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
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Hic-5, Mechanical stress, Smooth muscle cell, CRP2, Collagen-gel contraction
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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., 1994). 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., 1998
; Matsuya et al., 1998
; Nishiya et al., 1999
; Nishiya et al., 2002
). 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., 1993
; Glenney and Zokas, 1989
; Salgia et al., 1995
). 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., 1997
). The Hic-5 expression level also decreased during immortalization of mouse embryo fibroblasts, whereas that of paxillin increased (Ishino et al., 2000
). 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., 1998
).
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., 2002; Shibanuma et al., 2003
). 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., 2003
; Woods et al., 2002
). 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., 2001
). 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., 2003). 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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., 1994). 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., 2003
). In brief, both pTRE and the Tet-Off cell line were purchased from Clontech Laboratories (Palo Alto, CA), and LD1mhic-5 cDNA and paxillin
-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 ml1 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., 1997
).
Expression plasmids and transfection
Mammalian HA-tagged expression plasmids for wild-type and mutant Hic-5 (Shibanuma et al., 2003; Nishiya et al., 1999
) 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., 2000). 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-FAK) and HA-tagged PTP-PEST (HA-PTP-PEST) were described previously (Nishiya et al., 1999
; Nishiya et al., 2001
).
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., 2000) 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 ml1 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--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., 2000). 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., 1999) 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 ml1) 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 ml1 and a final cell density of 1 x105 cells ml1. 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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., 2003; Shibanuma et al., 2004
), 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., 2002). 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., 2003
). 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).
|
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., 1998), vinculin (Thomas et al., 1999
), GIT1 (Nishiya et al., 2002
) and PTP-PEST (Nishiya et al., 1999
). 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).
|
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, 2003). CRP1 is expressed in most tissues and cell types, including SM cells (Liebhaber et al., 1990
). CRP2 is predominantly expressed in arterial SM cells (Henderson et al., 1999
; Jain et al., 1996
). CRP3 is expressed only in striated-muscle cells (Arber et al., 1994
; Jain et al., 1996
; Jain et al., 1998
). 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
-actinin, a binding partner of CRPs. Under stretch stimuli,
-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.
|
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 ).
|
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 ).
|
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.
|
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.
|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Several genes, such as those for calponin, SM22 and SM
-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
, one of the most established SM-cell markers; SM22
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, 1995
). Sequences flanking the CArG elements are also necessary for the specificity of the expression (Chang et al., 2001
; Strobeck et al., 2001
). 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-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, 2003b
; Inoh et al., 2002
). 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, 2003a
; Katsumi et al., 2004
; Tang et al., 2002
), 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., 1999
; Tang and Gunst, 2001
; Tang et al., 2002
).
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., 1999). 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., 1999
). 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., 1994; Weiskirchen and Gunther, 2003
). 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., 1992). 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., 2003). 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., 2004
). 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 |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arber, S., Halder, G. and Caroni, P. (1994). Muscle LIM protein, a novel essential regulator of myogenesis, promotes myogenic differentiation. Cell 79, 221-231.[Medline]
Birge, R. B., Fajardo, J. E., Reichman, C., Shoelson, S. E., Songyang, Z., Cantley, L. C. and Hanafusa, H. (1993). Identification and characterization of a high-affinity interaction between v-Crk and tyrosine-phosphorylated paxillin in CT10-transformed fibroblasts. Mol. Cell. Biol. 13, 4648-4656.[Abstract]
Brunskill, E. W., Witte, D. P., Yutzey, K. E. and Potter, S. S. (2001). Novel cell lines promote the discovery of genes involved in early heart development. Dev. Biol. 235, 507-520.[CrossRef][Medline]
Burridge, K. and Chrzanowska-Wodnicka, M. (1996). Focal adhesions, contractility, and signaling. Annu. Rev. Cell. Dev. Biol. 12, 463-518.[CrossRef][Medline]
Chang, P. S., Li, L., McAnally, J. and Olson, E. N. (2001). Muscle specificity encoded by specific serum response factor-binding sites. J. Biol. Chem. 276, 17206-17212.
Fujita, H., Kamiguchi, K., Cho, D., Shibanuma, M., Morimoto, C. and Tachibana, K. (1998). Interaction of Hic-5, a senescence-related protein, with focal adhesion kinase. J. Biol. Chem. 273, 26516-26521.
Glenney, J. R., Jr and Zokas, L. (1989). Novel tyrosine kinase substrates from Rous sarcoma virus-transformed cells are present in the membrane skeleton. J. Cell Biol. 108, 2401-2408.[Abstract]
Hagmann, J., Grob, M., Welman, A., van Willigen, G. and Burger, M. M. (1998). Recruitment of the LIM protein Hic-5 to focal contacts of human platelets. J. Cell Sci. 111, 2181-2188.
Hasegawa, K., Arakawa, E., Oda, S., Yanai, N., Obinata, M. and Matsuda, Y. (1997). Novel smooth muscle cell lines from transgenic mice harboring temperature-sensitive SV40 large T-antigen gene. Temperature-dependent expression of smooth muscle myosin heavy chain-1 and calponin genes. J. Mol. Cell. Cardiol. 29, 2177-2186.[CrossRef][Medline]
Henderson, J. R., Macalma, T., Brown, D., Richardson, J. A., Olson, E. N. and Beckerle, M. C. (1999). The LIM protein, CRP1, is a smooth muscle marker. Dev. Dyn. 214, 229-238.[CrossRef][Medline]
Ingber, D. E. (2003a). Mechanosensation through integrins: cells act locally but think globally. Proc. Natl. Acad. Sci. USA 100, 1472-1474.
Ingber, D. E. (2003b). Tensegrity II. How structural networks influence cellular information processing networks. J. Cell Sci. 116, 1397-1408.
Inoh, H., Ishiguro, N., Sawazaki, S., Amma, H., Miyazu, M., Iwata, H., Sokabe, M. and Naruse, K. (2002). Uni-axial cyclic stretch induces the activation of transcription factor nuclear factor kappaB in human fibroblast cells. FASEB J. 16, 405-407.
Ishino, K., Kaneyama, Shibanuma, M. and 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.[CrossRef][Medline]
Jain, M. K., Fujita, K. P., Hsieh, C. M., Endege, W. O., Sibinga, N. E., Yet, S. F., Kashiki, S., Lee, W. S., Perrella, M. A., Haber, E. et al. (1996). Molecular cloning and characterization of SmLIM, a developmentally regulated LIM protein preferentially expressed in aortic smooth muscle cells. J. Biol. Chem. 271, 10194-10199.
Jain, M. K., Kashiki, S., Hsieh, C. M., Layne, M. D., Yet, S. F., Sibinga, N. E., Chin, M. T., Feinberg, M. W., Woo, I., Maas, R. L. et al. (1998). Embryonic expression suggests an important role for CRP2/SmLIM in the developing cardiovascular system. Circ. Res. 83, 980-985.
Katsumi, A., Orr, A. W., Tzima, E. and Schwartz, M. A. (2004). Integrins in mechanotransduction. J. Biol. Chem. 279, 12001-12004.
Kim-Kaneyama, J., Shibanuma, M. and Nose, K. (2002). Transcriptional activation of the c-Fos gene by a LIM protein, Hic-5. Biochem. Biophys. Res. Commun. 299, 360-365.[CrossRef][Medline]
Liebhaber, S. A., Emery, J. G., Urbanek, M., Wang, X. K. and Cooke, N. E. (1990). Characterization of a human cDNA encoding a widely expressed and highly conserved cysteine-rich protein with an unusual zinc-finger motif. Nucleic Acids Res. 18, 3871-3879.[Abstract]
Matsuya, M., Sasaki, H., Aoto, H., Mitaka, T., Nagura, K., Ohba, T., Ishino, M., Takahashi, S., Suzuki, R. and Sasaki, T. (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.
Misawa, K., Nosaka, T., Morita, S., Kaneko, A., Nakahata, T., Asano, S. and Kitamura, T. (2000). A method to identify cDNAs based on localization of green fluorescent protein fusion products. Proc. Natl. Acad. Sci. USA 97, 3062-3066.
Morita, S., Kojima, T. and Kitamura, T. (2000). Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther. 7, 1063-1066.[CrossRef][Medline]
Nishiya, N., Iwabuchi, Y., Shibanuma, M., Cote, J. F., Tremblay, M. L. and Nose, K. (1999). Hic-5, a paxillin homologue, binds to the protein-tyrosine phosphatase PEST (PTP-PEST) through its LIM 3 domain. J. Biol. Chem. 274, 9847-9853.
Nishiya, N., Tachibana, K., Shibanuma, M., Mashimo, J. I. and 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.
Nishiya, N., Shirai, T., Suzuki, W. and Nose, K. (2002). Hic-5 interacts with GIT1 with a different binding mode from paxillin. J. Biochem. 132, 279-289.[Abstract]
Ohba, M., Shibanuma, M., Kuroki, T. and Nose, K. (1994). Production of hydrogen peroxide by transforming growth factor-beta 1 and its involvement in induction of Egr-1 in mouse osteoblastic cells. J. Cell Biol. 126, 1079-1088.[Abstract]
Salgia, R., Brunkhorst, B., Pisick, E., Li, J. L., Lo, S. H., Chen, L. B. and Griffin, J. D. (1995). Increased tyrosine phosphorylation of focal adhesion proteins in myeloid cell lines expressing p210BCR/ABL. Oncogene 11, 1149-1155.[Medline]
Schmidt, C., Pommerenke, H., Durr, F., Nebe, B. and Rychly, J. (1998). Mechanical stressing of integrin receptors induces enhanced tyrosine phosphorylation of cytoskeletally anchored proteins. J. Biol. Chem. 273, 5081-5085.
Shibanuma, M., Mashimo, J., Kuroki, T. and 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., Oshimura, M. and Nose, K. (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]
Shibanuma, M., Kim-Kaneyama, J. R., Ishino, K., Sakamoto, N., Hishiki, T., Yamaguchi, K., Mori, K., Mashimo, J. and Nose, K. (2003). Hic-5 communicates between focal adhesions and the nucleus through oxidant-sensitive nuclear export signal. Mol. Biol. Cell 14, 1158-1171.
Shibanuma, M., Kim-Kaneyama, J. R., Sato, S. and Nose, K. (2004). A LIM protein, Hic-5, functions as a potential coactivator for Sp1. J. Cell. Biochem. 91, 633-645.[CrossRef][Medline]
Shore, P. and Sharrocks, A. D. (1995). The MADS-box family of transcription factors. Eur. J. Biochem. 229, 1-13.[Abstract]
Shyy, J. Y. and Chien, S. (1997). Role of integrins in cellular responses to mechanical stress and adhesion. Curr. Opin. Cell Biol. 9, 707-713.[CrossRef][Medline]
Smilenov, L. B., Mikhailov, A., Pelham, R. J., Marcantonio, E. E. and Gundersen, G. G. (1999). Focal adhesion motility revealed in stationary fibroblasts. Science 286, 1172-1174.
Strobeck, M., Kim, S., Zhang, J. C., Clendenin, C., Du, K. L. and Parmacek, M. S. (2001). Binding of serum response factor to CArG box sequences is necessary but not sufficient to restrict gene expression to arterial smooth muscle cells. J. Biol. Chem. 276, 16418-16424.
Tang, D. D. and Gunst, S. J. (2001). Depletion of focal adhesion kinase by antisense depresses contractile activation of smooth muscle. Am. J. Physiol. Cell. Physiol. 280, C874-C883.
Tang, D., Mehta, D. and Gunst, S. J. (1999). Mechanosensitive tyrosine phosphorylation of paxillin and focal adhesion kinase in tracheal smooth muscle. Am. J. Physiol. 276, C250-C258.[Medline]
Tang, D. D., Wu, M. F., Opazo Saez, A. M. and Gunst, S. J. (2002). The focal adhesion protein paxillin regulates contraction in canine tracheal smooth muscle. J. Physiol. 542, 501-513.
Thomas, S. M., Hagel, M. and Turner, C. E. (1999). Characterization of a focal adhesion protein, Hic-5, that shares extensive homology with paxillin. J. Cell Sci. 112, 181-190.
Tomasek, J. J., Haaksma, C. J., Eddy, R. J. and Vaughan, M. B. (1992). Fibroblast contraction occurs on release of tension in attached collagen lattices: dependency on an organized actin cytoskeleton and serum. Anat. Rec. 232, 359-368.[Medline]
Turner, C. E. (2000a). Paxillin and focal adhesion signalling. Nat. Cell Biol. 2, E231-E236.[CrossRef][Medline]
Turner, C. E. (2000b). Paxillin interactions. J. Cell Sci. 113, 4139-4140.
Wang, N. and Ingber, D. E. (1994). Control of cytoskeletal mechanics by extracellular matrix, cell shape, and mechanical tension. Biophys. J. 66, 2181-2189.[Abstract]
Wang, N., Butler, J. P. and Ingber, D. E. (1993). Mechanotransduction across the cell surface and through the cytoskeleton. Science 260, 1124-1127.[Medline]
Weiskirchen, R. and Gunther, K. (2003). The CRP/MLP/TLP family of LIM domain proteins: acting by connecting. BioEssays 25, 152-162.[CrossRef][Medline]
Woods, A. J., Roberts, M. S., Choudhary, J., Barry, S. T., Mazaki, Y., Sabe, H., Morley, S. J., Critchley, D. R. and Norman, J. C. (2002). Paxillin associates with poly(A)-binding protein 1 at the dense endoplasmic reticulum and the leading edge of migrating cells. J. Biol. Chem. 277, 6428-6437.
Yuminamochi, T., Yatomi, Y., Osada, M., Ohmori, T., Ishii, Y., Nakazawa, K., Hosogaya, S. and Ozaki, Y. (2003). Expression of the LIM proteins paxillin and Hic-5 in human tissues. J. Histochem. Cytochem. 51, 513-521.