ARTICLE |
Correspondence to: Yutaka Yatomi, Dept. of Laboratory Medicine, Yamanashi Medical University, Nakakoma, Yamanashi 409-3898, Japan. E-mail: yatomiy@res.yamanashi-med.ac.jp
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Summary |
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The LIM domain is a proteinprotein interaction motif critically involved in a variety of fundamental biological processes, including cytoskeletal organization, cell lineage specification, and organ development. In this study we examined the expression of the LIM proteins paxillin and Hic-5 in adult human tissues by immunohistochemistry and immunoblotting. Paxillin expression was widespread and observed both in non-muscle and muscle tissues. Of the latter, paxillin was mainly expressed in multinuclear striated muscle. In contrast, Hic-5 showed restricted expression and was expressed in muscle tissues, mainly in mononuclear smooth muscle. Taken together with previous findings, it appears likely that the counterbalance between paxillin and Hic-5 may be deeply involved in muscle differentiation. (J Histochem Cytochem 51:513521, 2003)
Key Words: paxillin, Hic-5, LIM protein, focal adhesion, cytoskeletal reorganization, striated muscle, smooth muscle, human
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Introduction |
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PAXILLIN plays an important role in the formation of focal adhesion complexes that link the actin cytoskeleton and regulate the intracellular signaling pathways, thereby coordinating cell spreading, movement, and gene expression (
Hic-5 was originally identified as a transforming growth factor (TGF)ß1- and hydrogen peroxide-inducible gene by differential hybridization (
Although it is now established that Hic-5 is a paxillin homologue localized to focal adhesion complexes, recent studies have shown that these LIM proteins have some distinct functional features (see Discussion). Furthermore, recent reports, including ours, have clearly shown that paxillin and Hic-5 may play distinct and exclusive roles in megakaryocytes and platelets. Paxillin, but not Hic-5, is expressed in megakaryocytes, and may play an important role in signaling pathways within the focal adhesions through interaction with Pyk2 (IIbß3-mediated outside-in signaling (
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Materials and Methods |
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Tissues
Cardiac muscle and iliopsoas muscle were obtained from adult autopsy cases. All other tissue samples were obtained from the surgical margins of various lesions. The sample collection was in accordance with the Yamanashi Medical University Guidelines. The tissues were immediately fixed in methanol for 2428 hr and then embedded in paraffin.
Antibodies
In this study, two anti-LIM protein antibodies (Transduction Laboratories; Lexington, KY) were used. Anti-paxillin MAb (clone 349) recognizes not only the 68-kD paxillin protein but also the 50-kD Hic-5 protein, while anti-Hic-5 MAb (clone 34) recognizes only Hic-5 (
Immunohistochemistry
Methanol-fixed, paraffin-embedded specimens were incubated with anti-paxillin MAb or anti-Hic-5 MAb (as the primary antibody) overnight at 4C. Binding of the primary antibody was detected using biotinylated anti-mouse IgG (ICN Pharmaceuticals; Aurora, OH) and streptavidinperoxidase conjugate (Dako LSAB Kit; Dako Japan, Kyoto, Japan), and visualized with diaminobenzidine. The specimens were counterstained with Mayer's hematoxylin.
Immunoblotting
Fresh samples were incubated with ice-cold lysis buffer [final concentrations: 1% Triton X-100, 0.5% Nonidet P-40, 50 mM Tris-HCl (pH 7.2), 1 mM EGTA, 1 mM Na3VO4, 0.5 mM PMSF, and 50 µg/ml of leupeptin] and homogenized. The lysates were centrifuged at 15,000 x g for 10 minutes (at 4C). The resultant supernatants were resolved on 8% SDS-PAGE and then electrophoretically transferred to PVDF membranes. The membranes were blocked with 1% BSA in PBS. After extensive washing with PBS containing 0.1% Tween-80, the immunoblots were incubated with 0.05 µg/ml of anti-paxillin MAb or anti-Hic-5 MAb for 2 hr at room temperature. Antibody binding was detected using peroxidase-conjugated goat anti-mouse IgG and visualized with ECL chemiluminescence reaction reagents (Amersham Pharmacia Biotech; Poole, UK).
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Results |
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The results are summarized in Table 1. For each result described in this table, sections from at least three tissue blocks were studied. The immunohistochemistry studies on different samples were highly consistent, with no major variations in the tissue distribution of anti-paxillin and anti-Hic-5 reactivities.
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Widespread Expression of Paxillin and Restricted Expression of Hic-5
Positive staining was observed with anti-paxillin antibody in a variety of samples, including cardiac muscle (Fig 1A), skeletal muscle (Fig 1D), vascular smooth muscle (Fig 1A and Fig 1D), organ smooth muscle (Fig 2A and Fig 2C), gland epithelium (Fig 2A, Fig 3A, and Fig 3C), skin epithelium (Fig 4A), peripheral nerve (Fig 4B), and brain (Fig 4C); see Table 1 for summary.
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In contrast, positive staining with anti-Hic-5 antibody was observed specifically in smooth muscle samples (Fig 1B, Fig 1E, Fig 2B, and Fig 2D); see Table 1 for summary. As described in Materials and Methods, anti-paxillin MAb (clone 349) recognizes both paxillin and Hic-5, whereas anti-Hic-5 MAb recognizes only the latter. Accordingly, a combination of positive staining with anti-paxillin antibody and negative staining with anti-Hic-5 antibody indicates the expression of paxillin (but not Hic-5), which is true of striated muscle, epithelium (from various origins), and nerves (Table 1). On the other hand, whether paxillin or Hic-5 is predominantly expressed cannot be determined in the case of positive staining with both anti-paxillin antibody and anti-Hic-5 antibody, which is true of smooth muscle (and myoepithelial cell) samples (Table 1).
Predominant Expression of Paxillin and Hic-5 in Striated Muscle and Smooth Muscle, Respectively
As described above, the relative expression of paxillin and Hic-5 in smooth muscle (and myoepithelial cell) samples cannot be determined solely by immunohistochemistry studies. Accordingly, immunoblotting studies were performed; paxillin and Hic-5 can be discriminated by the difference between their molecular weights. As shown in Fig 5, not only the 68-kD paxillin protein but also the 50-kD Hic-5 protein was confirmed to be recognized by immunoblotting with anti-paxillin MAb. Only the 50-kD Hic-5 protein was recognized by immunoblotting with anti-Hic-5 MAb. Muscle samples were analyzed by this procedure.
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As expected from the immunohistochemistry studies, striated muscle samples, i.e., cardiac muscle and skeletal muscle (iliopsoas), were confirmed to express mainly paxillin (Fig 5). Hic-5 was not detected in cardiac muscle (Fig 5). Although this LIM protein was detected in iliopsoas, its expression was very weak (Fig 5). This weak Hic-5 detection may be due to cross-contamination from the vasculature, which abundantly expresses Hic-5. In contrast, smooth muscle samples, including myometrium, arterial and venous vascular smooth muscle tissues, and gastric smooth muscle, were found to strongly express Hic-5 (Fig 5). Paxillin expression was much weaker than Hic-5 expression in gastric and uterine smooth muscle cell (Fig 5). Furthermore, paxillin detection was almost negligible in vascular smooth muscle tissues. Paxillin was detected only very weakly in the artery and not at all in the vein (Fig 5).
Possible Expression of Hic-5 in Myoepithelial Cells
When mammary (Fig 3A and Fig 3B), sweat, and salivary (Fig 3C and Fig 3D) glands were studied by immunohistochemistry, positive staining with both anti-paxillin antibody and anti-Hic-5 antibody was observed in myoepithelial cells, which surround ductal and acinar epithelium of these glandular organs. It appears likely that myoepithelial cells express mainly Hic-5 because these cells are strongly stained with anti-Hic-5 antibody and because myoepithelial cells are of a smooth muscle phenotype, which is related to the predominant expression of Hic-5 (not paxillin). However, further studies are needed to clearly determine the relative expression of paxillin and Hic-5 in these cells. It was impossible to perform immunoblotting studies with the use of myoepithelial cells due to the difficulty in isolating them purely.
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Discussion |
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Proteins containing LIM domains play important roles in a variety of fundamental biological processes, including cytoskeletal organization, cell lineage specification, and organ development. The LIM domain is a proteinprotein interaction motif that is critically involved in these processes (
Although it is established that Hic-5 is a paxillin homologue localized to focal adhesion complexes (see Introduction), recent studies have shown that these LIM proteins have some distinct functional features. Hic-5 expression is increased during cell senescence of normal human fibroblasts and is decreased during immortalization of mouse embryo fibroblasts (
The contrasting expression of the LIM proteins in muscle tissues may be worthy of notice. Paxillin is mainly expressed in multinuclear striated muscle, whereas Hic-5 is in mononuclear smooth muscle, at least in adult human tissues. Recently, these LIM proteins have been shown to be involved in muscle differentiation. During muscle development, myoblasts function as muscle stem cells and, in response to the loss of trophic support, exit the cell cycle, fuse, and upregulate a variety of genes that commit them to form striated skeletal muscle. Those cells that fail to activate appropriate survival and differentiation programs instead rapidly die by apoptosis (
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Acknowledgments |
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Supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
We are indebted to Dr T. Takafuta (Yamanashi Medical University) for helpful discussions.
Received for publication April 1, 2002; accepted October 2, 2002.
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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Bach I (2000) The LIM domain: regulation by association. Mech Dev 91:5-17[Medline]
Brunskill EW, Witte DP, Yutzey KE, Potter SS (2001) Novel cell lines promote the discovery of genes involved in early heart development. Dev Biol 235:507-520[Medline]
Denhez F, WilcoxAdelman SA, Baciu PC, Saoncella S, Lee S, French B, Neveu W et al. (2002) Syndesmos, a syndecan-4 cytoplasmic domain interactor, binds to the focal adhesion adaptor proteins paxillin and Hic-5. J Biol Chem 277:12270-12274
Fujita H, Kamiguchi K, Cho D, Shibanuma M, Morimoto C, Tachibana K (1998) Interaction of Hic-5, a senescence-related protein, with focal adhesion kinase. J Biol Chem 273:26516-26521
Hagmann J, Grob M, Welman A, van Willigen G, Burger MM (1998) Recruitment of the LIM protein hic-5 to focal contacts of human platelets. J Cell Sci 111:2181-2188
Hiregowdara D, Avraham H, Fu Y, London R, Avraham S (1997) Tyrosine phosphorylation of the related adhesion focal tyrosine kinase in megakaryocytes upon stem cell factor and phorbol myristate acetate stimulation and its association with paxillin. J Biol Chem 272:10804-10810
Hu Y, Cascone PJ, Cheng L, Sun D, Nambu JR, Schwartz LM (1999) Lepidopteran DALP, and its mammalian ortholog HIC-5, function as negative regulators of muscle differentiation. Proc Natl Acad Sci USA 96:10218-10223
Ishino K, Kaneyama J, Shibanuma M, Nose K (2000) Specific decrease in the level of Hic-5, a focal adhesion protein, during immortalization of mouse embryonic fibroblasts, and its association with focal adhesion kinase. J Cell Biochem 76:411-419[Medline]
Lipsky BP, Beals CR, Staunton DE (1998) Leupaxin is a novel LIM domain protein that forms a complex with PYK2. J Biol Chem 273:11709-11713
Matsuya M, Sasaki H, Aoto H, Mitaka T, Nagura K, Ohba T, Ishino M et al. (1998) Cell adhesion kinase beta forms a complex with a new member, Hic-5, of proteins localized at focal adhesions. J Biol Chem 273:1003-1014
Nishiya N, Tachibana K, Shibanuma M, Mashimo J, Nose K (2001) Hic-5-reduced cell spreading on fibronectin: competitive effects between paxillin and Hic-5 through interaction with focal adhesion kinase. Mol Cell Biol 21:5332-5345
Nosaka S, Onji T, Shibata N (1984) Enhancement of actomyosin ATPase activity by tropomyosin. Recombination of myosin and tropomyosin between muscles and platelet. Biochim Biophys Acta 788:290-297[Medline]
Oda A, Ochs HD, Lasky LA, Spencer S, Ozaki K, Fujihara M, Handa M et al. (2001) CrkL is an adapter for Wiskott-Aldrich syndrome protein and Syk. Blood 97:2633-2639
Osada M, Ohmori T, Yatomi Y, Satoh K, Hosogaya S, Ozaki Y (2001) Involvement of Hic-5 in platelet activation: integrin alphaIIbbeta3-dependent tyrosine phosphorylation and association with proline-rich tyrosine kinase 2. Biochem J 355:691-697[Medline]
Sastry SK, Lakonishok M, Wu S, Truong TQ, Huttenlocher A, Turner CE, Horwitz AF (1999) Quantitative changes in integrin and focal adhesion signaling regulate myoblast cell cycle withdrawal. J Cell Biol 144:1295-1309
Schaller MD (2001) Paxillin: a focal adhesion-associated adaptor protein. Oncogene 20:6459-6472[Medline]
Shibanuma M, Mashimo J, Kuroki T, Nose K (1994) Characterization of the TGF beta 1-inducible hic-5 gene that encodes a putative novel zinc finger protein and its possible involvement in cellular senescence. J Biol Chem 269:26767-26774
Shibanuma M, Mochizuki E, Maniwa R, Mashimo J, Nishiya N, Imai S, Takano T et al. (1997) Induction of senescence-like phenotypes by forced expression of hic-5, which encodes a novel LIM motif protein, in immortalized human fibroblasts. Mol Cell Biol 17:1224-1235[Abstract]
Takeuchi K (1985) Properties of porcine platelet myosin. I. Similarity between vertebrate smooth muscle and nonmuscle myosins in their binding properties with F-actin. J Biochem 97:295-305[Abstract]
Thomas SM, Hagel M, Turner CE (1999) Characterization of a focal adhesion protein, Hic-5, that shares extensive homology with paxillin. J Cell Sci 112:181-190
Turner CE (1991) Paxillin is a major phosphotyrosine-containing protein during embryonic development. J Cell Biol 115:201-207[Abstract]
Turner CE (2000) Paxillin and focal adhesion signalling. Nature Cell Biol 2:E231-236[Medline]
Turner CE, Glenney JR, Jr, Burridge K (1990) Paxillin: a new vinculin-binding protein present in focal adhesions. J Cell Biol 111:1059-1068[Abstract]
Wheeler GN, Hynes RO (2001) The cloning, genomic organization and expression of the focal contact protein paxillin in Drosophila. Gene 262:291-299[Medline]
Zhang J, Zhang LX, Meltzer PS, Barrett JC, Trent JM (2000) Molecular cloning of human Hic-5, a potential regulator involved in signal transduction and cellular senescence. Mol Carcinogen 27:177-183[Medline]