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J. Biol. Chem., Vol. 277, Issue 46, 43968-43972, November 15, 2002

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Mitogen-activated Protein Kinase-activated Protein (MAPKAP) Kinase 2 Deficiency Protects Brain from Ischemic Injury in Mice^*

Xinkang Wang^§, Lin Xu, Hugh Wang, Peter R. Young, Matthias Gaestel^¶, and Giora Z. Feuerstein

From the  Department of Cardiovascular Sciences, Bristol-Myers Squibb Company, Wilmington, Delaware 19880-0400 and ^¶ Medical School Hannover, Institute of Biochemistry, 30625 Hannover, Germany

Received for publication, July 9, 2002, and in revised form, August 26, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mitogen-activated protein (MAP) kinase-activated protein kinase 2 (MK2) is one of several kinases directly regulated by p38 MAP kinase. A role of p38 MAP kinase in ischemic brain injury has been previously suggested by pharmacological means. In the present study, we provide evidence for a role of MK2 in cerebral ischemic injury using MK2-deficient (MK2^/) mice. MK2^/ mice subjected to focal ischemia markedly reduced infarct size by 64 and 76% after transient and permanent ischemia, respectively, compared with wild-type mice. Furthermore, MK2^/ mice had significant reduction in neurological deficits. Real-time PCR analysis identified a significantly lower expression in interleukin-1 mRNA (53% reduction) but not in tumor necrosis factor- mRNA in MK2^/ mice over wild-type animals after ischemic injury. The significant reduction in interleukin-1 was also confirmed in MK2^/ mice by enzyme-linked immunosorbent assay. The marked neuroprotection from ischemic brain injury in MK2^/ mice was not associated with the alteration of hemodynamic or systemic variables, activation of caspase-3, or apoptosis. Our data provide new evidence for the involvement of MAP kinase pathway in focal ischemic brain injury and suggest that this effect might be associated with the expression of interleukin-1 in the ischemic brain tissue.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The three mitogen-activated protein (MAP)¹ kinase families include the extracellular regulated kinases, the c-jun N-terminal kinase/stress-activated protein kinases and the p38 MAP kinases. These MAP kinases have been implicated in a variety of cellular functions such as proliferation, differentiation, and survival (1). MAP kinase-activated protein kinase 2 (MK2) is one of several kinases that are regulated exclusively through direct phosphorylation by p38 MAP kinase in response to stress stimuli. Mice deficient in MK2 showed a reduction in bacterial lipopolysaccharide-induced biosynthesis of tumor necrosis factor (TNF)-, interferon-, interleukin (IL)-1, IL-6, and nitric oxide (2), suggesting a critical role of MK2 in inflammatory cytokine production.

Cerebral ischemia is a pathophysiological condition caused by decrease in blood supply to the brain and hence the deprivation of oxygen and glucose in the ischemic brain tissue, which eventually leads to cell death (necrosis and apoptosis) and inflammation (3). Concomitant activation of extracellular regulated kinase, c-jun N-terminal kinase, and p38 MAP kinase has been reported in both gerbil and rat models of transient brain ischemia (4, 5). Furthermore, certain MAP kinases have been implicated in cerebral ischemic injury. For example, inhibition of extracellular regulated kinase 1/2 by a selective MAP kinase/extracellular-regulated kinase kinase 1 inhibitor revealed significant neuroprotection after transient cerebral ischemia in mice (6, 7). Similarly, inhibition of p38 MAP kinase reduced brain injury and neurological deficits after permanent occlusion of the middle cerebral artery (MCAO) in rats (8). Because MK2 is an immediate downstream kinase of the p38 MAP kinase signaling pathway, we hypothesized that MK2 might also be directly associated with ischemic brain injury after MCAO. Therefore, mice genetically deficient in MK2 were used to test this hypothesis in both transient and permanent focal stroke induced by MCAO. In this model, histological and functional variables have been explored along with biochemical markers of inflammation and apoptosis.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Focal Brain Ischemia-- MK2^/ mice were on a mixed 129v × C57BL/6 background as described previously (2) and the colony further expanded by Charles River Laboratories (Wilmington, MA). Genotyping of MK2^/ was carried out using a three-primer PCR with the oligonucleotides 5'-cgtgggggtggggtgacatgctggttgac (5'MK2), 5'-ggtgtcaccttgacatcccggtgag (3'MK2), and 5'-tgctcgctcgatgcgatgtttcgc (Neo). A fragment length of about 500 bp indicates wild type and 800 bp indicates the gene disruption.

Adult MK2^/ and C57BL/6 mice (18-22 g, paired for gender and weight) were used throughout the experiments. Animals were housed and cared for in accordance with the Guide for the Care and Use of Laboratory Animals (29). Procedures using laboratory animals were approved by the Institutional Animal Care and Use Committee of Bristol-Myers Squibb Company.

Mice were anesthetized with gas inhalation, which is composed of 30% oxygen (0.3 liter/min) and 70% nitrous oxide (0.7 liter/min). The gas was passed through an isoflurane vaporizer set to deliver 3-4% isoflurane during initial induction and 1.5-2% during surgery. Under such conditions, an incision of the skin was made directly on top of the right common carotid artery region, and the bifurcation of the external common carotid and internal common carotid was identified. A small incision was made on the external common carotid, and a 5-0 mono-filament suture (9-11 mm long with a round tip) was threaded into the internal common carotid via the external common carotid. The suture was advanced toward the middle cerebral artery to create focal ischemia. In the case of permanent brain ischemia, the suture was not removed, whereas for transient brain ischemia, the suture was removed 30 min after MCAO. Sham operation was performed using the same procedure except that no suture was inserted into the carotid artery. At the end of the study, mice were anesthetized with gas inhalation and forebrains were removed at various times after ischemia, reperfusion, or sham surgery as indicated in each figure legend. For biochemical analysis, the entire ipsilateral and contralateral hemispheres were dissected and immediately frozen in liquid nitrogen and stored at 80 °C for later use.

To measure the infarct volume, brains were removed 24 h after MCAO and evaluated using 2,3,5-triphenyltetrazolium chloride (TTC) staining of 2-mm thick brain slices. The stained brain tissue was fixed in 10% formalin in phosphate-buffered saline. The image was captured using a Microtek ScanMaker 4 DUO scanner (Microtek, Carson, CA) and quantitated using Image Pro Plus 4.1 software (Media Cybernetics, Silver Spring, MD).

Neurological Deficits-- Neurological deficits were examined at days 1 and 3 after MCAO (n = 10) using a five-point scale adapted and modified from Zhang et al. (9) (no neurological deficit, 0; right Horner's syndrome, 1 point; failure to extend left forelimb and hindlimb, 1 point each; turning to left, 1 point; and circling to left, 1 point.

Physiological Parameters-- The physiological parameters were measured and confirmed under two anesthesia conditions (i.e. gas inhalation as described above and pentobarbital (50 mg/kg, intraperitoneally)). In randomly selected animals, regional cerebral blood flow (CBF) was measured with a laser Doppler perfusion monitor (Moor Instruments Inc., Wilmington, DE). After anesthesia, a small incision was made at the midpoint between the right orbit and the external auditory canal. The temporalis muscle was retracted and the underlying fascia cleared. The laser Doppler probe was placed 1.5 mm posterior and 3.5 mm lateral to the bregma on the ipsilateral hemisphere. CBF was carefully monitored (to avoid any large vessel) before, during (15 min), and after (30 min) MCAO. Relative CBF was calculated as percentage relative to levels before MCAO.

The arterial blood pressure and heart rate were measured by connecting tubing through the femoral artery using an MP100 workstation and analyzed using an AcqKnowledge software (BIOPAC Systems, Inc., Santa Barbara, CA) according to the manufacturer's specifications. Femoral arterial blood samples were analyzed for pH, oxygen, and carbon dioxide by direct collection through a PE-50 tubing into an i-STAT G3+ cartridge and processed with a portable clinical analyzer (Abbott).

Real-time RT-PCR-- Total RNA was isolated from ipsilateral and contralateral brain tissues (n = 8) after transient MCAO or after sham operation using an RNA isolation kit from QIAGEN (Valencia, CA). The primers and probes (Table I) used for real-time RT-PCR were designed using Primer-Express 1.0 software from Applied Biosystems (Foster City, CA). The specificity of PCR primers for IL-1, TNF, and a housekeeping gene, ribosomal protein L32, was tested using a standard PCR protocol in an ABI thermocycler (model 9600) before TaqMan quantitation and confirmed by gel electrophoresis. Real-time PCR was performed as described previously in detail (10) with the following modifications: One-step RT-PCR was performed using a Platinum Taq System (Invitrogen) according to the manufacturer's specification. The reaction started with 0.5-1 µg of total RNA in a 25-µl reaction volume. The reaction mixture contained 12.5 µl of 2× reaction mix, 0.6 µl of 50 mM MgSO₄, 0.125 µl of RNase inhibitor, 0.5 µl each of the 10 µM forward and reverse primers, 0.5 µl of the 5 µM probe, and 0.3 µl of RT/Taq mix. The mixture was incubated in 50 °C for 30 min and 95 °C for 5 min, and then the PCR cycles were started at 95 °C for 15 s and 60 °C for 60 s for 40 cycles. Each RT-PCR was done in duplicate and performed simultaneously. Data were analyzed using the Sequence Detector v1.6.3 program (Applied Biosystems).

View this table: [in this window] [in a new window]   Table I Primers and TaqMan probes used in the real-time PCR PCR primers (F, forward; R, reverse) and probes were synthesized according to the mouse TNF- (GenBank accession no. M13049), IL-1 (GenBank accession no. M15131), and rpL32 (GenBankTM accession number AK002353) cDNA sequences, respectively. TaqMan probes contain 6-carboxyfluorescein for IL-1 and TNF at 5'-end and VIC, an ABI dye, for the rpL32. All the probes have a quencher dye, 6-carboxytetramethylrhodamine, at the 3' end.

Enzyme-linked Immunosorbent Assay for IL-1-- Tissue lysate from ipsilateral and contralateral brain samples (15 h after MCAO for the peak expression of IL-1, n > 7) were pulverized using a porcelain mortar and pestle under liquid nitrogen. The pulverized brain tissues were incubated in a lysis buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100) and 5 µl/ml of protease inhibitor mixture (P-8340; Sigma) for 1 h at 4 C°. After a 10-min centrifugation at 10,000 × g, the supernatant of tissue lysate was collected and aliquoted for enzyme-linked immunosorbent assay (ELISA) and protein concentration measurement using a detergent-compatible protein assay kit (Bio-Rad). The levels of IL-1 protein in the brain tissue were measured using an ELISA kit for mouse IL-1 (Endogen) following the manufacturer's specification. Tissue extracts (50 µl) were applied to each well for the ELISA and the final measure was read out using a plate reader at 450 nm. The concentration of IL-1 protein in each sample was determined according to the standard (recombinant mouse IL-1 protein) provided with the kit. All the measured IL-1 concentrations were at the linear part of the standard curve. Each sample was normalized by its total protein concentration in milligrams.

Western Blot Analysis-- Western blot analysis was used to evaluate the levels of the active form of caspase-3 in MK2^/ (n = 8) and wild-type (n = 9) mice 24 h after transient MCAO. The pulverized brain tissues were lysed and processed as described in the previous section. The soluble component of the tissue lysate was used for Western blot (100 µg of protein/lane) using a mouse monoclonal IgG against caspase-3 (sc-7272) as described in detail previously (11). The blot was stripped and re-probed to a goat polyclonal anti-actin antibody (sc-1616; Santa Cruz Biotechnology, Inc.).

Apoptosis Analysis-- Apoptosis was measured by quantitating the DNA fragmentation in MK2^/ (n = 8) and wild-type (n = 9) mice 24 h after transient MCAO using a cell death detection ELISA kit (Roche Molecular Biochemicals). This sandwich-enzyme immunoassay provides a quantitative determination of histone-associated DNA fragments (mono- and oligonucleosomes) based on a photometric reaction using monoclonal antibodies directed against both DNA and histones. Frozen, pulverized brain tissue was lysed using the lysing buffer provided by the kit (30 min at room temperature) and pelleted (200 × g). Aliquots of the supernatant were used in the assay according to the manufacturer's protocol.

Statistical Analysis-- Data in text and figures are mean ± S.E. for the indicated number (n) of animals. Statistical comparisons were made by analysis of variance (Fisher's protected least squares difference), and values were considered to be significant at p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Physiological Parameters in MK2^/ and Wild-type Mice after Cerebral Ischemia-- Cerebral blood flow, heart rate, arterial blood pressure, pH, blood oxygen, and blood carbon dioxide were measured in MK2^/ and wild-type mice before and after transient MCAO (Table II). No significant difference was observed in CBF, heart rate, and blood gases between MK2^/ and wild-type mice before and after MCAO. The only significant difference was the 11% increase in the mean arterial blood pressure in MK2^/ mice compared with wild-type mice 30 min after reperfusion (p < 0.05; Table I). However, this small increase in blood pressure is within the normal range in mice.

View this table: [in this window] [in a new window]   Table II Physiological conditions in the wild-type and MK2/ mice after MCAO with reperfusion Mice were subjected to 30 min of MCAO followed by reperfusion. The physiological data were measured before, during (15 min after start of MCAO), or after (30 min of reperfusion) MCAO. CBF, cerebral blood flow (arbitrary units, the relative arbitrary unit before MCAO was illustrated as 100%); HR, heart rate (per min); MABP, mean artery blood pressure (mm Hg); pCO2, blood carbon dioxide; pO2, blood oxygen.

MK2^/ Provides Partial Protection from Ischemic Brain Injury-- As shown in Fig. 1, significant reduction in infarct size was observed after transient (64% reduction, n = 13, p < 0.05) and permanent (76% reduction, n = 10, p < 0.01) MCAO in MK2^/ mice compared with the paired wild-type mice. The resistance of MK2^/ mice to ischemic brain injury was also supported by the reduction in neurological deficits (Fig. 2). The neurological deficits were not significantly reduced in MK2^/ mice until 3 days after transient MCAO (34% reduction compared with wild-type mice, n = 14, p < 0.01). In contrast, a significant reduction in neurological deficits was observed 24 h after permanent MCAO (52% reduction in MK2^/ mice compared with the wild-type mice, n = 10, p < 0.01) (Fig. 2). The 3-day neurological deficit data were not collected after permanent MCAO because these animals were processed for infarct size evaluation at 24 h.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   MK2 deficiency reduced infarct size after transient and permanent cerebral ischemia. MK2/ (KO) or paired wild-type (C57BL/6; WT) mice were subjected to transient or permanent MCAO as described in detail under "Experimental Procedures." Brain tissues were collected 24 h after MCAO and 2-mm brain slices were stained with TTC. Representative images of the MK2/ mice and wild-type mice after transient MCAO were illustrated where the infarct area was labeled (A). Total ischemic lesion in the MK2/ mice and wild-type mice was measured and summarized as means ± S.E. (B) after transient (top, TMCAO, n = 13) or permanent (bottom, PMCAO, n = 10) MCAO. *, p < 0.05; **, p < 0.01 compared with the wild-type animals.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Neurological deficits in MK2/ and wild-type mice after transient and permanent MCAO. For transient MCAO (TMCAO), MK2/ (KO) and wild-type (WT) mice were subjected to 30-min MCAO followed by reperfusion, and neurological deficits were evaluated at days 1 and 3 after reperfusion (n = 14). For permanent MCAO (PMCAO), neurological deficits were examined at day 1 (24 h; n = 10) after ischemia using the same groups of animals for infarct size measurement (illustrated in Fig. 1). Neurological deficits were measured using a five-point scale as described under "Experimental Procedures." **, p < 0.01 compared with the wild-type animals.

Cytokine Gene Expression in Ischemic Brain of MK2^/ and Wild-type Mice after MCAO-- Fig. 3 depicts the mRNA expression of two key inflammatory cytokines, IL-1 and TNF, in MK2^/ and wild-type mice 12 h after transient MCAO. Significant induction was observed for both cytokine mRNAs in the ipsilateral (ischemic) over the contralateral brain tissue in wild-type mice (with 4.3- and 3.4-fold increases for TNF and IL-1 mRNA, respectively). However, in MK2^/ mice, significant induction was seen only in TNF mRNA (3.6-fold increase in the ipsilateral brain tissue) but not IL-1 mRNA (1.6-fold increase) after MCAO (Fig. 3). The levels of IL-1 mRNA expression in the ischemic brain tissue were significantly lower in MK2^/ than in wild-type mice (p < 0.05, n = 8).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Real-time PCR analysis of TNF and IL-1 mRNA expression in the brain after transient MCAO. MK2/ (KO) and wild-type (WT) mice were subjected to 30-min MCAO followed by reperfusion. Brain tissues were collected at 12 h after MCAO and used for real-time PCR analysis as described under "Experimental Procedures." The levels of TNF or IL-1 mRNA expression were measured and normalized with rpL32 mRNA. Data are illustrated as the ratio of ipsilateral (ischemic; I) to contralateral (nonischemic; C) samples for TNF and IL-1 mRNA (n = 8). *, p < 0.05 compared with the wild-type animals.

ELISA analysis showed that levels of IL-1 expression were increased 3.3-fold (n = 11, p < 0.05) and 7.9-fold (n = 7, p < 0.01) in the ischemic brain tissue over the nonischemic (contralateral) tissue in wild-type mice 15 h after transient and permanent MCAO, respectively. Similar to its mRNA induction profile, the levels of IL-1 expression after brain ischemia were significantly lower in MK2^/ mice (Fig. 4) (i.e. only 49% (n = 9, p < 0.05) and 21% (n = 8, p < 0.05) compared with the wild-type mice after transient and permanent MCAO, respectively).

View larger version (26K): [in this window] [in a new window]   Fig. 4.   ELISA analysis of IL-1 expression in the brain after transient and permanent MCAO. MK2/ (KO) and wild-type (WT) mice were subjected to 30 min of MCAO followed by reperfusion (TMCAO) or permanent MCAO (PMCAO). Brain tissues were collected at 15 h after MCAO and processed for "sandwich" ELISA analysis as described under "Experimental Procedures." The levels of IL-1 protein in the brain (picograms per milliliter) were determined according to the recombinant IL-1 protein standard. Data are illustrated as mean ± S.E. after normalizing with total protein concentration applied to the assay. *, p < 0.05, compared with the ipsilateral (ischemic) sample of the wild-type animals.

Comparative Analysis of Caspase-3 Activation and Apoptosis in MK2^/ and Wild-type Mice after MCAO-- Because MAP kinase has been implicated in cell survival as well as apoptosis after cerebral ischemic injury, we evaluated key markers of apoptosis (i.e. activation of caspase-3 (assessed for the expression of active caspase-3) and DNA fragmentation). Western analysis was used to detect the expression of active caspase-3 (p20) in the brain after MCAO. The levels of active caspase-3 in the ischemic brain were significantly elevated 24 h after MCAO in both MK2^/ and wild-type mice, showing 1.8- and 2.0-fold increases, respectively, over the contralateral tissue. However, no significant difference was observed between these two experimental groups (Fig. 5A).

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Comparative analysis of apoptosis in the MK2/ and wild-type mice after MCAO. A, Western analysis of active caspase-3 (p20) expression. MK2/ (KO) (n = 9) and wild-type (WT) (n = 11) mice were subjected to 30 min of MCAO followed by reperfusion. Brain tissues were collected at 24 h after MCAO and used for Western blot to measure the expression of active caspase-3 (p20). Data are illustrated as the ratio of ipsilateral (I)/contralateral (C) brain tissues for mean ± S.E. after normalizing with a housekeeping gene, actin. B, DNA fragmentation measurement by ELISA. Data are illustrated as mean ± S.E. values of the I/C ratio after normalizing with total protein concentration applied to the assay. OD, optical density.

Similarly, although 3.9- and 4.3-fold increases in DNA fragmentation were observed in MK2^/ and wild-type mice, respectively, after cerebral ischemia, as evaluated by measurement of DNA fragmentation using an ELISA method, no significant difference was noted between these two groups (Fig. 5B).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the present study, we have demonstrated that ischemic brain injury was significantly reduced in MK2-deficient mice compared with that of wild-type mice after either transient or permanent MCAO. Permanent MCAO results in the depletion of energy and oxygen to brain tissue, which ultimately leads to cell death; transient MCAO results in ischemic brain injury primarily because of cellular toxicity caused by oxygen radicals and other mediators involved with reperfusion injury (12). Both permanent and transient MCAO result in an inflammatory reaction as one of the key pathophysiological events occurring after ischemic brain injury. Activation of the p38 MAP kinase pathway after transient and permanent MCAO has been demonstrated (5), and the inhibition of p38 MAP kinase by a specific small molecule inhibitor was shown to protect the brain from ischemic injury after permanent MCAO in rats (8). However, the involvement of a particular downstream component of p38 MAP kinase in ischemic brain injury has not been previously elucidated. In the present study, we provide direct evidence for the potential role of MK2, which is regulated by the p38 MAP kinase, in the ischemic brain injury using a genetic model of MK2 deficiency.

Mice deficient in MK2 showed no difference in several key hemodynamic, hematologic, and biochemical parameters compared with wild-type animals under normal conditions or post stroke. However, mice deficient in MK2 gene were protected from both transient and permanent MCAO, as evidenced by smaller infarct size and improved neurological function. Although only TTC staining was used to assess infarct size in this study, the validity of this method as an authentic representation of tissue injury was demonstrated by the tight correlation of data generated with TTC staining quantitation of infarct size using hematoxylin and eosin staining (13, 14). Furthermore, TTC staining was also corroborated by magnetic resonance imaging (14-16). Finally, and more importantly, the relative resistance of the MK2^/ mice to stroke was manifested not only by reduction in infarct size but also by improvement in motor function (Figs. 1 and 2).

The specific mechanism by which MK2 may promote ischemic brain injury is unknown. Because inflammation is a notable pathophysiological consequence after cerebral ischemia (17) and MK2 has been implicated in regulation of cytokine production (2), the expression of two key proinflammatory cytokines, IL-1 and TNF, was evaluated in MK2^/ and wild-type mice after MCAO. Interestingly, whereas TNF mRNA was significantly induced by ischemia in both MK2^/ and wild-type mice, the levels of IL-1 mRNA were significantly lower in MK2^/ mice compared with the wild-type mice. The corresponding reduction in IL-1 protein was also noted in MK2^/ mice compared with wild-type animals. These data are in agreement, at least in part, with a previous report that one class of p38 antagonists, known as cytokine-suppressing anti-inflammatory drugs, has the capacity to inhibit IL-1 production in response to lipopolysaccharide stimulation in human monocytes (18, 19). Unfortunately, because of lack of proper reagents to study mouse TNF in tissue, the levels of TNF protein could not be determined in the present study. We wish to offer our speculation that because TNF mRNA expression was not altered in MK2^/ mice compared with wild-type mice, similar response in protein levels might be expected.

Increase in IL-1 expression has been observed in several types of brain injury including excitotoxicity and lipopolysaccharide-induced brain injury as well as brain trauma and ischemia (17, 20). In particular, the significant up-regulation of IL-1 is recognized to play a detrimental role after brain ischemia, because the blockade of IL-1 by interleukin-1 receptor antagonist has been shown to be neuroprotective (21). IL-1 was recently shown to be associated with inflammatory cell activation and the induction of cyclooxygenase-2, IL-1, and IL-6 in other models of brain injury (22). Therefore, the suppression of IL-1 production in the ischemic brain tissue of MK2^/ mice might be associated with reduced ischemic damage.

Apoptosis represents another potential mechanism associated with ischemia-induced neuronal death (23). In particular, p38 MAP kinase has been implicated in neuronal apoptosis in response to various stimuli (24). Therefore, we investigated the potential involvement of apoptosis in MK2-deficient mice subjected to MCAO. Whereas apoptosis has been clearly demonstrated after cerebral ischemia, as monitored by active caspase-3 and DNA fragmentation, no difference was found between MK2^/ mice and wild-type mice in these apoptotic parameters after MCAO. In addition, no difference was observed in gliosis and microglial activation as evidenced by immunohistochemical evaluation.² Gene expression array also failed to detect differences between MK2-deficient and control mice in the activation of specific signaling pathways using the "Pathway Finder" panel (Super Array, Inc.) (data not shown). It should also be pointed out that several downstream targets have been reported to be phosphorylated and regulated by MK2, including the 27-kDa heat shock protein (25), lymphocyte-specific protein 1 (26), tyrosine hydroxylase (27) and 5-lipoxygenase (28); however, because of lack of reagents that cross-react with mouse protein, the specific activation of these MK2 substrates could not be determined.

In conclusion, our study provides further evidence for the involvement of the p38/MK2 pathway in focal ischemic brain injury. The diminution of IL-1 expression in the MK2^/ mice may suggest that MK2 might be a key modulating pathway of certain inflammatory cytokines activated in brain injury.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed: Dept. of Cardiovascular Sciences, Bristol-Myers Squibb Company, Experimental Station, E400/3418, Wilmington, DE 19880-0400. Tel.: 302-467-5080; Fax: 302-467-6831; E-mail: xinkang.wang@bms.com.

Published, JBC Papers in Press, September 4, 2002, DOI 10.1074/jbc.M206837200

² X. Wang, L. Xu, H. Wang, P. R. Young, M. Gaestel, and G. Z. Feuerstein, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: MAP, mitogen-activated protein; MK2, mitogen-activated kinase-activated protein kinase 2; TNF, tumor necrosis factor; MCAO, occlusion of the middle cerebral artery; TTC, 2,3,5-triphenyltetrazolium chloride; CBF, cerebral blood flow; RT, reverse transcription; ELISA, enzyme-linked immunosorbent assay; IL, interleukin; rpL32, ribosomal protein L32.

	REFERENCES

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