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Genes to Cells (2004) 9, 653-660. doi:10.1111/j.1356-9597.2004.00749.x
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Interaction of Rho-kinase with myosin II at stress fibres

Saeko Kawabata1, Jiro Usukura2, Nobuhiro Morone2, Masaaki Ito3, Akihiro Iwamatsu4, Kozo Kaibuchi1 and Mutsuki Amano1,*

1 Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, Nagoya, Aichi 466-8550, Japan
2 Department of Anatomy and Cell Biology, Graduate School of Medicine, Nagoya University, Nagoya, Aichi 466-8550, Japan
3 First Department of Internal Medicine, Mie University School of Medicine, Tsu, Mie 514-8507, Japan
4 Central Laboratories for Key Technology, Kirin Brewery Company Limited, Yokohama, Kanagawa 236-0004, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Rho-kinase and myosin phosphatase cooperatively regulate the phosphorylation level of myosin light chain and are involved in the formation of stress fibres and smooth muscle contraction. Rho-kinase has been known to be localized at stress fibres, but little is known about the mechanism of its localization. Here we identified non-muscle myosin heavy chain IIA and IIB as the pleckstrin homology domain-interacting molecules by affinity column chromatography. The pleckstrin homology domain of Rho-kinase binds to myosin II directly in in vitro cosedimentation assay. The C-terminal region of the pleckstrin homology domain was important for this interaction, and the point mutations in the pleckstrin homology domain mutant (W1170A, W1340L) resulted in a decrease in the binding. We also found that the pleckstrin homology domain, but not the pleckstrin homology domain mutant (W1170A, W1340L), was localized at stress fibres in fibroblasts. These results indicate that Rho-kinase is localized at stress fibres through binding of the pleckstrin homology domain to myosin II.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Rho-associated kinase (Rho-kinase/ROCK/ROK) and myosin-binding subunit (MBS) of myosin phosphatase are effectors of Rho small GTPase. Rho-kinase and MBS have been implicated in many cellular processes downstream of Rho, including stress fibre and focal adhesion formation, smooth muscle contraction, cytokinesis, neurite retraction, and cell migration (for reviews, see Kaibuchi et al. 1999; Amano et al. 2000; Riento & Ridley 2003). Rho-kinase regulates the phosphorylation level of myosin light chain (MLC) by the direct phosphorylation of MLC (Amano et al. 1996) and by the inactivation of myosin phosphatase, which is composed of MBS, a 37-kDa type 1 phosphatase catalytic subunit, and a 20-kDa subunit, through the phosphorylation of MBS (Kimura et al. 1996). In addition to MLC, adducin and the Ezrin/Radixin/Moesin (ERM) family proteins are the substrates of both Rho-kinase and myosin phosphatase. Rho-kinase phosphorylates ERM and adducin, and myosin phosphatase that interacts with ERM and adducin through MBS dephosphorylates ERM and adducin phosphorylated by Rho-kinase (Fukata et al. 1998; Kimura et al. 1998; Retzer & Essler 2000). Thus, Rho-kinase and MBS appear to cooperatively control the phosphorylation level of the subset of substrates and regulate the cytoskeletal organization in vivo.

Rho-kinase is composed of the N-terminal catalytic domain, the central coiled-coil domain, and the C-terminal pleckstrin homology (PH) domain interrupted by the cysteine-rich domain. The Rho-binding region of Rho-kinase is situated at the C-terminal portion of the coiled-coil domain. Deletion of the C terminus results in the activation of Rho-kinase, and the C-terminal fragment containing Rho-binding and PH domains serves as a dominant negative form. We have previously demonstrated that this C-terminal fragment directly interacts and inhibits the constitutively activated catalytic fragment (Amano et al. 1999), indicating that the C terminus is a negative regulatory region of Rho-kinase. Rho and possibly arachidonic acid are supposed to cancel this negative regulation (Araki et al. 2001). Chen et al. (2002) showed that ROK exists as oligomers through the interaction between coiled-coil domains, as well as between the catalytic and the C-terminal regions. Thus, these domains are considered to be important for regulation of the kinase activity.

The PH domain is generally known to be involved in the lipid-protein and protein-protein interactions. The lipid-binding properties of the PH domain ensure recruitment of the protein to membrane phospholipids or lipid second messengers. The PH domain of Rho-kinase may bind to certain lipids or proteins other than Rho-kinase itself, which regulate the activity or localization of Rho-kinase. However, little is known about the roles of the PH domain of Rho-kinase for the domain's regulation and localization through interaction with other molecules.

There have been several observations concerning the cellular localization of Rho-kinase. Rho-kinase has been reported to be distributed throughout cytoplasm and to be partly translocated to membrane fraction in a RhoA-dependent manner (Leung et al. 1995; Matsui et al. 1996). Though a large fraction of Rho-kinase seems to exist as a soluble form, the localization of Rho-kinase at the cleavage furrow during cytokinesis (Kosako et al. 1999), stress fibres (Katoh et al. 2001; Chen et al. 2002), and vimentin intermediate filaments (Sin et al. 1998) has also been reported. The underlying mechanism of the cellular localization of Rho-kinase is still unclear.

We here identified the PH domain-interacting molecules to examine the mode of localization of Rho-kinase and found that myosin II directly bound to the PH domain of Rho-kinase in vitro, indicating that myosin II is the anchoring molecule of Rho-kinase to stress fibres.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Identification of Rho-kinase-interacting molecules

To search for Rho-kinase-PH domain-interacting molecules, porcine brain membrane extract was loaded on to a glutathione-Sepharose affinity column on which Glutathione-S-transferase (GST) or GST-Rho-kinase-PH was immobilized. The proteins bound to the affinity columns were eluted by the addition of 10 mM glutathione. Two proteins with apparent molecular masses of about 230 kDa were detected in the elution fraction from the GST-Rho-kinase-PH affinity column, but not from the GST affinity column (Fig. 1). Because these two bands were very close to each other and hardly separated on the sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) gel, they were subjected to further analysis and referred to together as p230. To identify p230, molecular mass analyses were performed. The molecular weights of peptides derived from p230 were determined and found to be identical to those from non-muscle myosin heavy chain IIA and IIB. The estimated molecular weights of human non-muscle myosin heavy chain IIA and IIB (226.5 and 228.9 kDa) were close to those of p230 as estimated by SDS-PAGE.



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  		Figure 1  Purification of Glutathione-S-transferase (GST)-Rho-kinase-pleckstrin homology (PH)-interacting molecules. Porcine brain extract was loaded on to a glutathione-Sepharose column containing GST or GST-Rho-kinase-PH. The bound proteins were eluted by addition of 10 mM glutathione. Aliquots of eluates were resolved by SDS-PAGE and followed by silver staining. Positions of non-muscle myosin heavy chain IIA and IIB (NMHC IIA/B), GST, and GST-Rho-kinase-PH (GST-PH) are indicated.

 
Direct interaction of the PH domain of Rho-kinase with myosin II

We performed myosin cosedimentation assay to examine whether Rho-kinase-PH domain interacts with myosin II directly. We used smooth muscle myosin II as the material of this assay, because smooth muscle myosin heavy chain is functionally similar to non-muscle myosin heavy chain (75% identical at the amino acid level) and because it was easily prepared. GST-Rho-kinase-PH, but not GST, went to the pellet with myosin II filaments (Fig. 2A). However, GST-Rho-kinase-PH did not sediment in the absence of myosin II. By electron microscopic analysis, the sedimented myosin II formed filamentous structure, from which myosin heads were observable at locations as twin short protrusions (arrows in Fig. 2B). Although GST or GST-Rho-kinase-PH was not identified strictly on the shadowed image, filaments incubated with GST-Rho-kinase-PH contained much more small blobs along the filament, comparing to the filaments incubated with GST alone (Fig. 2B). Such small blobs appear to be GST-Rho-kinase-PH. These results indicate that the PH domain of Rho-kinase directly interacts with myosin II filaments.



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  		Figure 2  Cosedimentation analysis of the pleckstrin homology (PH)-domain of Rho-kinase. GST or GST-Rho-kinase-PH was cosedimented with or without myosin II. (A) Aliquots of the supernatant (S) and pellet (P) were subjected to SDS-PAGE and followed by silver staining. Positions of myosin heavy chain (MHC), GST, and GST-Rho-kinase-PH (GST-PH) are indicated. The results are representative of at least three independent experiments. (B) Shadowed images of myosin filaments incubated with GST (upper) and GST-Rho-kinase-PH (lower). The filaments incubated with GST-Rho-kinase-PH were more decorated with small blobs. Arrows indicate twin myosin head.

 
Determination of the region responsible for myosin binding

To determine which region of the PH domain of Rho-kinase is involved in the binding to myosin II, several deletion and point mutation constructs were tested (Fig. 3A). The C-terminally truncated Rho-kinase-PH fragments, GST-Rho-kinase-PH{Delta}1 and GST-Rho-kinase-PH{Delta}2, were not cosedimentated with myosin II. The amount of GST-Rho-kinase-PH{Delta}3, lacking the N-terminal 27 amino acids of the Rho-kinase-PH, in the pellet was more than that of Rho-kinase-PH. The amount of GST-Rho-kinase-PH{Delta}4, lacking the N-terminal 135 amino acids of the Rho-kinase-PH, in the pellet was less than that of Rho-kinase-PH. GST-Rho-kinase-PH (AL) with the substitutions of conserved Trp residues in the Rho-kinase-PH, Trp-1170 for Ala and Trp-1340 for Leu, showed minimal effect on this ability (Fig. 3B). These results indicate that the C-terminal region and Trp-1170/Trp-1340 of the Rho-kinase-PH are necessary for the binding to myosin II.



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  		Figure 3  Cosedimentation analysis of the various deletion and point mutants of the pleckstrin homology (PH) domain of Rho-kinase. (A) Schematic representation of the PH domain of Rho-kinase. The structure of the PH domain of Rho-kinase, various deletion and point mutants are presented. GST-Rho-kinase-PH (PH) (1125-1388 amino acids), GST-Rho-kinase-PH{Delta}1 (PH{Delta}1) (1125-1237 amino acids), GST-Rho-kinase-PH{Delta}2 (PH{Delta}2) (1125-1337 amino acids), GST-Rho-kinase-PH{Delta}3 (PH{Delta}3) (1152-1338 amino acids), GST-Rho-kinase-PH{Delta}4 (PH{Delta}4) (1261-1388 amino acids), and GST-Rho-kinase-PH (AL) (PH (AL)) (1125-1338 amino acids, with the substitutions of Trp-1170 for Ala and Trp-1340 for Leu). Abbreviations: PHD, pleckstrin homology domain; CRD, cysteine rich domain. (B) The PH-domain of Rho-kinase and various deletion and point mutants were cosedimented with or without myosin II. Aliquots of the pellet were subjected to SDS-PAGE and analysed by Coomassie Brilliant Blue staining. The results are representative of at least three independent experiments.

 
Localization of Rho-kinase-PH domain at stress fibres in fibroblasts

Several groups have shown previously that Rho-kinase associates with stress fibres (Katoh et al. 2001; Chen et al. 2002), which leads to the possibility that Rho-kinase is localized at stress fibres through binding to myosin. As previously reported, immunofluorescence staining showed that endogenous Rho-kinase was localized at the stress fibres (Fig. 4). We investigated the intracellular localization of green fluorescent protein (GFP)-Rho-kinase-PH. Immunofluorescent staining showed that the PH domain of Rho-kinase was co-localized with myosin filaments (Fig. 5A). We also confirmed that the PH domain of ROCK1/ROKß, an isoform of Rho-kinase, was localized at stress fibres (data not shown). We further investigated the intracellular localization of mutants of the PH domain of Rho-kinase (Fig. 5B). Immunofluorescent staining showed that GFP-Rho-kinase-PH{Delta}1, GFP-Rho-kinase-PH{Delta}2, and GFP-Rho-kinase-PH (AL) were barely localized at stress fibres, whereas GFP-Rho-kinase-PH{Delta}3 and GFP-Rho-kinase-PH{Delta}4 were localized at stress fibres. By doubly staining with GFP-antibody and myosin heavy chain antibody, it was confirmed that GFP-Rho-kinase-PH and GFP-Rho-kinase-PH{Delta}3, but not GFP-Rho-kinase-PH (AL), were co-localized with myosin filaments (data not shown). The localization at stress fibres of each mutant of the PH-domain of Rho-kinase showed a similar ability to bind to myosin by in vitro cosedimentation assay (Fig. 5C). Similar results were obtained when these mutants were expressed in REF52 cells (data not shown). These results indicate that Rho-kinase is localized at the stress fibres through binding of the PH domain to myosin.



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  		Figure 4  Localization of Rho-kinase at stress fibres in fibroblast. REF52 cells were doubly stained with anti-Rho-kinase-COIL (coiled-coil) antibody (anti-COIL) or anti-Rho-kinase-RB (Rho-binding) antibody (anti-RB) and anti-actin antibody.

 


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  		Figure 5  Localization of green fluorescent protein (GFP)-Rho-kinase-pleckstrin homology (PH) and -PH mutants in NIH3T3 cells. (A) NIH3T3 cells were transfected with pEGFP (GFP) (a, c) or pEGFP-Rho-kinase-PH (PH) (b, d), and doubly stained with anti-GFP antibody (anti-GFP) and anti-myosin heavy chain antibody (anti-myosin). (B) NIH3T3 cells were transfected with pEGFP-Rho-kinase-PH (a), pEGFP-Rho-kinase-PH{Delta}1 (b), pEGFP-Rho-kinase-PH{Delta}2 (c), pEGFP-Rho-kinase-PH{Delta}3 (d), pEGFP-Rho-kinase-PH{Delta}4 (e), or pEGFP-Rho-kinase-PH (AL) (f) and stained with anti-GFP antibody. (C) The percentages of cells in which mutants of the PH-domain of Rho-kinase were localized at stress fibres were examined. Data are means ± SD of at least triplicate determinations.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
In this study, we demonstrated that the PH domain of Rho-kinase directly interacted with smooth muscle myosin II and, presumably, with non-muscle myosin IIA and IIB. It has been demonstrated that Rho, Rho-kinase, and MBS of myosin phosphatase are included in isolated stress fibres and that they contract the fibres in a calcium-independent manner (Katoh et al. 2001). Our present data help us to elucidate the mechanism for Rho-kinase to be localized at stress fibres. Substitution of conserved Trp residues in the PH-domain, Trp-1170 for Ala and Trp-1340 for Leu, resulted in the loss of myosin II binding ability. The effects of these mutations in vivo have been already reported by Chen et al. (2002), who demonstrated that the C-terminal fragment of ROK{alpha} (Rho-kinase) containing these mutations is able to interact with and inhibit the catalytic domain but does not exert the dominant negative effects on the stress fibre organization that the intact C-terminal fragment possesses. This could be explained by the loss of myosin II-binding ability of the PH domain resulting from these mutations, and it further supports our hypothesis. We also examined the localization of the PH domain at the cleavage furrow where Rho-kinase is co-localized with myosin II, but it was not recruited to the cleavage furrow. The Rho-binding region, but not the Rho-binding ability, was necessary for recruitment to the cleave furrow; suggesting that there exists another anchoring molecule other than myosin II (unpublished observations). Furthermore, the spatiotemporal Rho signalling pathway from receptors at plasma membrane to organization of stress fibres in the cytoplasm is still unclear: after Rho is activated at the plasma membrane in the vicinity of receptors, where and when does it interact with and activate/inactivate Rho-kinase and MBS of myosin phosphatase to organize stress fibres? This raises the similar question of whether Rho-kinase is associated with myosin constitutively or only when it is activated. The latter is supported by our preliminary observations that the catalytic domain of Rho-kinase directly bound to myosin II in in ivtro cosedimentation assay and that the C-terminal fragment of Rho-kinase containing the PH domain interfered with the interaction between the catalytic domain of Rho-kinase and myosin II. Further analyses are necessary to address these questions.

The interaction between Rho-kinase and myosin II appears to form the functional complex composed of Rho-kinase, myosin II, myosin phosphatase, and Rho and to ensure efficient phosphorylation of MLC. This would be especially important to maintain stress fibres, as well as to form them on stimulation with extracellular signals. The treatment of cells with Rho-kinase inhibitors, such as Y-27632 and HA1077, not only inhibits the lysophosphatidic acid-induced stress fibre and focal adhesion formation but also disrupts the pre-existing stress fibres and focal adhesions in the presence of lysophosphatidic acid or serum, indicating that the Rho-kinase activity is needed to maintain the fibres and adhesions by keeping MLC phosphorylation. The fact that there remain stress fibres containing the MLC mutant following the substitution of Ser-18 and Thr-19 for Asp, which mimics the phosphorylated form of MLC, after the long-term serum depletion also supports this idea (Amano et al. 1998). It is most likely that myosin II is one of anchoring molecules of Rho-kinase, and that this interaction helps to keep stress fibres.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Materials and chemicals

GST-Rho-kinase-PH (1125-1388 amino acids), GST-Rho-kinase-PH{Delta}1 (1125-1237 amino acids), GST-Rho-kinase-PH{Delta}2 (1125-1337 amino acids), GST-Rho-kinase-PH{Delta}3 (1152-1338 amino acids), GST-Rho-kinase-PH{Delta}4 (1261-1388 amino acids), and GST-Rho-kinase-PH (AL; 1125-1338 amino acids, 1170 A, 1340 L), were produced in Escherichia coli and purified on a glutathione-Sepharose 4B column purchased from Amersham Bioscience (NJ, USA). Smooth muscle myosin II was prepared as described (Ikebe & Hartshorne 1985). Anti-myosin heavy chain polyclonal antibody was purchased from Biomedical Technologies, Inc. (MA, USA). Anti-actin monoclonal antibody was purchased from CHEMICON International, Inc. (CA, USA). Rabbit polyclonal antibodies against Rho-kinase-coiled-coil (COIL) and against Rho-kinase-Rho-binding (RB) were prepared as described (Katoh et al. 2001). Anti-GFP monoclonal antibody was purchased from Roche Diagnostics Corp. (Indianapolis, IN). Fluorolink Cy3-labelled goat anti-rabbit IgG and Cy2-conjugated donkey anti-mouse IgG were purchased from Amersham Bioscience (NJ, USA) and Jackson (PA, USA), respectively. All materials used in the nucleic acid study were purchased from Takara Shuzo (Kyoto, Japan). Other materials and chemicals were obtained from commercial sources.

Plasmid constructs

To obtain deletion mutants of the PH domain of Rho-kinase, the oligonucleotides used for polymerase chain reaction amplifications had the following sequences: oligonucleotides 5'-AAATGGATCCGCCTTGCACATTGGTTTGGAT-3' and 5'-AAGGATCCTTATTCTCCTTCGTTGGCATACAG-3' for Rho-kinase-PH{Delta}1, 5'-AAATGGATCCGCCTTGCACATTGGTTTGGAT-3' and 5'-AAGGATCCTTACTGCTCTTCTGTAGAATTTGCC-3' for Rho-kinase-PH{Delta}2, 5'-AAGGATCCAGACTAGAAGGATGGCTTTCA-3' and anti-5'-ATAAGGATCCCACAGAAGGCAGTTAGCTTGG-3' for Rho-kinase-PH{Delta}3, 5'-AAGGATCCCATGAATTTATTCCTACTCTGTAT-3' and anti-5'-ATAAGGATCCCACAGAAGGCAGTTAGCTTGG-3' for Rho-kinase-PH{Delta}4, and 5'-CTAAGAAATTTGGAGCGGTTAAAAAGTATG-3', anti-5'-CATACTTTTTAACCGCTCCAAATTTCTTAG-3', 5'-GAGCAGCAAAAGTTGGTTAGTCGGTTA-3', and anti-5'-TAACCGACTAACCAACTTTTGCTGCTC-3' for Rho-kinase-PH (AL).

Affinity column chromatography

Sixty grams of porcine brain grey matter was homogenized in 180 mL of buffer A (20 mM Tris/HCl (pH 8.0), 1 mM dithiothreitol, 150 mM NaCl, 1 mM EDTA, and 1% Igepal CA-630) containing 0.05 mM amidinophenylmethanesulphonyl fluoride hydrochloride, 2 µg/mL leupeptin and 1 µg/mL Aprotinin. The homogenate is centrifuged at 100 000 g for 1 h at 4 °C. The supernatant was used as the membrane extract. This extract of porcine brain grey matter (4.5 mL, 73 mg of proteins) was applied to a 0.25-mL glutathione-Sepharose 4B column containing 5 nmol of GST or GST-Rho-kinase-PH. After washing the columns three times with 0.825 mL of buffer A, the bound proteins were eluted by the addition of 0.825 mL of buffer A containing 10 mM glutathione. The first eluate was subjected to SDS-PAGE and proteins were detected by silver staining.

Identification of Rho-kinase-PH domain-interacting molecules

The affinity-purified Rho-kinase-PH domain-interacting molecules were dialysed three times against distilled water and concentrated by freeze-drying. The concentrated samples were separated by SDS-PAGE and transferred on to a polyvinylidene difluoride membrane (Problot, Applied Biosystems, Foster City, CA, USA). The immobilized proteins were reduced, S-carboxymethylated, and digested in situ with Achromobacter protease I (a Lys-C) (Iwamatsu 1992). Molecular mass analyses of Lys-C fragments were performed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry using a PerSeptive Biosystem Voyager-Delayed Extraction/RP (Jensen et al. 1996). Proteins were identified by comparing the molecular weights determined by MALDI-TOF/Mass Spectrometry.

Myosin cosedimentation assay

GST, GST-Rho-kinase-PH, or GST-Rho-kinase-PH mutants (0.2 mg/mL) were mixed with or without purified myosin II (0.6 mg/mL) in the presence of 10 mM MOPS (pH 7.5), 1 mM dithiothreitol, 65 mM KCl, 1 mM MgCl2, and 0.5 mg/mL bovine serum albumin. This mixture was incubated at 25 °C for 10 min and centrifuged at 45 000 g for 10 min at 25 °C. The pellet and supernatant were subjected to SDS-PAGE.

Electron microscopic analysis of the PH domain of Rho-kinase binding to myosin filaments

Rotary shadowing was employed for morphological confirmation of the binding of GST-Rho-kinase-PH with myosin filaments. The pellets of myosin cosedimentation assay were mixed with mica flakes according to the previous protocol (Heuser 1983), and immediately frozen by plunging them on to the copper block cooled with liquid helium. Frozen specimens were brought into freeze-etching device (FR-9000, Hitachi Science Co. Mito Japan), and shadowed with platinum and carbon after freeze drying at –90 °C for 10 min. Shadowed samples were removed from mica surface by floating on 48% hydro-fluoric acid solution, and then washed twice with distilled water prior to electron microscopic observation.

Immunofluorescence analysis

For the staining of endogenous Rho-kinase, REF52 cells were treated with phosphate-buffered saline (PBS) containing 0.02% Triton X-100 for 1 min at 37 °C, followed by fixation with methanol for 15 min and washing with PBS on ice. After fixation, the cells were double stained with anti-RB or -COIL polyclonal antibody and Cy3-conjugated-anti-rabbit antibody and with anti-actin monoclonal antibody and Cy2-conjugated-anti-mouse antibody.

The cells were fixed with methanol for 15 min and washed with PBS on ice. After fixation, the cells were doubly stained with anti-GFP monoclonal antibody and Cy2-conjugated-anti-mouse antibody, and anti-myosin heavy chain polyclonal antibody and Cy3-conjugated-anti-rabbit antibody. After being washed with PBS, the cells were examined using a Zeiss axiophoto microscope.

Transfection into NIH3T3 and REF52 cells

NIH3T3 cells were seeded at a density of 1.6 x 104 cells on to 13-mm glass coverslips in Dulbecco's modified eagle's medium (DMEM) with 10% calf serum and cultured overnight. The medium was renewed 2 h before transfection. Transfection of plasmids into NIH3T3 cells was carried out using Lipofectamine Plus reagent purchased from Invitrogen Corp. (CA, USA) according to the manufacturer's protocol. A Lipofectamine Plus/DNA mixture was prepared and added to the dish with gentle agitation. After a 4-h incubation, the cells were grown in DMEM with 10% calf serum for 1 day. REF52 cells were seeded at a density of 1.4 x 105 cells on to 13-mm glass coverslips in DMEM with 10% foetal bovine serum and cultured overnight. The medium was renewed 2 h before transfection. Transfection of plasmids into REF52 cells was carried out using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocol. A Lipofectamine 2000/DNA mixture was prepared and added to the dish with gentle agitation. After a 4-h incubation, the cells were grown in DMEM with 10% foetal bovine serum for 1 day.


   Acknowledgements
 
We are grateful to T. Ishii and Y. Kanazawa for secretarial and technical assistance. We also thank Dr K. Katoh (Jichi Medical School) for helpful discussion. This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan; Special coordination funds for promoting Science and Technology; the Pharmaceuticals and Medical Devices Agency; Uehara Memorial Foundation; and the Mochida Memorial Foundation for Medical and Pharmaceutical Research.


   Footnotes
 
Communicated by: Noriko Osumi

* Correspondence: E-mail: m-amano{at}med.nagoya-u.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Received: 14 March 2004
Accepted: 27 April 2004




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