compared to cells treated with NMDA alone.

View larger version (55K): [in this window] [in a new window]   Figure 2. OsM attenuates NMDA-mediated excitotoxic injury in vivo. The striatal region of mice was injected with NMDA alone or in the presence of vehicle (PBS+0.025% BSA) or 5 µg/ml OsM. 24 h later, the lesion volume was determined in 40 µm coronal sections that spanned the entire lesion. A) Representative sections showing the lesion produced after injection with NMDA alone (left panel) and with NMDA+OsM (right panel). The lesion appears as a darkened zone and the injection site (white arrows). White horizontal line in left image: 2 mm scale bar. B) Composite data showing the attenuating effect of OsM on NMDA-induced lesion volume (n=10 for NMDA; n=4 for NMDA+vehicle [PBS+ .025% BSA]; n=8 for NMDA+OsM). *Significant difference in lesion volume of NMDA+OsM treated group compared to mice injected with NMDA alone (P<0.001) and NMDA+vehicle (P<0.05).

View larger version (38K): [in this window] [in a new window]   Figure 3. Schematic representation of the proposed neuroprotective mechanism of OsM. OsM binds to an OSMRßbeta;/gp130 receptor complex, initiating signaling pathways via JAK-STAT and MAPK. The activated receptor interacts with the NMDA receptor complex blocking NMDA-mediated excitotoxicity. OsM-mediated down-regulation of NR2C might lead to reduced responsiveness to NMDA. OsM-mediated increase in OSMRßbeta; expression could provide a positive feedback loop while up-regulation of NS-1 and TIMP-1 would result in inhibition of t-PA and matrix-metalloproteinase activity, respectively. These proteases have been shown to have detrimental effects during excitotoxic injury. JAK, janus kinase; STAT, signal transducer and activator of transcription; MAPK, mitogen-activated protein kinase.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-5850fje

This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-5850fjev1 20/13/2369    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Weiss, T. W. Articles by Medcalf, R. L. Search for Related Content PubMed PubMed Citation Articles by Weiss, T. W. Articles by Medcalf, R. L.