Lymphocyte recruitment and activation have been implicated in the progression of cerebral ischemia-reperfusion (I/R) injury, but the roles of specific lymphocyte subpopulations and cytokines during stroke remain to be clarified. Here we demonstrate that the infiltration of T cells into the brain, as well as the cytokines interleukin-23 (IL-23) and IL-17, have pivotal roles in the evolution of brain infarction and accompanying neurological deficits. Blockade of T cell infiltration into the brain by the immunosuppressant FTY720 reduced I/R-induced brain damage. The expression of IL-23, which was derived mostly from infiltrated macrophages, increased on day 1 after I/R, whereas IL-17 levels were elevated after day 3, and this induction of IL-17 was dependent on IL-23. These data, together with analysis of mice genetically disrupted for IL-17 and IL-23, suggest that IL-23 functions in the immediate stage of I/R brain injury, whereas IL-17 has an important role in the delayed phase of I/R injury during which apoptotic neuronal death occurs in the penumbra. Intracellular cytokine staining revealed that gammadeltaT lymphocytes, but not CD4(+) helper T cells, were a major source of IL-17. Moreover, depletion of gammadeltaT lymphocytes ameliorated the I/R injury. We propose that T lymphocytes, including gammadeltaT lymphocytes, could be a therapeutic target for mitigating the inflammatory events that amplify the initial damage in cerebral ischemia.

These results indicate that PAR-6/aPKC selectively interacts with either mLgl or PAR-3 under the control of aPKC activity to regulate epithelial cell polarity.

Previously, we identified a new mammalian sHSP, MKBP, as a myotonic dystrophy protein kinase-binding protein, and suggested its important role in muscle maintenance (Suzuki, A., Sugiyama, Y., Hayashi, Y., Nyu-i, N., Yoshida, M., Nonaka, I., Ishiura, S., Arahata, K., and Ohno, S. (1998) J. Cell Biol. 140, 1113-1124). In this paper, we develop the former work by performing extensive characterization of five of the six sHSPs so far identified, that is, HSP27, ␣B-crystallin, p20, MKBP/ HSPB2, and HSPB3, omitting lens-specific ␣A-crystallin. Tissue distribution analysis revealed that although each sHSP shows differential constitutive expression in restricted tissues, tissues that express all five sHSPs are only muscle-related tissues. Especially, the expressions of HSPB3, identified for the first time as a 17-kDa protein in this paper, and MKBP/HSPB2 are distinctly specific to muscles. Moreover, these sHSPs form an oligomeric complex with an apparent molecular mass of 150 kDa that is completely independent of the oligomers formed by HSP27, ␣B-crystallin, and p20. The expressions of MKBP/HSPB2 and HSPB3 are induced during muscle differentiation under the control of MyoD, suggesting that the sHSP oligomer comprising MKBP/ HSPB2 and HSPB3 represents an additional system closely related to muscle function. The functional divergence among sHSPs in different oligomers is also demonstrated in several ways: 1) an interaction with myotonic dystrophy protein kinase, which has been suggested to be important for the maintenance of myofibril integrity, was observed only for MKBP/HSPB2; 2) a myotube-specific association with actin bundles was observed for HSP27 and ␣B-crystallin, but not for MKBP/ HSPB2; and 3) sHSPs whose mRNAs are induced by heat shock are ␣B-crystallin and HSP27. Taken together, the results suggest that muscle cells develop two kinds of stress response systems composed of diverged sHSP members, and that these systems work independently in muscle maintenance and differentiation.Heat shock and numerous other stress conditions lead to the rapid induction of several genes whose protein products, collectively called heat shock proteins (HSPs), 1 play protective roles in cell survival (1). Considering that muscles are frequently subjected to severe conditions caused by heat, oxidative, and mechanical stresses, especially during exercise (2), these HSPs may be especially important in this particular tissue. In fact, several HSPs, including HSP60, 70, and 90, have been shown to be induced after exhaustive exercise (3). In addition, the inducible isoform of HSP70 has been shown to be constitutively expressed in a certain type of skeletal muscle fiber (4), suggesting that muscle cells are chronically ready to respond to frequent stresses. However, there have been limited numbers of studies that focus on HSPs in muscle cells.HSPs with low molecular masses of 15-30 kDa are called small heat shock proteins (sHSPs); they commonly share a homologous sequence of about 80 amino acids called the "␣-crystallin domain" (5). Among ...

Focal adhesions (FAs) are essential structures for cell adhesion, migration, and morphogenesis. Integrin-linked kinase (ILK), which is capable of interacting with the cytoplasmic domain of β1 integrin, seems to be a key component of FAs, but its exact role in cell–substrate interaction remains to be clarified. Here, we identified a novel ILK-binding protein, affixin, that consists of two tandem calponin homology domains. In CHOcells, affixin and ILK colocalize at FAs and at the tip of the leading edge, whereas in skeletal muscle cells they colocalize at the sarcolemma where cells attach to the basal lamina, showing a striped pattern corresponding to cytoplasmic Z-band striation. When CHO cells are replated on fibronectin, affixin and ILK but not FA kinase and vinculin concentrate at the cell surface in blebs during the early stages of cell spreading, which will grow into membrane ruffles on lamellipodia. Overexpression of the COOH-terminal region of affixin, which is phosphorylated by ILK in vitro, blocks cell spreading at the initial stage, presumably by interfering with the formation of FAs and stress fibers. The coexpression of ILK enhances this effect. These results provide evidence suggesting that affixin is involved in integrin–ILK signaling required for the establishment of cell–substrate adhesion.

Background: PAR-6, aPKC and PAR-3 are polarity proteins that co-operate in the establishment of cell polarity in Caenorhabditis elegans and Drosophila embryos. We have recently shown that mammalian aPKC is required for the formation of the epithelia-specific cellcell junctional structure. We have also revealed that a mammalian PAR-6 forms a ternary complex with aPKC and ASIP/PAR-3, and localizes at the most apical end of the junctional complex in epithelial cells.