No abstract

The high mobility group box-1 (HMGB1), originally identified as an architectural nuclear protein, exhibits an inflammatory cytokine-like activity in the extracellular space. Here we show that treatment with neutralizing anti-HMGB1 monoclonal antibody (mAb; 200 microg, twice) remarkably ameliorated brain infarction induced by 2-h occlusion of the middle cerebral artery in rats, even when the mAb was administered after the start of reperfusion. Consistent with the 90% reduction in infarct size, the accompanying neurological deficits in locomotor function were significantly improved. Anti-HMGB1 mAb inhibited the increased permeability of the blood-brain barrier, the activation of microglia, the expression of TNF-alpha and iNOS, and suppressed the activity of MMP-9, whereas it had little effect on blood flow. Intracerebroventricular injection of HMGB1 increased the severity of infarction. Immunohistochemical study revealed that HMGB1 immunoreactivity in the cell nuclei decreased or disappeared in the affected areas, suggesting the release of HMGB1 into the extracellular space. These results indicate that HMGB1 plays a critical role in the development of brain infarction through the amplification of plural inflammatory responses in the ischemic region and could be an outstandingly suitable target for the treatment. Intravenous injection of neutralizing anti-HMGB1 mAb provides a novel therapeutic strategy for ischemic stroke.

Mitotic chromosomal dynamics is regulated by the coordinated activities of many mitotic kinases, such as cyclin-dependent kinase 1 (Cdk1), Aurora-B or Polo-like kinase 1 (Plk1), but the mechanisms of their coordination remain unknown. Here, we report that Cdk1 phosphorylates Thr 59 and Thr 388 on inner centromere protein (INCENP), which regulates the localization and kinase activity of Aurora-B from prophase to metaphase. INCENP depletion disrupts Plk1 localization specifically at the kinetochore. This phenotype is rescued by the exogenous expression of INCENP wild type and INCENP mutated at Thr 59 to Ala (T59A), but not at Thr 388 to Ala (T388A). The replacement of endogenous INCENP with T388A resulted in the delay of progression from metaphase to anaphase. We propose that INCENP phosphorylation by Cdk1 is necessary for the recruitment of Plk1 to the kinetochore, and that the complex formation of Plk1 and Aurora-B on INCENP may play crucial roles in the regulation of chromosomal dynamics.

14‐3‐3 proteins control various cellular processes, including cell cycle progression and DNA damage checkpoint. At the DNA damage checkpoint, some subtypes of 14‐3‐3 (β and ζ isoforms in mammalian cells and Rad24 in fission yeast) bind to Ser345‐phosphorylated Chk1 and promote its nuclear retention. Here, we report that 14‐3‐3γ forms a complex with Chk1 phosphorylated at Ser296, but not at ATR sites (Ser317 and Ser345). Ser296 phosphorylation is catalysed by Chk1 itself after Chk1 phosphorylation by ATR, and then ATR sites are rapidly dephosphorylated on Ser296‐phosphorylated Chk1. Although Ser345 phosphorylation is observed at nuclear DNA damage foci, it occurs more diffusely in the nucleus. The replacement of endogenous Chk1 with Chk1 mutated at Ser296 to Ala induces premature mitotic entry after ultraviolet irradiation, suggesting the importance of Ser296 phosphorylation in the DNA damage response. Although Ser296 phosphorylation induces the only marginal change in Chk1 catalytic activity, 14‐3‐3γ mediates the interaction between Chk1 and Cdc25A. This ternary complex formation has an essential function in Cdc25A phosphorylation and degradation to block premature mitotic entry after DNA damage.

The small GTPase Rho and one of its targets, Rhoassociated kinase (Rho-kinase), are implicated in a wide spectrum of cellular functions, including cytoskeletal rearrangements, transcriptional activation and smooth muscle contraction. Since Rho also plays an essential role in cytokinesis, Rho-kinase may possibly mediate some biological aspects of cytokinesis. Here, using a series of monoclonal antibodies that can speci®cally recognize distinct phosphorylated sites on glial ®brillary acidic protein (GFAP) and vimentin, phosphorylation sites by Rho-kinase in vitro were revealed to be identical to in vivo phosphorylation sites on these intermediate ®lament (IF) proteins at the cleavage furrow in dividing cells. We then found, by preparing two types of antiRho-kinase antibodies, that Rho-kinase accumulated highly and circumferentially at the cleavage furrow in various cell lines. This subcellular distribution during cytokinesis was very similar to that of ezrin/radixin/ moesin (ERM) proteins and Ser 19 -phosphorylated myosin light chain. These results raise the possibility that Rhokinase might be involved in the formation of the contractile ring by modulating these F-actin-binding proteins during cytokinesis and in the phosphorylation and regulation of IF proteins at the cleavage furrow.

Chk1, one of the critical transducers in DNA damage/replication checkpoints, prevents entry into mitosis through inhibition of Cdk1 activity. However, it has remained unclear how this inhibition is cancelled at the G 2 /M transition. We reported recently that Chk1 is phosphorylated at Ser 286 and Ser 301 by Cdk1 during mitosis. Here, we show that mitotic Chk1 phosphorylation is accompanied by Chk1 translocation from the nucleus to the cytoplasm in prophase. This translocation advanced in accordance with prophase progression and was regulated by Crm-1-dependent nuclear export. Exogenous Chk1 mutated at Ser 286 and Ser 301 to Ala (S286A/S301A) was observed mainly in the nuclei of prophase cells, although such nuclear accumulation was hardly observed in wild-type Chk1. Induction of S286A/S301A resulted in the delay of mitotic entry. Biochemical analyses using immunoprecipitated cyclin B 1 -Cdk1 complexes revealed S286A/S301A expression to block the adequate activation of Cdk1. In support of this, S286A/S301A expression retained Wee1 at higher levels and Cdk1-induced phosphorylation of cyclin B 1 and vimentin at lower levels. A kinase-dead version of S286A/S301A also localized predominantly in the nucleus but lost the ability to delay mitotic entry. These results indicate that Chk1 phosphorylation by Cdk1 participates in cytoplasmic sequestration of Chk1 activity, which releases Cdk1 inhibition in the nucleus and promotes mitotic entry.

Summary Figure   Working model for the ERBIN–p0071 interaction at cell‐cell adhesions. ERBIN is associated with p0071 at adherens junctions and desmosomes. p0071 is thought to interact with classic cadherins or desmosomal cadherins (Hatzfeld 1999). The subcellular localization of ERBIN may be regulated by the Rho family and other signals controlling cell polarity. Background: ERBIN, an ErbB2 receptor‐interacting protein, belongs to a recently described family of proteins termed the LAP [leucine‐rich repeats and PSD‐95/dLg‐A/ZO‐1 (PDZ) domains] family which has essential roles in establishment of cell polarity. Results: To identify new ERBIN‐binding proteins, we screened a yeast two‐hybrid library, using the carboxyl‐terminal fragment of ERBIN containing PDZ domain as the bait, and we isolated p0071 (also called plakophilin‐4) as an ERBIN‐interacting protein. p0071 is a member of the p120 catenin family, which are defined as proteins with 10 armadillo repeats, and localizes along the cell‐cell border. The ERBIN PDZ domain binds the COOH‐terminus of p0071 containing the PDZ domain‐binding sequence. Endogenous ERBIN was co‐immunoprecipitated with p0071. In fully polarized Madin–Darby canine kidney (MDCK) cells, ERBIN co‐localized largely with β‐catenin and partly with desmoplakin along the lateral plasma membrane domain. At these cell‐cell contact regions, ERBIN co‐localizes with p0071. Over‐expression of the dominant active forms of Cdc42, Rac1 or RhoA, Rho family small GTPases, resulted in a marked accumulation of ERBIN at the cell‐cell contacts of MDCK and HeLa cells. Conclusion: These results show that ERBIN interacts in vivo with p0071 and that it may be involved in the organization of adherens junctions and the desmosomes of epithelia. In addition, we demonstrated that the subcellular localization of ERBIN might be regulated by Rho family small GTPases.

yocardial infarction is a disease that remains highly lethal despite recent advanced medical treatment. It is caused by the sudden interruption of coronary flow by occlusion of the coronary artery, which sequentially causes irreversible cardiomyopathy, tissue loss, and scar formation. Various pharmacological or surgical therapies have been reported for ischemia-reperfusion (I/R) injury, 1 and recent investigations have revealed the involvement of nitric oxide in myocardial injury during coronary reperfusion. 2 Moreover, I/R induces an inflammatory response and cytokine release from inflammatory cells, such as monocytes, macrophages and leukocytes, 3 but there is still no satisfactory protective therapy for I/R injury by regulating inflammation.High-mobility group box 1 protein (HMGB1) is a nonhistone DNA-binding, multifunctional protein that has both nuclear and extracellular functions. HMGB1 was originally identified as a DNA-binding protein, and was described as a multifunction transcriptional factor (amphoterin) that interacted with DNA. 4 In addition, HMGB1 has a binding site for the receptor for advanced glycation end-products (RAGE), which was originally identified as a receptor for the AGEs that accumulate during hyperglycemia. 5,6 Consequently, HMGB1-deficient mice die of hypoglycemia within 24 h of birth, suggesting that HMGB1 is associated with glycemic homeostasis. 7 Nuclear HMGB1 is released into the extracellular space during inflammatory responses that markedly and extensively activate macrophages and monocytes, as well as from necrotic cells, being released in sepsis. [8][9][10] Recent animal model studies have shown that HMGB1 is associated with I/R injury in several organs. 11 In liver I/R injury in the rat, plasma HMGB1 levels were increased at the early time point of 1 h after reperfusion and thereafter further increased in a time-dependent manner for 24 h. Treatment with a neutralizing antibody to HMGB1 significantly decreased liver damage after reperfusion injury. 12,13 In cerebral I/R injury in the rat, extracellular HMGB1 prevented expansion of the lesion during acute neuronal damage processes, and treatment with anti-HMGB1 antibody improved brain infarction post-treatment. 14 In I/R injury of the heart, cardiomyocyte inflammatory receptors are stimulated by pro-inflammatory mediators. 15 Recent studies have suggested that I/R injury may be attenuated through therapeutic targeting of these inflammatory receptors, such as RAGE, toll-like receptor (TLR) 2, and TLR4. [16][17][18] These observations predict that blocking the HMGB1 signal may protect the heart from I/R injury. However, another study of HMGB1 showed that exogenous HMGB1 induced myocardial cell proliferation and differentiation in the infarcted heart tissue. 19 In the present study, neutralizing anti-HMGB1 monoclonal antibody (mAb) was used to elucidate the role of HMGB1 in cardiac I/R injury. Background Coronary ischemia-reperfusion (I/R) injury causes cardiomyocyte necrosis in a multi-step process that includes an inflammatory ...