Uni-axial stretching regulates intracellular localization of Hic-5 expressed in smooth-muscle cells in vivo

Joo-ri Kim-Kaneyama1, Wataru Suzuki1, Kiyoko Ichikawa1, Takahiro Ohki1, Yoko Kohno2, Masataka Sata3, Kiyoshi Nose1 and Motoko Shibanuma1,*

1 Department of Microbiology, Showa University School of Pharmaceutical Sciences, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
2 Department of Oral Pathology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
3 Department of Cardiovascular Medicine, University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan

* Author for correspondence (e-mail: smotoko{at}pharm.showa-u.ac.jp)

Accepted 14 December 2004


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Hic-5 is a focal adhesion protein belonging to the paxillin LIM family that shuttles in and out of the nucleus. In the present study, we examined the expression of Hic-5 among mouse tissues by immunohistochemistry and found its expression only in smooth-muscle cells in several tissues. This result is consistent with a previous report on adult human tissues and contradicts the relatively ubiquitous expression of paxillin, the protein most homologous to Hic-5. One factor characterizing smooth-muscle cells in vivo is a continuous exposure to mechanical stretching in the organs. To study the involvement of Hic-5 in cellular responses to mechanical stress, we exposed mouse embryo fibroblasts to a uni-axial cyclic stretching and found that Hic-5 was relocalized from focal adhesions to stress fibers through its C-terminal LIM domains during the stress. In sharp contrast to this, paxillin did not change its focal-adhesion-based localization. Of the factors tested, which included interacting partners of Hic-5, only CRP2 (an only-LIM protein expressed in vascular smooth-muscle cells) and GIT1 were, like Hic-5, localized to stress fibers during the cyclic stretching. Interestingly, Hic-5 showed a suppressive effect on the contractile capability of cells embedded in three-dimensional collagen gels, and the effect was further augmented when CRP2 co-localized with Hic-5 to fiber structures of those cells. These results suggested that Hic-5 was a mediator of tensional force, translocating directly from focal adhesions to actin stress fibers upon mechanical stress and regulating the contractile capability of cells in the stress fibers.

Key words: Hic-5, Mechanical stress, Smooth muscle cell, CRP2, Collagen-gel contraction


    Introduction
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 Introduction
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Mechanical force is an important biological regulator of cellular functions, affecting cellular morphology and adhesiveness, especially in smooth-muscle cells (SM cells), which surround contractile organs and are always exposed to mechanical stress. Transmembrane receptors of integrins consist of {alpha} and ß subunits, and are considered to play a key role in SM-cell mechanotransduction (Schmidt et al., 1998Go; Shyy and Chien, 1997Go; Wang et al., 1993Go; Wang and Ingber, 1994Go). The {alpha} subunit of integrins mainly determines binding specificity for the extracellular matrix (ECM), whereas the ß subunit initiates intracellular signaling. In cultured cells, the attachment of integrins to the ECM is known to induce the aggregation of integrins to form focal adhesions, which contain actin fibers as well as cytoskeleton-associated proteins such as FAK, vinculin, paxillin and talin (Turner, 2000aGo; Turner, 2000bGo). The sites presumably transmit force between the intracellular contractile apparatus and the ECM, and the focal-adhesion proteins are good candidates for the mediators of mechanical signals to the cytoskeleton. In contrast to cultured cells, the molecular machinery that mediates the attachment of integrins to the ECM in vivo is poorly understood, whereas, in SM cells, transmembrane-associated dense plaques are structurally related to the focal adhesions of cultured cells (Burridge and Chrzanowska-Wodnicka, 1996Go).

Hic-5, a focal-adhesion protein that belongs to the paxillin family, was originally isolated as a gene induced by transforming growth factor ß1 (TGF-ß1) and hydrogen peroxide (Shibanuma et al., 1994Go). It has been reported that Hic-5 and paxillin share many structural characteristics and binding partners. Their four C-terminal LIM domains interact with protein-tyrosine-phosphatase/PEST (PTP-PEST), whereas the LD motifs in their N-terminal half contain binding sites for focal-adhesion tyrosine kinase (FAK), proline-rich tyrosine kinase 2 (PYK2), vinculin and GIT1 (Fujita et al., 1998Go; Matsuya et al., 1998Go; Nishiya et al., 1999Go; Nishiya et al., 2002Go). Despite such closely related structures and common interacting factors, Hic-5 and paxillin have different effects on some biological processes. Paxillin was identified as a tyrosine-phosphorylated protein in v-Src-, BCR-Abl- and v-Crk-transformed cells, and has been associated with cell growth (Birge et al., 1993Go; Glenney and Zokas, 1989Go; Salgia et al., 1995Go). By contrast, forced expression of Hic-5 induced cellular-senescence-like phenotypes in human fibroblasts. Consistent with this observation, its expression was decreased in several different human tumor cell lines and Ras-transformed cells, suggesting its association with growth arrest (Shibanuma et al., 1997Go). The Hic-5 expression level also decreased during immortalization of mouse embryo fibroblasts, whereas that of paxillin increased (Ishino et al., 2000Go). Moreover, Hagmann et al. reported that a switch from paxillin expression to Hic-5 expression must occur late in the maturation of megakaryocytes into platelets (Hagmann et al., 1998Go).

At a molecular level, we recently demonstrated that Hic-5 shuttles between focal adhesions and the nucleus via an oxidant-sensitive nuclear export signal (NES) and that the nucleus-localized Hic-5 transactivated c-Fos and p21WAF1. Under oxidative stress, Hic-5 accumulated in the nucleus and actually played a role in the transcriptional upregulation of the endogenous c-Fos gene (Kim-Kaneyama et al., 2002Go; Shibanuma et al., 2003Go). Although paxillin also shuttles between these two compartments, the process is insensitive to oxidants and appears not to be related to transcriptional regulation (Shibanuma et al., 2003Go; Woods et al., 2002Go). Thus, the specific function of Hic-5 might be as a redox-responsive cofactor for gene expression in the nucleus. At focal adhesions, Hic-5 inhibits cell spreading through competition with paxillin (which promotes cell spreading) for FAK, and subsequent prevention of downstream signal transduction (Nishiya et al., 2001Go). Overall, these results suggest that Hic-5 and paxillin act in opposite or distinctive ways and, consequently, that the relative balance of these homologs might have profound effects on cellular behavior such as adhesive, migratory and proliferative behavior, as well as transcriptional activities in the nucleus.

Recently, the expression of paxillin and Hic-5 in adult human tissues was examined (Yuminamochi et al., 2003Go). The expression patterns of paxillin and Hic-5 were quite different: paxillin expression was widespread and observed in both non-muscle and muscle tissues, whereas Hic-5 showed a restricted expression in mononuclear smooth muscle. In the present study, aiming to understand the function of Hic-5 in SM cells, we examined the subcellular distribution of Hic-5 during mechanical stress and its involvement in the contractile capability of a cell.


    Materials and Methods
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Immunohistochemistry
The large intestine, uterus and aorta from ICR mice (4 weeks old) in Optimal Cutting Temperature Compound (Sakura Finetechnical, Tokyo, Japan) were snap frozen in liquid nitrogen. Sections 4 µm thick were made from the frozen specimen using a Zeiss Cryostat HM500M. Individual sections were fixed with 3.7% formaldehyde in 1x TBS and blocked for 60 minutes in DAKO Protein Block, then incubated with monoclonal mouse antibodies against smooth-muscle {alpha}-actin (Sigma, 1:500) or Hic-5 (Transduction Laboratories, 1:200). Normal mouse IgG (DAKO) was used in place of the specific primary antibody as a negative control. The secondary antibody was the anti-mouse IgG coupled to horseradish peroxidase (Amersham Biosciences, 1:500). Bound IgG was revealed by incubation with diaminobenzidene (DAB) for 10 minutes. Counterstaining was carried out with Harris hematoxylin (Sigma). Controls were routinely included. Slides were covered with ENTELLAN (Merck) and cover slips, and viewed on an Axioskop microscope (Zeiss, Tokyo, Japan). Images were acquired with a digital color camera and corresponding software (Axiocam).

Cell culture
Mouse embryo fibroblast and COS7 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) as reported previously (Ohba et al., 1994Go). The Tet-Off/LD1mhic-5 cell line and Tet-Off/paxillin were established from the mouse embryo fibroblast Tet-Off cell line as described previously (Shibanuma et al., 2003Go). In brief, both pTRE and the Tet-Off cell line were purchased from Clontech Laboratories (Palo Alto, CA), and LD1mhic-5 cDNA and paxillin{alpha}-encoding cDNA were provided by H. Sabe (Osaka Biomedical Institute, Osaka, Japan) was introduced into the cloning site of pTRE. These cell lines were maintained in MEM supplemented with 10% FCS and 5 ng ml–1 doxycycline (Dox). In the Tet-Off cell lines, hemagglutinin (HA)-tagged LD1mhic-5 and HA-tagged paxillin were induced 24 hours after the removal of Dox and used for the experiments. SVS30, a SM cell line that retains the expression of SM-cell markers, was generously provided by Vessel Research Laboratory (Tokyo, Japan) and grown in medium 199 (Gibco BRL) supplemented with 10% FCS as reported previously (Hasegawa et al., 1997Go).

Expression plasmids and transfection
Mammalian HA-tagged expression plasmids for wild-type and mutant Hic-5 (Shibanuma et al., 2003Go; Nishiya et al., 1999Go) were as follows: wild type; pCG-LD1mhic-5, chimeric proteins of Hic-5 with paxillin (N-paxillin/C-Hic and N-Hic/C-paxillin); (pCG-pax/hic and -hic/pax), deletion (delL2-4, LIM) and point mutants (mL1, mL2, mL3, delL4); (pCG-LD1mhic/delL2,3,4, pCG-hhicLIM) and (pCG-LD1mhic/mL1, /mL2, /mL3, /delL4).

For the construction of Flag-CRP2, mouse cDNA encoding CRP2 was isolated from a mouse 17-day embryo marathon-ready cDNA library (Clontech) by PCR, verified by sequencing and cloned into the multicloning sites of pcDNA3 with a Flag-tag insertion.

The retrovirus expression vector for CRP2 was generated based on the pMXs-IG vector developed by Misawa et al. (Misawa et al., 2000Go). The CRP2 cDNA was excised from Flag-CRP2 with Flag-tag and inserted into the cloning sites of pMXs-IG.

The plasmids for expression of HA-tagged paxillin (pCG-pax), myc-tagged FAK (pSR{alpha}-FAK) and HA-tagged PTP-PEST (HA-PTP-PEST) were described previously (Nishiya et al., 1999Go; Nishiya et al., 2001Go).

The transient transfection of plasmids was performed using a conventional calcium-phosphate precipitation method. As an exception, we used retroviral transfection in the experiment with collagen gel contraction. Plat-E packaging cells (Morita et al., 2000Go) were transfected with the retroviral construct of CRP2 by a conventional calcium-phosphate precipitation method to produce culture supernatants containing virus. The #38/Tet-Off/LD1mhic-5 cells were infected with virus by culturing the cells for 24 hours in 1:1 Plat-E-conditioned medium: fresh DMEM, 10% FCS, supplemented with 8 µg ml–1 Polybrene (Sigma). SVS30 cells were transiently transfected with plasmids using Amaxa nucleofection technology (Amaxa, Koeln, Germany) in a solution from nucleofector kit V, following the Amaxa guidelines for cell line transfection. Briefly, 100 µl of 1x106 cell suspension mixed with 2.5 µg plasmid was transferred to the provided cuvette and nucleofected with an Amaxa Nucleofector apparatus (Amaxa). Cells were transfected using a U-25 pulsing parameter.

Antibodies
The monoclonal anti-actinin, anti-vinculin, anti-vimentin, anti-{alpha}-SM-actin and anti-Flag (M2) antibodies, and the polyclonal anti-Flag antibody were purchased from Sigma (St Louis, MO). The monoclonal anti-HA antibody (12CA5) was from Boehringer Mannheim (Mannheim, Germany), the polyclonal anti-FAK (A-17) and anti-HA (Y-11) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The monoclonal anti-GIT, anti-Hic-5 and anti-paxillin antibodies were from BD Biosciences, and the anti-Myc (9E10) monoclonal antibody was obtained from Upstate Biotechnology (Lake Placid, NY).

Application of mechanical stretching
The cells attached to the fibronectin-coated silicone chamber bottom were incubated for 24 hours in DMEM supplemented with 10% FCS before the mechanical stretching. Then, mechanical stretching was applied (120% peak to peak, at 1 Hz) by using a uni-axial stretching system (NS-550, Scholertec) at 37°C, 5% CO2.

Immunofluorescence
Immunofluorescence labeling was performed as described previously (Ishino et al., 2000Go). The anti-mouse Alexa-568-conjugated IgG and anti-rabbit Alexa-488-conjugated IgG antibodies were from Molecular Probes, and the anti-rabbit TRITC-conjugated IgG was purchased from DAKO, for detecting the primary antibodies. Fluorescence microscopy was carried out using an Axioskop microscope (Zeiss, Tokyo, Japan).

Electron-microscopic observation
The aortas of ICR mice were immersed in 4% paraformaldehyde solution including 0.1% glutaraldehyde and 0.05% Triton X-100, and embedded in glycol methacrylate. Ultrathin sections were incubated with 1% bovine serum albumin (BSA) in 0.01 M PBS for 1 hour and rinsed with 0.01 M PBS for 15 minutes. The sections were incubated overnight with an anti-Hic-5 polyclonal antibody (number 1024) (Nishiya et al., 1999Go) and an anti-CRP2 antibody at 4°C. After the sections were washed with 0.01 M PBS, 10-nm-gold-labeled goat anti-rabbit IgG (EY Laboratories, San Mateo, CA) and 15-nm-gold-labeled sheep anti-mouse IgG (EY Laboratories, San Mateo, CA) secondary antibodies were applied for 2 hours. The sections were counterstained with uranyl acetate and Reynold's lead citrate, and examined under an electron microscope (JEOL, JEM-1200EXII).

Immunoprecipitation and immunoblotting
Cells were lysed at 4°C in lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM NaF, 0.5% Nonidet P-40) containing protease inhibitor cocktail (Sigma) and phosphatase inhibitors (1 mM sodium orthovanadate). Lysates were clarified by centrifugation at 18,000 g for 10 minutes. The supernatant protein was immunoprecipitated for 2 hours at 4°C with 2 µg protein-A/Sepharose conjugated to polyclonal anti-HA antibody (Y-11, Santa Cruz Biotechnology) or normal rabbit IgG as a control. After four washes with lysis buffer with 0.1% BSA, the immunoprecipitates were eluted by boiling for 5 minutes in 30 µl SDS-PAGE sample buffer. The resulting immunoprecipitates were electrophoresed through a SDS-polyacrylamide gel, transferred onto a polyvinylidene difluoride membrane and immunoblotted with each antibody. The blots were developed with chemiluminescence reagents according to the manufacturer's protocol (PerkinElmer Life Sciences).

Collagen gel contraction
Cells were removed from dishes by trypsin treatment, washed with the culture medium, counted and suspended at an appropriate density. Solubilized collagen solution (type I; Koken, Tokyo, Japan) containing the cells was prepared by mixing 1.8 ml neutralized collagen solution (3 mg ml–1) with 3.6 ml 3 xDMEM, 0.8 ml FCS and 2.8 ml cell suspension in DMEM plus 10% FCS at 4°C to give a final collagen concentration of 1.0 mg ml–1 and a final cell density of 1 x105 cells ml–1. Immediately after mixing, the solution was transferred into Petri dishes and warmed to 37°C. After 30 minutes, when gels had formed, culture medium was added. The day after the gels were poured, to initiate floating matrix contraction, matrices were gently released from the underlying culture dish into DMEM using a spatula. To quantify the contraction, the diameter of the gels was measured and recorded.


    Results
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 Results
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 References
 
Distribution of Hic-5 in adult mouse tissues
Immunohistochemistry was performed to identify the Hic-5-expressing cell type in adult mouse tissues. The immunoreactivity was localized to SM cells of several tissues such as the large intestine, uterus, ovary, bronchial airway and blood vessels (Fig. 1A and data not shown). Using an antibody against {alpha}-smooth-muscle actin ({alpha}-SM-actin), which is a SM-cell marker, we confirmed that staining for Hic-5 was seen in the same area as that for {alpha}-SM-actin (Fig. 1A). In a previous study, it was reported that Hic-5 was present specifically in the SM cells and myoepithelial cells of mammary, sweat and salivary glands in adult human tissues (Yuminamochi et al., 2003Go). From these observations, Hic-5 appeared to be expressed in the cell types that are commonly subjected to continuous mechanical changes, including SM and myoepithelial cells, implying a possible involvement of Hic-5 in the cellular response to mechanical stress.



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Fig. 1. Distribution of Hic-5 in adult mouse tissues. (A) Immunoreactivity with anti-Hic-5 monoclonal antibody (middle), anti-{alpha}-SM-actin monoclonal antibody (bottom) and control IgG (top) in the aorta (left) and the large intestine (right). (B) The colocalization of endogenous and transfected Hic-5. The cells were transfected with the expression vector of HA-tagged Hic-5 (pCG-LD1mhic-5) subjected to uni-axial cyclic stretch at 1 Hz for 60 minutes (stretch +) or not (stretch –) and immunostained with both anti-HA (Y-11) antibody and anti-Hic-5 antibody. The anti-Hic-5 antibody stained the same types of structures in the transfected as in the neighboring untransfected cells, supporting the colocalization of the endogenous and exogenous proteins. The arrow indicates the direction of stretch. Bars, 5 µm.

 

Mechanical stretching induces translocation of Hic-5 to actin stress fibers
Given that Hic-5 changed its cellular distribution depending on cellular conditions (Shibanuma et al., 2003Go; Shibanuma et al., 2004Go), we examined whether mechanical stretching induced a change in the intracellular localization of Hic-5. First, we confirmed the co-localization of endogenous and transfected Hic-5. The cells were transfected with the expression vector for HA-tagged Hic-5 and immunostained simultaneously with an anti-HA antibody and an anti-Hic-5 antibody. Fig. 1B shows the co-localization of endogenous and transfected Hic-5.

For the application of mechanical stretching, a uni-axial stretching system was used to reproduce a mechanical situation observed in situ in many cell types (Inoh et al., 2002Go). We applied uni-axial cyclic stretching to the Tet-Off cell line of a mouse embryo fibroblast in which HA-tagged Hic-5 was induced by removal of Dox) from the culture medium (Shibanuma et al., 2003Go). Immunostaining of Hic-5 with an anti-HA antibody demonstrated that Hic-5 localized mainly to the focal adhesions of the unstretched cells (Fig. 2A). By contrast, Hic-5 staining was prominent in the stress-fiber structure in cells subjected to the cyclic stretching for 60 minutes (Fig. 2A), indicating the translocation of Hic-5 from focal adhesions to stress fibers in response to the mechanical stretching. Interestingly, paxillin, the most homologous to Hic-5 of the focal-adhesion proteins, remained at focal adhesions during the cyclic stretch in a similar system of mouse embryo fibroblast Tet-Off cells for paxillin (Fig. 2B).



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Fig. 2. Translocation of Hic-5 from focal adhesions to actin stress fibers during uni-axial cyclic stretch. The Tet-Off cell lines of Hic-5 (A) and paxillin (B) were incubated without Dox for 24 hours, subjected to uni-axial cyclic stretching at 1 Hz for 60 minutes (+) or not (–) and stained with the antibody 12CA5 against HA-tag of Hic-5 (A, top) and paxillin (B, top). The cells were also stained with FITC-conjugated phalloidin (middle) and merged images of phalloidin and HA tag of Hic-5 or paxillin are shown on the right. SVS30 SM cells were subjected to uni-axial cyclic stretching at 1 Hz for 60 minutes (+) or not (–), and stained with the antibody against Hic-5 and with FITC-conjugated phalloidin (C) and with the antibody against paxillin and FITC-conjugated phalloidin (D). The arrow indicates the direction of stretching. Bars, 10 µm.

 

We also examined the relocalization of endogenous Hic-5 using the SM cell line SVS30, which was established from aortas of a transgenic mouse harboring gene encoding a temperature-sensitive mutant of the SV40 large T-antigen. When the SVS30 cells were similarly exposed to mechanical stretching and immunostained with the antibody to endogenous Hic-5 and paxillin, essentially the same results were obtained, although faint stress-fiber staining was sometimes observed in the paxillin-stained cells (Fig. 2C,D). Because the anti-paxillin antibody cross-reacts with Hic-5, signals appeared on the stress fiber owing to this cross-reactivity. Notably, the Hic-5 staining promptly shifted from stress fibers to focal adhesions in almost all cells within 30 minutes of removal of the stretch stimulus (data not shown).

Intracellular distribution of Hic-5-binding proteins under mechanical stretching
Hic-5, like paxillin, has been reported to bind to several signaling and structural molecules. In the present study, we investigated the stretch-induced intracellular distribution of Hic-5-interacting factors such as FAK (Fujita et al., 1998Go), vinculin (Thomas et al., 1999Go), GIT1 (Nishiya et al., 2002Go) and PTP-PEST (Nishiya et al., 1999Go). The cells subjected to the cyclic stretching for 60 min were stained with antibodies against endogenous vinculin and GIT1. Regarding FAK and PTP-PEST, expression vectors for myc-tagged FAK or HA-tagged PTP-PEST were introduced into cells approximately 24 hours before the stretch and the intracellular distribution was examined by using antibodies against the tags. The actin stress fibers were visualized using FITC-conjugated-phalloidin labeling. Vinculin and FAK were localized mainly to the focal-adhesion sites of the unstretched cells. Upon the stretch stimulus, most of them were redistributed throughout the cytoplasm, with residual vinculin at focal contacts (Fig. 3A,B). PTP-PEST was mostly distributed throughout the cytoplasm in both the unstretched and the stretched cells although it was partially localized to large bundles of actin in the stretched cells (Fig. 3C). GIT1 was localized to actin fibers in unstretched and stretched conditions (Fig. 3D).



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Fig. 3. Intracellular distribution of Hic-5-binding proteins during uni-axial cyclic stretch. Mouse embryo fibroblast cells subjected to the uni-axial cyclic stretch for 60 minutes (+) or not (–), as in Fig. 2, were stained with antibodies against endogenous vinculin (A, top) and GIT1 (D, top). (B,C) The expression vector encoding myc-tagged FAK (B) or HA-tagged PTP-PEST (C) was introduced into mouse embryo fibroblast cells and, at 24 hours after transfection, the cells were subjected to cyclic stretching as above and stained with antibodies against tags [9E10 (B) and 12CA5 (C)]. The distribution of actin stress fibers was visualized by FITC-conjugated phalloidin labeling (A-D, middle). The arrow indicates the direction of stretching. Bars, 10 µm.

 

In addition to Hic-5-interacting proteins, we investigated the localization of other LIM proteins, focusing on the cysteine-rich proteins (CRP), which belong to the family of LIM-only proteins and consist of two double zinc-finger LIM domains followed by a glycine-rich region. In vertebrates, four members have been identified (CRP1, CRP2, CRP3 and TLP). These four show high sequence homology with each other, although they differ in their expression patterns (Weiskirchen and Gunther, 2003Go). CRP1 is expressed in most tissues and cell types, including SM cells (Liebhaber et al., 1990Go). CRP2 is predominantly expressed in arterial SM cells (Henderson et al., 1999Go; Jain et al., 1996Go). CRP3 is expressed only in striated-muscle cells (Arber et al., 1994Go; Jain et al., 1996Go; Jain et al., 1998Go). In the present study, the intracellular distributions of CRP1 and CRP2, which are expressed in SM cells, were investigated by introducing Flag-tagged expression vectors into cells. CRP2 was localized along the actin-fiber structures in unstretched cells, which became prominent under mechanical stretch (Fig. 4A). By contrast, CRP1 was distributed in the cytoplasm and the nucleus but not on actin stress fibers (data not shown). We also investigated the localization of {alpha}-actinin, a binding partner of CRPs. Under stretch stimuli, {alpha}-actinin was localized periodically (not continuously like Hic-5 and CRP2) along actin stress fibers (Fig. 4B). In summary, among the factors examined, only GIT1 and CRP2 were localized to actin stress fibers in the cells like Hic-5 during the cyclic stretch (Fig. 4C). Thus, GIT1 and/or CRP2 are possible candidates involved in the localization of Hic-5 to stress fibers.



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Fig. 4. Intracellular distribution of CRP2 and {alpha}-actinin during uni-axial cyclic stretch. Mouse embryo fibroblast cells were transfected with Flag-tagged CRP2 expression vector, subjected to cyclic stretching (+) or not (–) and stained with the polyclonal antibody against the Flag tag (A) or with that against endogenous {alpha}-actinin (B). The insets in the merged images are magnification of a part of a cell's rectangular frame. The white arrows in the inset of B show a periodic pattern of {alpha}-actinin staining. Bars, 10 µm. (C) The Hic-5 domains interacting with the factors and presence (+) or absence (–) of the factors on stress fiber during the cyclic stretching.

 

Hic-5 is localized to stress fibers through its LIM domains
To investigate which domain is crucial to the localization of Hic-5 to stress fibers, we introduced plasmids expressing the HA-tagged mutant deleted of domains LIM2-4 (del L2-4) or the LIM-only region of Hic-5 (LIM) (Fig. 5A) into cells and examined their localization under cyclic stretching. We confirmed the expression of the Hic-5 mutants at the expected sizes (Fig. 5B). As indicated in Fig. 5C (stretch +), LIM but not delL2-4 was able to localize to actin stress fibers during the cyclic stretching, suggesting that the localization of Hic-5 to stress fibers occurs through its LIM domains and ruling out the direct involvement of GIT1, which interacted with the LD3 motif of Hic-5, in the localization. In unstretched cells, LIM was localized in the focal-adhesion sites and nucleus, whereas del LIM2-4 was not localized to focal adhesions but diffusely in the cytoplasm (Fig. 5C, stretch –).



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Fig. 5. The C-terminal region of Hic-5 is sufficient for the stress-fiber-restricted localization under cyclic stretching. (A) The HA-tagged Hic-5 deletion mutants used in this experiment. (B) The expression plasmids of the Hic-5 mutants were transfected into mouse embryo fibroblast cells; 24 hours later, western blotting was carried out using the antibody to the HA tag. (C) Mouse embryo fibroblast cells were transfected with each expression vector, subjected to cyclic stretching (+) or not (–) and immunostained with the 12CA5 antibody against the HA tag (left, red). (C, middle) F-actin stained by FITC-conjugated phalloidin (green); (C, right) merged images. The arrow indicates the direction of stretching. Bars, 5 µm.

 

Next, the distribution of Hic-5 mutants (mL1-mL3 and delL4) in which each of the LIM domains was disrupted by point mutations (LIM1-LIM3) or a deletion (LIM4) (Fig. 6A) was compared with that of the wild type (WT). The point mutations at LIM2 and LIM3 abolished the ability of Hic-5 to localize to actin stress fibers (Fig. 6C, stretch +), indicating that LIM2 and LIM3 were fundamentally required for the stretching-induced translocation of Hic-5 to the actin stress fibers. Interestingly, mL2 and mL3 were also unable to localize to focal adhesions in the unstretched cells (Fig. 6C, stretch –).



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Fig. 6. The LIM2 and LIM3 domains are crucial for Hic-5 to be localized to stress fibers. (A) The HA-tagged Hic-5 mutants in which each of the LIMs was disrupted by point mutations (mL1-mL3) or a deletion (delL4). The expression plasmids for each mutant have been previously described (Shibanuma et al., 2003Go). (B) The expression plasmids were transfected into mouse embryo fibroblast cells; 24 hours later, western blotting was carried out using the antibody against the HA tag. (C) Mouse embryo fibroblast cells were transfected with each expression vector, subjected to cyclic stretching (+) or not (–) and immunostained with the 12CA5 antibody against the HA tag. (C, left) Staining of the Hic-5 mutants (red) with the 12CA5 antibody to the HA tag; (C, middle) F-actin stained by FITC-conjugated phalloidin (green); (C, right) merged images. The arrow indicates the direction of stretch. Bars, 5 µm.

 

Hic-5 associates with CRP2 through its LIM domains
As described above, CRP2 was, like Hic-5, found in actin stress fibers during cyclic stretching. Here, we examined the protein-protein interaction between CRP2 and Hic-5. To this end, we introduced expression vectors that encode Flag-tagged CRP2 with those for HA-tagged Hic-5 or paxillin into COS7 cells and immunoprecipitated the cell lysate with anti-HA antibody, before performing immunoblotting with the anti-Flag antibody. As shown in Fig. 7A (Hic-5 in lane 1, paxillin in lane 4), the interaction of CRP2 with Hic-5 but not with paxillin was observed. In a similar experiment using chimeric proteins (Fig. 7B), in which the N- and C-terminal halves of Hic-5 and paxillin were mutually exchanged, the chimeric protein carrying the LIM domains of Hic-5 but not those of paxillin were able to interact with CRP2 (Fig. 7A, lanes 2 and 3), suggesting that Hic-5 interacted with CRP2 though its LIM domains in the C-terminal half. Consistent with this, the LIM-only region of Hic-5 was sufficient to interact with CRP2 (data not shown). In addition, co-immunostaining of exogenously expressed HA-tagged Hic-5 and Flag-tagged CRP2 showed that both proteins were present in stress fibers in the stretched cells (Fig. 7C). Furthermore, immunoelectron microscopy of the mouse aorta confirmed the colocalization of Hic-5 and CRP2 in an in vivo setting, showing that a proportion of Hic-5 was found at filamentous structures in SM cells with CRP2 (Fig. 7D, smaller gold particles are Hic-5, larger ones are CRP2). These results indicate that Hic-5 interacted with CRP2 in vitro and in vivo; in addition, Hic-5 and CRP2 were localized together along fibers, implying their cooperation in the localization onto actin stress fibers and/or in function.



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Fig. 7. Hic-5 associates and localizes with CRP2 to actin stress fibers. (A) Flag-tagged CRP2 was expressed with HA-tagged Hic-5 wild-type and mutants in COS7 cells: lane 1, wild-type; lane 2, N-paxillin/C-Hic; lane 3, N-Hic/C-paxillin; lane 4, paxillin. 24 hours after transfection, the cells were lysed and subjected to immunoprecipitation with polyclonal anti-HA antibody (lane H) or normal rabbit IgG (lane C) as a control, followed by immunoblotting with monoclonal anti-HA (top) or anti-Flag (M2) (bottom) antibodies. (B) The wild-type and chimeric proteins of Hic-5 and paxillin studied in A. (C) Hic-5 (pCG-LD1mhic-5) and CRP2 (Flag-CRP2) co-expressed in mouse embryo fibroblast cells were co-immunostained using the monoclonal anti-HA (12CA5) and polyclonal anti-Flag antibodies after being exposed to the cyclic stretching. The top, middle and bottom images show Hic-5, CRP2 and a merged image, respectively. The arrow indicates the direction of stretching. Bar, 2 µm. (D) Immunogold electron microscopy of a mouse aorta. (top) L, lumen; ET, endothelial cell; EL, elastic lamina; SMC, smooth-muscle cell. (bottom) Magnified image of a part of the smooth-muscle cell (framed rectangle at top). Arrows indicate immunogold (10 nm) labeling of Hic-5 and arrowheads indicate immunogold (15 nm) labeling of CRP2. (middle) A similar field exposed to control serum. Bars, 2 µm (top) and 100 nm (middle and bottom).

 

Involvement of Hic-5 and CRP2 in the regulation of cell contraction force
To address the biological significance of the actin-cytoskeleton localization of Hic-5 during cyclic stretching, we investigated whether Hic-5 is involved in the contractile capability of a cell by using a three-dimensional collagen gel. The collagen was first mixed in solution with mouse embryo fibroblast Tet-Off cells in the presence or absence of Dox; the gel was allowed to polymerize and then released from culture dishes to initiate contraction (floating matrices). Observation of the contraction process revealed that, when the gel contained cells expressing exogenous Hic-5, the process was significantly slower on removal of Dox than in the control (Fig. 8A, top). By contrast, the cells expressing exogenous paxillin enhanced the contraction of the collagen gel (Fig. 8A, bottom). With regard to actin fiber localization, the collagen-gel contraction was measured in cells expressing the Hic-5 mutants mL2 and mL3, which had lost the ability to translocate to actin stress fibers under stretched conditions. In contrast to the wild-type Hic-5, the suppressive effect on the collagen-gel contraction was abolished in the gels that included the cells expressing mL2 and mL3 mutants (Fig. 8B, bottom). This result suggests the importance of the localization on actin stress fiber of Hic-5 in regulating the contractile force of cells. Additionally, mL1, which was localized to stress fibers, retained the ability to suppress the gel contraction, whereas del4 lost this ability in spite of its localization to stress fibers (data not shown). It is well known that Hic-5 is an adaptor protein that interacts with various factors. Thus, it is likely that del4 is a mutant defective in the interaction with a certain factor crucially involved in the regulation of gel contraction. Together, these results suggest that, in response to cyclic stretching, Hic-5 is relocalized to actin stress fibers and negatively regulates the contractile force of cells.



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Fig. 8. Effects of Hic-5, paxillin and CRP2 on collagen-gel contraction. (A) The Tet-Off/LD1mhic-5 (top) and the MEF/Tet-Off/paxillin (bottom) cell lines incubated with (Dox +) or without (Dox –) doxycycline for 24 hours were embedded in collagen gels and the gel contraction was assessed by measuring the diameters at each indicated time. The means and standard deviations of four parallel measurements are shown. (B, top) The Tet-Off/LD1mhic-5 cell line incubated with (Dox +) or without (Dox –) doxycycline for 24 hours was infected with the retrovirus of CRP2 (CRP +) or control virus (CRP –) for the next 24 hours and then embedded in collagen gel. The gel contraction was assessed as in A and the pictures were taken 56 hours after floating the collagen gels (C). (B, bottom) Expression plasmids encoding the mL2 and mL3 Hic-5 mutants were introduced into SVS30 smooth-muscle cells by electroporation and, 24 hours later, the cells were embedded in collagen gels and observed as in A. (D) Intracellular distribution of Hic-5 and CRP2 in the cells embedded in collagen gel. The cells in collagen gel were prepared as in C and stained with anti-Hic-5 (red) and anti-Flag polyclonal (CRP2) (green) antibodies, and DAPI (blue). Bar, 10 µm.

 

Subsequently, we investigated the role of CRP2 in the regulation of cell contractile force by infection with a retrovirus expressing CRP2 simultaneously with green fluorescent protein (GFP). Producing at least half GFP-positive cells was considered to reflect a successful infection. We infected mouse embryo fibroblast Tet-Off cells with the retrovirus with or without Dox in the medium. Compared with control cells (Fig. 8B, top, Fig. 8C, Dox+), cells expressing exogenous CRP2 reduced the contraction of the gel (Fig. 8B, top, Fig. 8C, Dox+ with CRP2+). Interestingly, when Hic-5 and CRP2 were co-expressed in the cells, the inhibitory effect on contraction was enhanced (Fig. 8B, top, Fig. 8C, Dox– with CRP2+). The experiment on the intracellular distribution of Hic-5 and CRP2 in the cells embedded in collagen gel indicated that CRP2 was co-localized with Hic-5 to fiber structures (Fig. 8D). These results showed that Hic-5 and CRP2 cooperated in the regulation of cell contractile force on actin stress fibers.


    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Expression of Hic-5 in adult SM and multiple myogenic lineages during embryogenesis
Hic-5, a focal-adhesion protein that belongs to the paxillin family, showed a restricted expression in SM cells in various tissues of an adult mouse (Fig. 1). This observation was consistent with a previous study on human tissues (Yuminamochi et al., 2003Go). Yuminamochi et al. also compared the expression pattern of Hic-5 with that of paxilllin and showed that, in contrast to the SM-cell-restricted expression of Hic-5, paxillin was expressed ubiquitously in both non-muscle and muscle tissues, and that, among muscle tissues, it was present mainly in striated muscle (Yuminamochi et al., 2003Go). During development, weak to moderate expression of Hic-5 was first detected in the developing heart tube at embryonic day 8.0 (E8.0) by in situ hybridization, and this continued until E16.5 (Brunskill et al., 2001Go). In addition, Hic-5 was expressed in the myotome layer of the developing somite in the early embryo and, in the later stage, in the SM and skeletal-muscle layers, and in the mesenchymal tissue of several developing tissues (Brunskill et al., 2001Go).

Several genes, such as those for calponin, SM22{alpha} and SM{alpha}-actin (which are considered markers for SM cells), are expressed in the developing cardiac tube as well as in differentiating SM cells during embryogenesis. Hic-5 resembles SM22{alpha}, one of the most established SM-cell markers; SM22{alpha} is expressed in cardiac, skeletal and SM cells in the embryo before its expression becomes restricted to SM cells in the adult mouse. The promoters for these SM-cell-specific genes contain several CArG elements that represent binding sites for the MADS-box transcription factor serum response factor (SRF) (Shore and Sharrocks, 1995Go). Sequences flanking the CArG elements are also necessary for the specificity of the expression (Chang et al., 2001Go; Strobeck et al., 2001Go). There are also CArG elements in the hic-5 promoter, suggesting that its expression is mediated in a similar way to these SM-cell marker genes.

Actin-stress-fiber localization of Hic-5 during mechanical stretch
Various types of molecule, such as FAK, paxillin, protein kinase C, ERK, Rho, stretch-sensitive ion channels, heterotrimeric G proteins, adenyl cyclase and NF-{kappa}B, are thought to be involved in the signaling that is elicited by a mechanical stimulus, although the exact mechanisms by which mechanical forces are transduced into a biological response have remained unclear (Ingber, 2003bGo; Inoh et al., 2002Go). Integrins, which regulate the linkage between the extracellular matrix and intracellular actin cytoskeleton, were thought to play an initial key role in the signal transduction of mechanical force. In fact, mechanical stress applied to integrins reportedly altered the cytoskeleton structure and activated signal-transduction pathways (Ingber, 2003aGo; Katsumi et al., 2004Go; Tang et al., 2002Go), suggesting that integrins can function as mechanotransducers that transmit the mechanical force between the contractile apparatus and the extracellular matrix by assembling a focal-adhesion complex upon their engagement. FAK and paxillin, two representative mediators of the integrin signals at focal adhesions, have been reported to regulate cytoskeletal reorganization and contractile events in response to mechanical stimuli. For example, both proteins undergo mechanosensitive tyrosine phosphorylation during the contractile activation of tracheal smooth muscle, the importance of which for the contraction of the cells was already evaluated (Tang et al., 1999Go; Tang and Gunst, 2001Go; Tang et al., 2002Go).

In the present study, we found that Hic-5, a member of the paxillin family, translocated from focal adhesions to actin-cytoskeleton components upon mechanical stretching in fibroblasts (Fig. 2A). Along with exogenously expressed Hic-5, we observed the same localization of endogenous Hic-5 using the SM cell line SVS30 (Fig. 2C), which was established from aortas of a transgenic mouse harboring a gene encoding a temperature-sensitive SV40 large T-antigen. Notably, this change in localization was not observed for paxillin (Fig. 2B,D). Instead, paxillin was reported to be phosphorylated as an important part of the mechanism for mechanotransduction at focal adhesions.

Our previous study indicated that LIM2 and LIM3 mutants of Hic-5 were unable to localize to focal adhesions (Nishiya et al., 1999Go). It was noteworthy that, in the present study, the same mutants could not be relocalized to the actin stress fibers in response to stretch stimuli, either (Fig. 6). From these findings, we speculate that the translocation of Hic-5 to the actin stress fibers during stretching is primarily controlled at focal adhesions that are presumed to undergo structural remodeling in response to stretch stimuli. Local tensional change is a potential initiator of the remodeling process, inducing the conformational alteration of the components of the structure. Actually, focal-adhesion remodeling occurs in a tension-dependent manner (Smilenov et al., 1999Go). Eventually, the process is expected to lead to the conversion of the local mechanical forces into biological signals. Thus, the proper localization of Hic-5 to focal adhesions under unstretched conditions is speculated to be prerequisite for the translocation of Hic-5 to stress fibers in response to stretch stimuli, so that Hic-5 senses the tensional change though the remodeling of the structure before the translocation to the actin stress fibers. Consequently, Hic-5 has the potential to mediate the tensional change directly from focal adhesions to actin stress fibers upon cellular mechanical stress as one of the effector molecules transmitting the mechanical signals.

We also investigated the factors involved in the localization of Hic-5 to the actin cytoskeleton during cyclic stretching and found that CRP2 was one possible candidate. CRP2 is an only-LIM protein belonging to the CRP family that displays a dual subcellular localization both in the nucleus and along actin-cytoskeleton elements in the cytoplasm (Arber et al., 1994Go; Weiskirchen and Gunther, 2003Go). Among family members, CRP2 was specifically expressed in arterial smooth muscle. We show here that, under mechanical stretching, CRP2 was mainly localized along the actin stress fibers (Fig. 4). Besides, CRP2 interacted with LIM domains in the C-terminal half of Hic-5 and was localized with Hic-5 to the actin stress fibers in the stretched cells (Fig. 7). By contrast, CRP2 did not interact with paxillin (Fig. 7), which did not translocate to the actin stress fibers following the stretch stimulus (Fig. 2). These results suggest that CRP2 played some role in the translocation of Hic-5 to the stress fibers during the cyclic stretching, although the mechanism of the translocation of Hic-5 to the actin stress fibers remains unclear.

Involvement of Hic-5 and CRP2 in the regulation of contractile capability of cells
Collagen lattices are an established model system for studying the mechanisms underlying the generation of tensile force by cells, which is known to depend on an organized actin cytoskeleton (Tomasek et al., 1992Go). Using this model system, we found that Hic-5 affected the contraction of cells and that CRP2 potentiated the effect of Hic-5 (Fig. 8). When the expression levels of Hic-5 were manipulated to increase in the fibroblasts embedded in the gels, the contraction of the gel was suppressed. CRP2 also showed the suppressive effect, and the effect was further augmented by co-expressing the proteins. In the cells in collagen gel, CRP2 was localized with Hic-5 to the fibers as in monolayer cells during the cyclic stretching. When CRP2 or Hic-5 was co-stained with phalloidin, a stress-fiber localization was confirmed for both factors (data not shown). These results suggested that Hic-5 in a complex with CRP2 on the stress fibers participates in the regulation of cell contractile force, providing biological significance to their localization on stress fibers.

Regarding the change in the intracellular localization of Hic-5, we recently reported that Hic-5 communicates between focal-adhesion sites and the nucleus through an oxidant-sensitive nuclear export signal (Shibanuma et al., 2003Go). In the nucleus, Hic-5 was suggested to participate in gene expression, being part of a multiprotein complex on the DNA that includes Sp1, Smad3 and p300 (Shibanuma et al., 2004Go). Thus, it was proposed to be a focal-adhesion protein shuttling between focal adhesions and the nucleus, depending on the cellular redox state, mediating the signal directly to the nucleus. In the present study, Hic-5 was shown to translocate to actin stress fibers in response to mechanical stress and to regulate the contractile capability of cells, and thus potentially transduce mechanical signals directly to the actin stress fibers. It is worth realizing that Hic-5 also accumulated in the nucleus on treatment with leptomycin B, a specific inhibitor of the nuclear export signal, in response to stretch stimuli (data not shown), suggesting that Hic-5 could also shuttle between focal adhesions and the nucleus under mechanical stretching instead of being immobilized on the stress fibers. The exact functions of Hic-5 on the stress fibers and its relationships to those at focal adhesions and in the nucleus are subjects for future study.

The regulation of mechanical force in cells though cytoskeletal reorganization might be crucial if cells are to deal with mechanical stress. The SM cells in blood vessels, as a typical example, are exposed continuously to contractile stimulation and are obliged to adapt to these conditions. Clarifying the mechanisms regulating the intracellular localization of Hic-5 to focal adhesions, the stress fibers and the nucleus, along with the functions of Hic-5 at each location, should lead to a better understanding of how the dynamism of the actin cytoskeleton could be coupled to contractile force and gene expression. As a result, it should hopefully provide a therapeutic tool for manipulating pathological conditions including vascular lesions affected by the malfunction of SM cells.


    Acknowledgments
 
We thank K. Kou and H. Funahashi for general assistance and advice in the electron microscopic study, and M. Murakami and M. Yamazaki for contributing to this work as the theme for their bachelor's degrees. We also thank Vessel Research Laboratory (Tokyo, Japan) for SVS30 cells. This work was supported in part by Grants-in-Aid for Scientific Research, the High-Technology Research Center Project, a Showa University Grant-in-Aid for Innovative Collaborative Research Projects and a Special Research Grant-in-Aid for Development of Characteristic Education from the ministry of Education, Culture, Sports, Science and Technology of Japan.


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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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